UNIVERSITA' DEGLI STUDI DI PADOVA

Facoltà di scienze MM.FF.NN.

Dipartimento di Biologia

DOTTORATO DI RICERCA IN :

FISIOLOGIA MOLECOLARE E BIOLOGIA STRUTTURALE

XIX CICLO

A COMBINED COMPUTATIA COMBINED COMPUTATIA COMBINED COMPUTATIA COMBINED COMPUTATIONAL AND EXPERIMENTAONAL AND EXPERIMENTAONAL AND EXPERIMENTAONAL AND EXPERIMENTAL L L L

APPROACH IN THE STRAPPROACH IN THE STRAPPROACH IN THE STRAPPROACH IN THE STRUCTURAL INVESTIGUCTURAL INVESTIGUCTURAL INVESTIGUCTURAL INVESTIGATIONS ATIONS ATIONS ATIONS

OFOFOFOF METALLOPROTEINSMETALLOPROTEINSMETALLOPROTEINSMETALLOPROTEINS

Coordinatore : CH.MO PROF. BENEDETTO SALVATO

Supervisore : CH.MO PROF. LUIGI BUBACCO

Correlatori : CH.MO PROF. MARIANO BELTRAMINI

DOTT. MAURIZIO BENFATTO

Dottorando : Stefano Marino

31 dicembre 2006

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INDEX:

1) Chapter1: General introduction pag. 5 1.1 Structure-function relationship in protein pag. 6 1.2 Methods in structural biology pag. 7 1.3 Computational methods in biology pag. 9 1.4 Scope of the thesis pag. 11 2) Chapter 2: structural features that govern enzymatic activity in carbonic anhydrase from a low temperature adapted fish Chionodraco hamatus pag. 15 3) Chapter3: Molecular mechanics investigation into S. antibioticus Tyrosinase activity toward chlorophenols and naphthols pag. 31 4) Chapter4: Role of tertiary structure in the diphenol oxydase activity of Octopus vulgaris Hemocyanin pag. 45 5) Chapter 5: A successive sub-grouping method for multiple sequence alignment analysis pag. 59 5.1 Introduction and Methods pag. 60 5.2 Applications pag. 65 5.3 Summary and perspectives pag. 71 6) Chapter 6: Summary and conclusions pag. 75

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CHAPTER 1

GENERAL INTRODUCTION

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Proteins are the most important building blocks of a living cell, being responsible for a huge variety of functions and implied in nearly all cellular structure, functions and regulations. They are regular, linear polymers composed of amino acids and they share a set of precise rules in their composition. As will be described later, the sequence of amino acids is usually sufficient to produce a well defined three-dimensional structure. This, in turn, serves to perform the most diverse functions in living organisms. The comprehension of the whole structure of the native fold of a protein is a fundamental step for understating how proteins accomplish their physiological tasks, and the achievement of this goal require a comparative approach, with results from different methods (from biophysics and biochemistry to bioinformatics) that have to be integrated for a successful investigations. Some of these approaches and methods, particularly important for the work of this thesis, will be briefly discussed in this introductory chapter,

1.1 Metalloproteins

It has been estimated that nearly one third of all proteins binds one or more metal ions as prosthetic groups. Many of these proteins contain metals like magnesium and calcium that often serve roles in structural stabilization and/or in protein regulation, so these metals have often great direct physiological concern, as it is evident for the variety of calcium dependent biological process and pathways (i.e. muscular proteins, Calcium dependent pumps, Ca-Voltage dependent channels and so on). A significant part of all metalloproteins contain transition metal ions such as iron, zinc, copper, nickel, molybdenum and vanadium. Several of these metals, in particular iron and copper, that are the most frequent, are redox active having a redox potential within the physiological range defined by the processes that occurs in biological systems. Proteins containing such metals are often entailed in processes involving exchange of electrons with the environment such as catalysis and electron transfer. The metals are bound to proteins through interactions with amino acid residues: most frequently they are bound (with different strengths that depend on both the properties of the ligand and of the metal ion) to Histidine, aspartic acid, asparagines, cysteine, methionine. The coordination geometry of the bound metal ion is determined by the position and orientation of the coordinating residues within the protein matrix and the preference for the metal ion. Thus the metal coordination sphere is pre-organized, which often leads to metal coordination geometries that differentiate from the energy minimum corresponding geometry (i.e. energy minimum corresponding configuration of the simplified mimetic models, 1) : this aspect implies some complication in fine structural analysis and will be faced in chapter 3. Additionally, the protein matrix plays an essential role in the intermolecular recognition between, for example, two proteins in electron transfer or protein-protein interactions that can modulate enzymatic activity. A strong modulation of metallic reaction centers reactivity will be faced in chapter (Hc) of the present work, in which an effective control on protein capabilities is carried out by modulating metallic centre accessibility with strong intra-molecular interactions. A differentiation have to be made between endogenous ligands and exogenous ligands: the former are amino acids of the protein that coordinate the metal (for example histidine in the case of the proteins

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investigated in this work) while the latter are represented by ligands external to the protein (solvent molecule , solutes or other various non amino acidic) that coordinate the metal. Chapter 2 deals with a Zinc protein for which the exogenous ligand is a water molecule; in chapter 3 and 4 are studied type-III copper containing proteins (Tyrosinase and Hemocyanin) for which the exogenous ligand is molecular oxygen. The properties of the coordinated metal may be modulate by the protein matrix and/or by a change in solution conditions (pH, water exchange rate in the enzymatic cleft, etc) or the binding or release of signaling molecules. For these reasons protein bound metals are allowed to fulfill a large repertoire of functions, while each metalloprotein is adapted to fulfill its function in a highly specific and selective manner. 1.2 Methods in structural biology

Bio-molecules are too small to be see in detail even with the most advanced light microscopes (principally for the marked difference between dimension of sample molecules and incident light used by microscopes). The experimental methods that structural biologists use to determine their structures generally involve measurements on vast numbers of identical molecules at the same time. These methods include among others: 1) X-ray diffraction techniques, like crystallography 2) spectroscopic techniques like NMR, EPR and X-ray absorption based techniques (XAS) 3) small angle scattering based methods (SAS) The differentiation of these methods derives from the nature of the physical phenomenon at the basis of each method: 1) for X-ray crystallography this is the diffraction of light by the electrons of the sample analyzed 2) for NMR and EPR is the modification on the state of respectively nuclei or electrons (within an external magnetic field) of the sample atoms after interaction with the radiation. For XAS techniques is the modification induced in the sample atoms after absorption of X-ray light (like promotion of inner level electrons to continuum state in XANES spectroscopy, discussed with more details below in this chapter) 3) for SAS is the scattering pattern after elastic interaction between beam of particles that is collected and analyzed for reconstruction of the overall shape of an object of investigation. The building up of a significant amount of structural data generated in decades of experimental work using the method mentioned above defined the basis of a theoretical approach, computational biology, that structural biologists now use to understand structure and structure function relationships in biological molecules. A wide variety of methods have been developed in these field. A distinction in two broad classes have to be made for those methods. The first class is centered on chemical/physical computations, more precisely defined chemo-informatics) and the second class dealing with pattern discovering and with sequence alignment problems (bioinformatics). The first class enlists methods of molecular modeling (Homology modeling, docking among others) that will be further discussed below. The second class include methods for treating multiple sequence alignment (like ClustalW), for pattern discovery on DNA sequences (for examples: consensus sequence characterizing promoter sequences) and for motifs individuation in protein sequences (for examples Zinc finger domains). This thesis mainly concern with chemo-informatics methods applied to protein structure and protein structure relationship investigations. While applying these methods to metalloproteins that have transition metal as prosthetic groups some complications are introduced, in particular for their

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theoretical treatment. In particular in the latter case the complications, due to the formulation for the basis sets for transition metal, are heavy and well confident solutions are not easy to obtain. In chapter 3, a light quantum chemical treatment of the type III copper binuclear centre of Tyrosinase is presented, and some aspects of the above mentioned difficulties will be discussed in detail. Below is presented first a short discussion of XANES experimental technique that have been employed in this work. XANES has been very informative for the case of the Zn protein carbonic anhydrase discussed in chapter 2. Then some basics, thought to be useful for the comprehension of the result presented in this thesis, on molecular modeling methods and principle are described.

1.2.1 XANES spectroscopy

XAS: The X-ray Absorption Spectroscopy (XAS) is currently a widely used technique giving information on the local structure and on the electronic states in gas-phase, molecular and condensed matter. X-ray absorption spectra are obtained by tuning the photon energy in a range where bound electrons can be excited (0.1-100 keV photon energy). This technique is usually applied at synchrotron radiation facilities providing intense and tunable x-ray beams. The spectral region near a core excitation (near-edge region) is usually called XANES (x-ray absorption near-edge structures). The latter techniques has been used in one chapter of this thesis, so few details will be given only for XANES energetic region of the X ray absorption spectra. XANES. XANES indicate the absorption peaks due to the photo absorption cross section in the X-ray Absorption Spectra (XAS) observed in the energy region, extending over a range of about 100 eV, between the edge region (energetic region by which the absorbing atom has its maximum value of absorbance, i.e. the energy of ionization of the absorber) and the EXAFS ( region extending from about 150 eV beyond the ionization potential, i.e for energetic regions beyond 150 eV from the edge). Edge energetic region is typical of the absorbing atom. In the absorption edge region of metals the photoelectron is excited to the first unoccupied level above the Fermi level. In the XANES region, starting about 5 eV beyond the absorption threshold, because of the low kinetic energy range (5-150 eV) the photoelectron backscattering amplitude by neighbor atoms is very large so that multiple scattering events become dominant in the XANES spectra. The different energy range between XANES and EXAFS can be simply explained by the comparison between the photoelectron wavelength λ and the interatomic distance of the photo absorber-back scatterer pair. The photoelectron kinetic energy is connected with the wavelength λ by the following relation:

that means that for high energy the wavelength is shorter than interatomic distances and hence the EXAFS region corresponds to a single scattering regime; while for lower E, λ is larger than interatomic distances and the XANES region is associated with a multiple scattering regime. Much chemical information can be extracted from the XANES region: formal valence (very difficult to experimentally determine in a nondestructive way); coordination environment (e.g., octahedral, tetrahedral coordination) and subtle geometrical distortions of it. Transitions to bound vacant states just above the Fermi level can be seen. Thus XANES spectra can be used as a probe of the unoccupied band structure of a material. As presented in chapter 2, with opportune computational tools it is possible to use XANES spectroscopy also for investigations on protein in solution: a detailed picture (relative geometries of atoms around the absorber) of the reaction centre, with considerable more resolution than that from X-ray diffraction techniques, can be obtained also for metalloproteins,

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otherwise difficult to treat spectroscopically, like EPR-silent containing metal atoms (for example Zn containing proteins).

1.3 Computational methods in biology: introduction

The increasing amount of information deriving for astonishing development of both molecular biology and computer sciences (disciplines with exponential growth starting form the 70s) has soon make it clear that a great efforts should have been dedicate to write biological specific algorithms and software. Bioinformatics and chemo-informatics are two branches of biology and chemistry that have reached ever more importance in the recent past, bringing to methods now universally used and well accepted like Blast and many molecular modeling tools currently employed in pharmaceutical research. As to a more physically oriented field, important methods with direct applications in biology and biochemistry have also been developed, aiming to investigate protein structure and reactivity taking into account electrons (14,16). Among these a particular citation goes to some quantum chemistry methods, like DFT, which from some years are quite successfully applied to biomolecules (12,13,15). In the last part of this introduction it will be presented a description of basic principles and some underlying theory of molecular modelling techniques employed in this thesis. In this work it will be used some of the most spread molecular modelling techniques, like homology modelling and Docking; in this section it will be faced some basic theoretical aspects (as well as some terminology and definitions used) of the modelling methods employed during the thesis work.

1.3.1 Molecular mechanic (MM) forcefield : The term molecular mechanics refers to the use of Newtonian mechanics to model molecular systems. The potential energy of all systems in molecular mechanics is calculated using force fields (see next). Molecular mechanics can be used to study small molecules as well as large biological systems or material assemblies with many thousands to millions of atoms. All-atomistic molecular mechanics methods have the following properties: 1. Each atom is simulated as a single particle 2. Each particle is assigned a radius (typically the van der Waals radius), polarizability, and a constant net charge (implemented in the force field) 3. Bonded interactions are treated as "springs" with an equilibrium distance equal to the experimental or calculated bond length forcefield (FF) definition. In the context of molecular mechanics, a force field refers to the functional form and parameter sets used to describe the potential energy of a system of particles (typically but not necessarily atoms). Force field functions and parameter sets are derived from both experimental work and high-level quantum mechanical calculations. Functional form. The basic functional form of a force field encapsulates both bonded terms relating to atoms that are linked by covalent bonds, and nonbonded (also called "noncovalent") terms describing the long-range electrostatic and van der Waals forces. The specific decomposition of the terms depends on the force field, but a general form for the total energy can be written as

The bond and angle terms are usually modeled as harmonic oscillators in force fields that do not allow bond breaking.

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The functional form is highly variable. It can include potentials for hydrogen bonds, an "improper torsion" term to account for the planarity of aromatic rings and other conjugated systems, and "cross-terms" that describe coupling of different internal variables, such as dihedral angles and bond lengths. The non-bonded terms are most computationally intensive because they include many more pair wise interactions per atom. The van der Waals term is usually computed with a Lennard-Jones potential and the electrostatic term with Coulomb's law, although both can be buffered or scaled by a constant factor to produce better agreement with experimental observation. Parametrization of the forcefield . In addition to the functional form of the potentials, a force field defines a set of parameters for each type of atom. For example, a force field would include distinct parameters for an oxygen atom in a carbonyl functional group and in a hydroxyl group. The parameter set includes polarizability, atomic mass, and partial charge for individual atoms, and equilibrium values of bond lengths and angles for pairs, triplets, and quadruplets of bonded atoms. Although many molecular simulations involve biological macromolecules such as proteins, the parameters for given atom types are generally derived from observations on small organic molecules that are more tractable for experimental studies and quantum calculations. Parameter sets and functional forms are defined by force field developers to be self-consistent. Because the functional forms of the potential terms vary extensively between even closely related force fields (or successive versions of the same force field), the parameters from one force field should never be used in conjunction with the potential from another.

1.3.2 Molecular Docking

Computational molecular docking is a research technique for predicting whether one molecule will bind to another. Protein-protein, protein-DNA and protein-ligand (i.e. the binding of an organic molecule to a protein) docking predictions are all performed, though the techniques employed in each area are highly various. In this work only the Protein-ligand docking is treated, and it’s done by modeling the interaction between protein and ligand: if the geometry of the pair is complementary and involves favorable biochemical interactions, the ligand will potentially bind the protein in vitro or in vivo. Given a refined structure of both protein and substrates to be tested, the success of a docking program depends on two components: the search algorithm and the scoring function. The search algorithm: The search space consists of all possible orientations and conformations of the protein paired with the ligand. With present computing resources, it is impossible to exhaustively explore the search space this would involve enumerating all possible distortions of each molecule (molecules are dynamic and exist in an ensemble of conformational states) and all possible rotational and translational orientations of the ligand relative to the protein at a given level of granularity. Most docking programs in use account for a flexible ligand, and several are attempting to model a flexible protein receptor. Each "snapshot" of the pair is referred to as a pose. There are many strategies for sampling the search space. Here are some examples: 1) Use a coarse-grained molecular dynamics simulation to propose energetically reasonable poses 2) Use a "linear combination" of multiple structures determined for the same protein to emulate receptor flexibility 3) Use a genetic algorithm to "evolve" new poses that are successively more and more likely to represent favorable binding interactions In this work (chapter 3) a genetic algorithm approach is used to explore the conformational space of the protein ligand complex. The scoring function. The scoring function takes a pose as input and returns a number indicating the likelihood that the pose represents a favorable binding interaction.

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Most scoring functions are physics-based molecular mechanics force fields that estimate the energy of the pose; a low (negative) energy indicates a stable system and thus a likely binding interaction. 1.3.3 Homology modeling

In protein structure prediction, homology modeling, also known as comparative modeling, is a class of methods for constructing an atomic-resolution model of a protein from its amino acid sequence (the "query sequence" or "target"). Almost all homology modeling techniques rely on the identification of one or more known protein structures (known as "templates" or "parent structures") likely to resemble the structure of the query sequence, and on the production of an alignment that maps residues in the query sequence to residues in the template sequence. The sequence alignment and template structure are then used to produce a structural model of the target. Because protein structures are more conserved than protein sequences, detectable levels of sequence similarity usually imply significant structural similarity. The quality of the homology model is dependent on the quality of the sequence alignment and template structure. 1.3.4 Electrostatic potential calculations

Poisson Boltzmann Equation (PBE) is one of the most popular continuum models for describing electrostatic interactions between molecular solutes in salty, aqueous media. Continuum electrostatics plays an important role in several areas of biomolecular simulation, including: 1) simulation of diffusional processes to determine ligand-protein and protein-protein binding kinetics, 2) implicit solvent molecular dynamics of biomolecules, 3) solvation and binding energy calculations to determine ligand-protein and protein-protein equilibrium binding constants and aid in rational drug design or other predictive tasks (17), 4) and biomolecular titration studies. The Poisson-Boltzmann equation is a differential equation that describes electrostatic interactions between molecules in ionic solutions. The equation is important in the fields of molecular dynamics and biophysics because it can be used in modeling implicit solvation, an approximation of the effects of solvent on the structures and interactions of proteins, DNA, RNA, and other molecules in solutions of different ionic strength. It is often difficult to solve the Poisson-Boltzmann equation for complex systems, but several computer programs have been created to solve it numerically. The equation can be written as:

where represents the distance-dependent dielectric, represents the electrostatic potential,

represents the charge density of the solute , represents the concentration of the ion i at an infinite distance from the solute , zi is the charge of the ion, q is the charge of a proton, k is the

Boltzmann constant, T is the temperature, and is a factor for the distance-dependent accessibility of position r to the ions in solution. If the potential is not large, the equation can be linearized to be solved more efficiently (8). In this work the computer programs used for calculating the electrostatic potential distribution are: 1) Hex 4.5 (11) which solves the Poisson differential equation:

(∂ 2/∂ x2 + ∂ 2/∂ y2 + ∂ 2/∂ z2) V = - ρ/ε0 thus not accounting for counter-ions effects 2) APBS (10) which solves numerically the full PBE

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1.4 Scope of the thesis

This thesis deals with applications of computational methods in combibnation with experiments to investigate structures and structure-function relationships in metalloproteins. In recent years a big effort has been made to developed appropriate theoretical methods for a feasible treatment of biological molecules (3); from both computer power and algorithm development notable improvement has been made that have allowed development and tuning of robust and faster forcefield and interaction models able to appreciably model biomolecules . This allowed molecular modeling to become in few years one of the most powerful and used tool in pharmaceutical (5,6,7) and biotechnological research (4). Great development in computational structural biology were also registered in comparative modeling of proteins with unknown structure: Powerful predictive tools have been developed in past 10-15 years, and now are currently used in several fields of research, in particular for building structural proteomes in sequenced yeast and bacteria, but also in superior organism. In this scenario, one of the key aspects of future research in the field will be a more exhaustive and precise investigation of how well all this predictive methods work. For this task the integration for comparative purposes of experimental data and theoretical ones is fundamental, as it has been demonstrated by the CASP approach (11): the different categories evaluated in CASP related with different aspects of the protein folding In addiction to that there is the need to build new integrated tools, that can integrate (with the maximum degree of automation) experimental data (even on part of the problem) and theoretical ones; this means that not a fully resolved structure can be always necessary to obtain a reasonable model, but that different partial structural information can be implemented in a model as restraint to the searches in the conformational space. I think this will be one of the leading future approaches in structural biology. This thesis and its scope is springs from these considerations: In fact, in spite of the fact that the primary goal of presented results was their biological meaning, the objective by which the employed tools were chosen was the attempt to prove transferability between computational and experimental data. This means that informatics tools were chosen to be as homogenous as possible with experiments, to both rationalize the obtained experimental results and provide the ground to design new experiments.

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REFERENCES 1. Malmstrom BG (1994) Rack-induced bonding in blue-copper proteins. Eur J Biochem. 223(3):711-8 2. Pocker Y, Miao CH. (1987) Molecular basis of ionic strength effects: interaction of enzyme and sulfate ion in CO2 hydration and HCO3- dehydration reactions catalyzed by carbonic anhydrase II. Biochemistry. Dec 15;26(25):8481-6 3. Di Ventura B, Lemerle C, Michalodimitrakis K, Serrano L. (2006) From in vivo to in silico biology and back. Nature. 2006 Oct 5;443(7111):527-33. 4. Clark J, Singer EM, Korns DR, Smith SS. (2004) Design and analysis of nanoscale bioassemblies.Biotechniques. 36(6):992-6, 998-1001. 5. Ooms F. (2000) Molecular modelling and computer aided drug design. Examples of their applications in medicinal chemistry. Curr Med Chem. 2000 Feb;7(2):141-58 6. Moro S, Spalluto G, Jacobson KA. (2005) Techniques: Recent developments in computer-aided engineering of GPCR ligands using the human adenosine A3 receptor as an example. Trends Pharmacol Sci. 2005 Jan;26(1):44-51. 7. Sarno S, Moro S, Meggio F, Zagotto G, Dal Ben D, Ghisellini P, Battistutta R, Zanotti G, Pinna LA. (2002) Toward the rational design of protein kinase casein kinase-2 inhibitors. Pharmacol Ther. 93(2-3):159-68 8. Fogolari F, Brigo A, Molinari H. (2002). The Poisson-Boltzmann equation for biomolecular electrostatics: a tool for structural biology. J Mol Recognit 15(6):377-92. 9. Hex software and manual in pdf can be find following this link: http://www.csd.abdn.ac.uk/hex/ 10. APBS (Adaptive Poisson Boltzmann Solver) software and manual can be find following this link: http://apbs.sourceforge.net/ 11. CASP (Critical Assessment of Techniques for Protein Structure Prediction) results can be find following this link: http://predictioncenter.org/ 12. Bassan A, Blomberg MR, Borowski T, Siegbahn PE. (2006) Theoretical studies of enzyme mechanisms involving high-valent iron intermediates. J Inorg Biochem. 100(4):727-43. 13. Linnanto J, Korppi-Tommola J. (2006) Quantum chemical simulation of excited states of chlorophylls, bacteriochlorophylls and their complexes. Phys Chem Chem Phys. 8(6):663-87. 14. Szilagyi RK, Solomon EI (2002) Electronic structure and its relation to function in copper proteins. Curr Opin Chem Biol. 6(2):250-8 15. Prabhakar R, Siegbahn PE, Minaev BF. (2003) A theoretical study of the dioxygen activation by glucose oxidase and copper amine oxidase. Biochim Biophys Acta. 2003 1647(1-2):173-8 16. Shao Y, Molnar LF, Jung Y, Kussmann J, Ochsenfeld C, Brown ST, Gilbert AT, Slipchenko LV, Levchenko SV, O'Neill DP, DiStasio RA Jr, Lochan RC, Wang T, Beran GJ, Besley NA, Herbert JM, Lin CY, Van Voorhis T, Chien SH, Sodt A, Steele RP, Rassolov VA, Maslen PE, Korambath PP, Adamson RD, Austin B, Baker J, Byrd EF, Dachsel H, Doerksen RJ, Dreuw A, Dunietz BD, Dutoi AD, Furlani TR, Gwaltney SR, Heyden A, Hirata S, Hsu CP, Kedziora G, Khalliulin RZ, Klunzinger P, Lee AM, Lee MS, Liang W, Lotan I, Nair N, Peters B, Proynov EI, Pieniazek PA, Rhee YM, Ritchie J, Rosta E, Sherrill CD, Simmonett AC, Subotnik JE, Woodcock HL 3rd, Zhang W, Bell AT, Chakraborty AK, Chipman DM, Keil FJ, Warshel A, Hehre WJ, Schaefer HF

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3rd, Kong J, Krylov AI, Gill PM, Head-Gordon M. (2006) Advances in methods and algorithms in a modern quantum chemistry program package. Phys Chem Chem Phys. 8(27):3172-91 17. Fogolari F, Tosatto SC. (2005) Application of MM/PBSA colony free energy to loop decoy discrimination: toward correlation between energy and root mean square deviation. Protein Sci. 14(4):889-901.

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CHAPTER 2

STRUCTURAL FEATURES THAT GOVERN ENZYMATIC ACTIVITY

IN CARBONIC ANHYDRASE FROM A LOW TEMPERATURE

ADAPTED FISH Chionodraco hamatus

Abstract

The carbonic anhydrase (CA1) family of zinc metalloenzymes includes many known

isozymes which have different subcellular distributions. The study described here is

focused on the identification of the structural features that define low temperature

adaptation in a Chionodraco hamatus protein, both at an atomic level for the reaction

center and for the tertiary structure of the protein. To this aim, a XANES/MXAN

analysis of the reaction center was undertaken for both a structurally characterized

Human CAII (CA2H) and for CA of C. hamatus (CAice). The higher structural levels

were analyzed by sequence comparison and homology modeling. To establish if the

structural insights acquired in fish CAs are general, theoretical models were generated by

homology modeling for three temperate climate-adapted fish CAs. The measured

structural differences between the two proteins are discussed in terms of the differences in

the electrostatic potential between hCAII and CAice. We conclude that modulation of the

interaction between the catalytic water molecule and the zinc ion could depend on the

effect of the electrostatic potential distribution.

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INTRODUCTION

The carbonic anhydrase (CA) family of zinc metalloenzymes includes many known isozymes which have different subcellular distributions: cytoplasmic (CAI, II, III, VII and XIII), plasma membrane associated (CAIV, IX, XII, XIV and XV), mitochondrial (CA VA and VB), or secreted (CAVI). CAs catalyze the hydration of carbon dioxide to yield bicarbonate ion and a proton. This reaction occurs via two distinct chemical steps (1). The first is a nucleophilic attack of zinc bound hydroxide to the substrate CO2 followed by the exchange of the product bicarbonate with a water molecule. The metal-bound hydroxide is regenerated from zinc-bound water by transfer of a proton to bulk solvent. The rate constant kcat generally reflects proton transfer between zinc-bound water and an active site “shuttle” residue in the second step. Subtle structural differences among the CA isozymes contribute to define a wide range of kcat values (10

3-106 s-1) measured for this step in different isozymes. Different residues may function as the intermediary proton shuttle between zinc-bound water and bulk solvent in isozymes. There are several crystal structures of CAs in the PDB and for vertebrate enzymes, most of them mammalian CAII (with or with out a bound inhibitor). Crystallographic structures are also available for both mammalian CAI (again, with or without a bound inhibitor) and CAIII (the S-glutathiolated form of rat CAIII). All of these structures provide key insights into structure-function relationships for this enzyme (2). The zinc cofactor is placed at the base of the conical active site cleft where it is coordinated to three histidine residues and one water molecule to yield a tetrahedral geometry. At physiological pH, the fourth zinc ligand is a hydroxide molecule which acts as a nucleophile to catalyze the hydrolysis of CO2 and esters (at a rate near to the limit posed by substrate diffusion) (3). Other important structural features relate to residues that either protrude into the catalytic cleft or contribute to generate a hydrophobic pocket in which the substrate seems to be held. Among the first, the proton shuttle mentioned above plays a key role in the catalytic process. For instance, H64 is the catalytic proton shuttle in CAII (kcat=10

6 s-1) (4,5). This residue is hydrogen-bonded to a zinc-bound water molecule through bridging solvent molecules across which the product proton has been transferred, as proposed by Hakansson et al.(6). The side chain of H200 is the best candidate for a proton shuttle in CAI (kcat =2x10

5 s-1) (7) due to its proximity to the zinc ion and the observed pKa value (8). In contrast, it seems that no proton shuttle is present in the active site of CAIII (kcat =1x10

4 s-1), in which the product proton is probably transferred directly to the bulk solvent (9). The active site cleft is split into hydrophobic and hydrophilic regions. Mutagenesis and structural analyses of the hydrophobic pocket adjacent to the zinc-bound hydroxide indicate this region as the site of CO2 association and that the hydrophobicity modulates the catalytic activity (3,10). The proposed association would place the substrate at about 3 Å from the zinc atom. There is a well-defined hydrophobic pocket adjacent to the zinc-bound hydroxide group. The amino acids (aa) involved are, W209, V121, and L/F198 that define the mouth and the side plus V143 that defines the bottom. The amino acids that define the hydrophobic pocket are completely conserved in carbonic anhydrase II (with L198) and CAIII (with F198). Steric effects occurring at the mouth of the pocket have been proposed to justify the lower catalytic activity measured for CAIII; these seem to depend on F198 (34). Among the polar residues of the active site, the hydroxyl group of the Tl99 side chain forms hydrogen bonds with the zinc-hydroxide group and the E106 side chain to form a Zn-OH-/T199/E106 hydrogen bond network (11,6), which is completely conserved in all animal carbonic anhydrase isozymes. The hydrogen bond between T199 and zinc-hydroxide has been postulated to be important to maintain the orientation and reactivity of the zinc-hydroxide group (12), catalyzing proton transfer, and discriminating between protic and aprotic anions (13).

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Amino acid substitutions at position 65 of CAII demonstrate that the size of the side chain is critical in the assembly of a bridging solvent network between the zinc-bound solvent and the shuttle residue H64. In particular, the presence in position 65 of both alanine (as in human CAII) or serine (as in almost all other vertebrate CAII) is compatible with a high proton shuttling rate for the adjacent His64 (5,14). Considering more physiological aspects, carbonic anhydrase plays a crucial role in the excretion of metabolic CO2 in all vertebrates. In fish, CO2 produced in the tissues is rapidly hydrated to HCO3

- by the action of erythrocyte CA. The generated HCO3- then diffuses into the blood stream,

where it accounts for almost 98 % of the total carbon dioxide stored and transported in the plasma (15,16). At the respiratory epithelia in gills or skin, CA catalyzes the rapid dehydration of HCO3

- to molecular CO2, which then diffuses passively into the ventilatory water stream. The CO2/HCO3

- system constitutes the most important physiological buffers for acid–base regulation (17). The Antarctic icefish of the family Channichthyidae (suborder Nothotheniodei), to which Chionodroco hamatus belong, are a unique example of adult vertebrates lacking hemoglobin and functionally active erythrocytes, and possessing only a small number of erythrocyte-like cells (18).The absence of hemoglobin does not represent a dramatic limitation to oxygen transport in the icefish. On the other hand, the very limited number of erythrocyte-like cells (and circulating CA) may compromise the CO2/HCO3

- equilibrium in the blood (19). However, the elevated solubility of CO2 in water at low temperatures, and the unusual characteristics of the circulatory system of ice fish, contribute to an efficient excretion of CO2. In teleosts, CA has been found in various tissues. It appears to be present in high concentrations in the gills (20), where it plays an important role in osmoregulation, nitrogen (ammonia) excretion, acid–base balance, and gas exchange (21). Comparisons between activity of C. hamatus gill CA (CAice), another icefish Treomatus bernachii (an Antarctic fish with hemoglobin and red blood cells) and the temperate-adapted fish Anguilla anguilla showed that CA activity for the fish lacking hemoglobin is high up to a temperature of 30 C°. Above this temperature, there is a dramatic decrease of activity that becomes virtually absent at 37 C°. At this temperature, A. anguilla CA is still at its maximum level of activity (22). With these data, we were particularly interested in the identification of the structural features that define low temperature adaptation in the C. hamatus protein, both at an atomic level for the reaction center as well as the tertiary structure of the protein. A XANES/MXAN analysis of the reaction center was undertaken for both a structurally characterized human CAII (CA2H) and for CAice. The higher structural levels were analyzed by sequence comparison and homology modeling. To evaluate the general applicability of the structural insights acquired by our study of CAs, we created some theoretical models using homology modeling, generated for three temperate climate-adapted fish CAs. MATERIAL AND METHODS

Protein purification - Cytoplasmic carbonic anhydrase of the icefish C. hamatus gill filaments (Ice-CA) was obtained as previously described (22). Ice-CA was purified by FPLC affinity chromatography on p-aminomethylbenzene-sulphonamide immobilized on cyanogen-bromide-activated agarose gel (23, 22). The gel column (1.6 x 20 cm), fitted to a AKTA-Pharmacia FPLC system, was equilibrated with: Tris 25 mM, Na2SO4 100 mM, adjusted to pH 8.7 with HCl and rinsed with Tris 25 mM, NaClO4 300 mM, adjusted to pH 8.7 with HCl; then the enzyme was eluted at 8 ml h-1 by CH3COOH 100 mM, NaClO4 500 mM, pH 5.6 at 4 °C. Protein elution was monitored by measuring the eluate absorbance at 280 nm, and all fractions containing CA activity, measured by an electrometric method (22), were pooled and concentrated by ultra filtration with YM10 membrane

18

(Amicon Corp, Lexington, U.S.A.), under nitrogen pressure (7x105 Pa). All purification steps were carried out at 4 °C. Sample preparation - Powdered solid solutions of the protein in saccharose were obtained starting from protein dialyzed against the appropriate buffer for 24 hour at 4° C. Buffers were prepared for a final concentration of 100 mM using secondary and tertiary ammonium bases, such as piperazine and triethanolamine, with glycine and aliphatic carboxylic acids (24). The protein solution, at a concentration of about 10 mg/ml and containing saccharose in a saccharose/protein ratio of 3:1 w/w (corresponding to a concentration of about 0.250 g of protein per gram of final solid-solution), was then rapidly frozen in liquid nitrogen and lyophilized. The solid samples of the CA form were obtained simply by lyophylization of the protein solution in the presence of the cryoprotectants. The specimens for XAS measurements were prepared by pressing about 50 mg of lyophilized powdered protein solid-solution under 140 atm in a small home-made press having a chamber with lateral dimensions of 2.2 x 0.3 cm; the slides obtained were about 0.2 cm thick. Solutions of irradiated proteins, used to evaluate the integrity of the samples, were obtained by dissolving the corresponding slide in milliQ® water and dialyzing against the appropriate buffer for 48 hours at 4°C. The XANES (X-ray Absorption Near Edge Structure) measurements were performed at the LURE synchrotron facility, in the D21 (EXAFS II) beamline. The Zn K-edge XANES signals were collected in fluorescence mode by a seven-element CANBERRA® Ge detector. A Si (311) crystal was used as a monochromator. The energy range was from 9600 to 9800 eV. (Calibration of energy was performed by means of a zinc foil reference). The energy resolution was 0.3 eV, with a counting time of 6 s/point. The absorption spectra were collected using a Perkin-Elmer Lambda 16 double beam spectrophotometer equipped with a thermostated cell holder. The XANES data analysis was performed by using the MXAN method (25) capable to fit the energy region from the edge up to 200 eV above the threshold in terms of selected structural parameters. The MXAN method performs the minimization of the residual function Rsq defined as:

∑=

−−=

m

i

ii

th

isq

m

yyR

1

21exp ])[( ε,

where m is the number of experimental data, yith and yi

exp are the theoretically computed and experimental measured values of the absorption coefficient, respectively. Our estimate of the experimental error is �i, considered constant in the fitting procedure, and in our case, considered equal to about 0,8 % of the experimental edge jump. Details of the MXAN procedure are described in (26,27,28,29).

The cluster of atoms used in the fitting procedure includes atoms up to 6.3 Å from the absorber and the muffin-tin radii are chosen according to the Norman criterion with a 5 % overlap. The number of atoms in the cluster used for the calculation was chosen on the basis of a convergence criterion. This cluster was generated starting from the PDB coordinates of the human CA II (PDB reference, 2cba). No core-hole was considered in the final state potential. Fitting Strategy The atomic positions are described using a polar coordinate system; the absorbing atom is placed at the origin of the coordinate frame. The atoms of one protein ligand or aa residue are considered as a perfectly rigid molecular group. Therefore, when the Zn-N distance is changed in the fitting, all atoms of the histidine rings move accordingly. In the fit, the bond of the Zn-O of the coordinated water molecule was allowed to vary in both in length and polar angles. The ionization energy Eo is 9660 eV (see figures 3 and 4), and the spectra were consistently normalized to 1. The contribution of the preedge energy region was subtracted using the standard procedure.

19

Modeling –The first step in choosing the appropriate templates was to use the structure prediction server BioInfoBank Meta Server (http://bioinfo.pl/meta/). The search can be customized choosing the prediction methods that have to be considered by 3D-jury; specifically, the threading method was ‘deselect’. Then, the option ‘model-to-all’ was chosen, which considers the ten best models from each server previously selected to be taken into account (30). For the modeling procedure, the Swiss Model method was used (http://swissmodel.expasy.org//SWISS-MODEL.html) in combination with the DeepView 3.7 (sp5) program (http://swissmodel.expasy.org/spdbv/). Electrostatic potential distributions were calculated by using numerical solution of the Non-Linear Poisson–Boltzmann equation implemented in APBS software (http://apbs.sourceforge.net/). All of the CA structures were superimposed and similarly oriented before the calculations. AMBER forcefield was used for partial atomic charges and radii, internal and external dielectric constant values of 2 and 78 were used, and solvent and ionic probe radii of 1.4 and 1.9 Å (with mono-valent salt concentration of 150mM) . For visualization PyMol (http://pymol.sourceforge.net/) with APBS plugin (APBS_tools.py) was used: APBS calculated values were plotted on a gaussian type surface, as it can be seen in Fig2 (according to the coding colour described in the text of Figure 2). Hex 4.5 (http://www.csd.abdn.ac.uk/hex ) was also used for electrostatic potential calculation, which solves the differential Poisson equation for electrostatic potential and which gives as output a value for the potential (in mV) for the tested protein. To test the models, the SAVS validation server was used (http://nihserver.mbi.ucla.edu/SAVS/) . Sequence analysis - For sequence analysis, Blast (http://www.ncbi.nih.gov/BLAST/), ClustalW (http://www.ebi.ac.uk/clustalw/, ) and custom designed software were used. RESULTS

Template structure for the MXAN analysis - The choice of an adequate structural template to be used in the MXAN analysis of the absorption k-edge data for CAice was carried out using the Metap server. The ranking algorithm used was 3D-jury (see Material and Methods section). The structural file resulted was made up of best-confidence predictions: (all hits were with a score above 226). 1flj, PDB code for S-gluathiolated Rattus norvegicus CAIII, with a resolution of 1.9Å (31). 1v9i, Bos taurus CAII , with a Q 253>C 253 mutation and a resolution of 2.5 Å. 2cba, Homo sapiens CAII (CA2h), with a high resolution of 1.54 Å (6) 12ca, Homo sapiens CAII with an Ala 121> Val 121 mutation and a resolution of 2.40 Å (3) 1hcb, Homo sapiens CAI complexed with bicarbonate and a resolution of 1.60 Å (32). Further criteria used in the selection of the template crystallographic structure were the absence of an exogenous ligand in the active site, wild type sequence, and the level of resolution of the crystal structure. The application of these criteria resulted in two plausible templates: 1flj and 2cba. In order to choose between these two templates, a sequence analysis was carried out among the genes of CAice, CA2h (that corresponds to the structure 2cba.pdb) and CA3r (that corresponds to the structure 1flj.pdb) (Figure 1). In addition to the general sequence homology, attention was given to the conserved residues that are believed to be involved in the reaction mechanism, the residues that in the crystal structures are close to the reaction center, and to the fully conserved residues throughout the sequence. Using the crystal structures 2cba and 1flj and the sequence alignments of these with CAice, it was possible to identify the amino acids in CAice that are likely to be within 10 Å from the zinc atom. The result of the comparison of the selected amino acids indicates that CAice has a higher similarity to (CA2h) 2cba, 88% aa identity than (CA3r) 1flj 76%, identity.

20

Figure 1: Alignment of C. hamatus CA, H. sapiens CAII, R. norvegicus CAIII. Label on the sequence: #, refers to the positions near the reaction center that are different in mammalian CAII and CAIII. Underlined are the amino acids placed (with one or more of their atoms) within 10 Å distance from the zinc atom.

A key residue is His64, which is the proton shuttle of the reaction mechanism in all mammalian CAIIs. This His is absent in the corresponding position of CAIIIr. CAice has a His residue in the position corresponding to 64 in CAII. Furthermore, this His residue in CAice is part of a pattern of four highly conserved residues (-GHSF-) present in mammalian CAII. As mentioned in the introduction, another relevant amino acid for the reactivity of CA is in position 198. This is a phenylalanine in all CAIIIs, but in both mammalian CAII and CAice, it is a leucine which provides further support for the proposed similarity between CAII and CAice. For the MXAN analysis, the cluster used to generate the simulated absorption spectra includes all atoms within 6.3 Å from the absorption center. Thus, a detailed comparison of all aa positions in this region in 2cba and 1flj was undertaken. An important difference is the presence of a Phe in position 95 in 1flj while a Leu is present in 2cba. This Leu, which is conserved in CAII, is also present in CAice. All atoms of residue 95, which is present in the sequence between the two Zn-coordinating histidines (94 and 96), were included in the cluster used for the MXAN analysis, making 2cba the stronger candidate as template. Furthermore, among all of the structures selected by the 3-D jury, 2cba had the highest resolution, confirming it as an ideal starting point for the structural simulations. CAice Modeling. Starting from the CAice sequence and using the 2cba structure as template (overall 62% of amino acidic identity), the Swiss Model server was used to generate a molecular model of the icefish protein. The refinement of the obtained model was carried out with DeepView 3.7. The resulting model was tested using the SAVS validation server. The obtained model for CAice was a starting point for other computations aimed to compare a few relevant physical and chemical properties of CAice and 2cba. The first property analyzed was the electrostatic potential distribution using, with undistinguishable results both Hex4.5 and APBS. The two proteins have a significantly different electrostatic potential distribution. CAice has a calculated overall negative potential of -0.22 mV (using Hex4.5). The corresponding value for 2cba (with no water, except the Zn- coordinated one) is +0.70 mV.

21

A visualization of the electrostatic potential plotted on the Gaussian-type surface is shown in figure 2 (generated using APBS). Different electrostatic potential distributions between the two proteins can be attributed to both a different propensity to the intermolecular interactions and to the properties of the reaction channel that leads the substrate to the zinc atom. To evaluate the possibility that the observation of a difference in the electrostatic potential distribution between the generated model of CAice and 2cba is more than coincidental, a broader set of high activity carbonic anhydrase (CAII-like) was considered. These were all from fish living in temperate climates: red blood cell (rbc) CA from Onchorinchus mykiss (CAIIoncho, >gi|32187014|gb|AAP73748.1|),rbc CA from Danio rerio (CA2Z, gi|35505160|gb|AAH57412.1]), cytoplasmic CA from Onchorinchus mykiss (CAIoncho, gi|41059441|gb|AAR99329.1). For all of the CAs mentioned above, a molecular model was generated (following the procedure described for icefish CA); the sequence homologies to 2cba were 63% with CA2Z, 61% with CAIIoncho, and 63% with CAIoncho. For each of the resulting molecular models, the electrostatic potential distribution was calculated. The calculated potential distributions for rbc CAs are similar to that calculated for human CAII, with an overall positive potential (figure 2) in an uneven distribution of neutral, negative and positive potential throughout the molecular surface. The unusual electrostatic potential distribution observed for CAice was also found in the molecular model generated for CAIoncho. This cytosolic isoform is also found in gills and has a catalytic activity lower than that of the rbc Onchorinchus mykiss CAII isoform (35). For comparative purposes, the electrostatic potential distribution was also calculated for the CA of the salt-tolerant unicellular green alga Dunaliella salina (pdb code 1y7w, 33).

A B C D E F

Figure 2: Electrostatic potential distribution mapped on the Gaussian-type surface calculated by APBS and visualized with PyMol for CA2Z (A), CA2h (B ), CA2Oncho ( C), CA1Oncho(D ), CAice ( E), and CAalga (F). The structures are graphically depicted looking down the active site cleft (first row) and in a 180° rotated view (second row). The potentials are contoured to range from -1.5 kT per electron (red) to +1.5 kT per electron (blue).

XANES/MXAN analysis on C. hamatus gill CA and Homo sapiens CAII - In figure 3, the normalized experimental k-edge absorption spectra are presented for both the human and the icefish carbonic

22

anhydrase. The two spectra do not show any chemical shift in the time course of the measurements. The most important difference between the two spectra is the feature at about 10 eV that is sharper in the ice fish protein (curve A in figure 3). The best fit of the k-edge absorption spectra of the human CAII is presented in figure 4A. The agreement between the experimental data and the calculated spectra is excellent over all spectral regions.

-20 0 20 40 60 80

B

Abs

orba

nce

(a.u

.)

E-Eo (eV)

A

Figure 3: Zn K-edge XANES experimental spectra for iceCA C. hamatus (A) and hCAII H. sapiens (B)

The value of the Rsq of the best fit is 4.06. The starting atomic coordinates for the fitting procedure were those obtained from the 2cba PDB file, obtained from the crystal structure of CAII from human erythrocytes. The one-shot calculation of the edge spectra using the crystallographic coordinates gives a Rsq value of 13.77. In Table 1 are reported the bond length values obtained from the fitting procedure for both proteins and, for comparison, also those obtained from the crystal structure of 2cba. The human protein has a greater asymmetric distribution of Zn-N distances which results in a statistically significant difference.

Figure 4: Comparison of the best-fit calculation (continuous line) and the experimental data (crosses) of CA2h (Panel A) and CAice (Panel B).

0 20 40 60 80

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

Abs

orba

nce

(a.u

.)

E-Eo (eV)

B

0 20 40 60 80

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

Abs

orba

nce

(a.u

.)

E-Eo (eV)

A

23

Furthermore, the B factors for both the Zn and the atoms of the metal ligands in the 2cba PDB file lead to an expected uncertainty for the bond lengths in the crystallographic data of about 0.25 Å. The reported Zn-O distance for the water molecule coordinated to the Zn atom (H2O 263 in 2cba) shows an even higher value of the B factors which further increases the uncertainty to 0.32 Å. In figure 4B is presented the best fit of the k-edge absorption spectra of cytosolic CA from C. hamatus. The agreement between the experimental data and the calculated spectra is again excellent over all spectral regions, and the value of the Rsq of the best fit is 4.44. The rationale for the choice of the starting coordinates for the MXAN analysis has been in part presented above. Further considerations relate to the result of the simulations of the edge spectrum based only on the coordinates of the crystal structure 2cba (Rsq = 27,36) and 1flj (Rsq = 38,79) which indicate a higher structural similarity of the considered atom cluster for the human enzyme. As mentioned above, a further advantage of the 2cba PDB file is its higher resolution. On these bases, 2cba was used as the template structure for the MXAN fitting procedure for both proteins. Among the structural parameters explored in the fitting procedure, the most important are the H2O263–zinc distance (figure 5a) and the theta angle (figure 5b) for the Zn-coordinated water molecule. The H2O263–zinc distance resulted to be the more relevant structural feature as can be seen in figure 5A. The theta angle for the Zn-coordinated water molecule shows a less pronounced minimum in the Rsq profile for both proteins. (For clarity, only the data for the human protein is reported in figure 5B ). The other determinant structural parameters were the theta angle and the distance from the Zinc atom and Oδ of Thr199. In comparing human CA and Caice, some important considerations are due on the interplay of zinc-coordinated water and Thr199. In the structural models of the active site obtained in the fitting procedure, a different position is observed for Thr199 (figure 6). The Zn-Oδ (T199) distance is shorter in Human CA as is the Zn-O (H2O263) distance. This is consistent with the observation that Thr199 and the coordinated H2O are involved in the H-bond network that represents one of the most conserved structural features of cytosolic carbonic anhydrases (CAI, CAII, CAIII).

Figure 5: Panel A) Rsq profile as function of the bond length of Zn-O of the H2O 263. Line a) CAIIh, line b) CAice. Panel B) Rsq profile as function of the theta angle between the z-axis and the Zn-O bond. For simplicity, the 0° value corresponds to the orientation of the Zn-O bond in the crystal structure of 2cba. The zinc-coordinated histidines, that in the fitting procedure were allowed to move, were not effective parameters in the optimization of the fit. The final structural model and the relative bond lengths are

A B

24

presented in figure 6 and in the table (Table 1), respectively. The histidines’s ligands are almost superimposable and are not likely to be responsible for the difference between the two proteins analyzed. The first observation is on the comparison between the structural data obtained from the fitting of the human protein and the available crystallographic structure2cba. As shown in the table, the differences for all considered parameters are within the values of the associated errors. These must be compared to the crystallographic data which are less precise. In comparing the structural models obtained for the human protein and the CAice, a general shortening of bond distances is observed in the reaction center that averages -0.05 Å. Taking into account the high resolution of the MXAN analysis, we observe that Zn-nitrogen distances are not equivalent for the coordinated histidine. This observation, at least for the human protein, seems to be in contrast with the crystallographic data. However, as previously observed, the magnitude of the differences in distance that generate this asymmetry are smaller than the error associated to the crystallographic data but larger than the estimated error in the MXAN analysis. The Zn-O distance for the coordinated water molecule is 0.048 Å, a value that seems to be very significant for the low errors associated to the MXAN distance determination. To further enforce this observation, a series of simulations were made to single out the effect of both the Zn-O distance and the theta angle on the χ2 value. Starting from the best fit structure for the two proteins, we generated individual simulations in which only the Zn-O distance was changed step-wise . A graph is shown in figure 5A . in which the χ2 value is plotted against the Zn-O distance. The individual curves for the two proteins show a well-defined difference in the minima for the explored parameter providing strong support to the claimed difference in distance for the coordinated water molecule in the two proteins. These two minima are not super-imposable, demonstrating that this difference can be considered highly significant from a statistical point of view. As will be discussed later, the implications of this difference are of importance in the functional properties of CA. A similar analysis was conducted on both proteins also for the coordinated water and the theta angle parameter. Dependence of the fit on this parameter is present only in the human protein. The results of this analysis are shown in figure 5B. A shallow minimum is observed around 15° (assuming the position derived from 2cba as 0°). However, this parameter has a lower impact on the fit when compared with the Zn-O distance. Superimposition of the best-fit corresponding structures for CAice and CA2h is shown in figure 6.

CAicefish CAhuman 2cba

d (Nε His94-Zn) 2,044 (±0,0001)* 1,996 (±0,035) 2,10 (±0,25)#

d (Nε His96-Zn) 2,076 (±0,033) 2,137 (±0,034) 2,12 (±0,25) #

d (Nδ His119-Zn) 2,084 (±0,046) 2,045 (±0,036) 2,11 (±0,25) #

d (HOH263-Zn) 2,044 (±0,0001)* 1,996 (±0.020) 2,05 (±0,32) #

d (OγThr199-Zn) 4,060 (±0,059) 3,913 (±0.038) 3,93 (±0,25) #

Table1: distances (in Angstrom) between atoms of the reaction centre for CAice, CAhuman, the crystallographic structure 2cba. * In considering the reported error value of the statistical error (evaluated by the MIGRAD subroutine of the MINUIT program (F. JAMES, CERN, Geneva)), it should be mentioned that the systematic error is on the order of 1%, as inferred for previous work on model systems. # The reported uncertainty in the distances represents the root mean square deviation calculated from the individual B values reported in the PDB file.

25

DISCUSSION

To define the molecular basis of cold adaptation, it is important to characterize the structure of those proteins that, for their physiological roles, are more likely to be modified in the process. Carbonic anhydrase is one of these potential target enzymes. In this study, starting from the primary sequence of CA from the Antarctic fish C. hamatus, a paradigm of cold adaptation, a comparison was made with all available CAs. Among the CA sequences of teleosts, an average identity of 75% was found. Specifically, the CA from C. hamatus was compared with the different isoforms known of Danio rerio and Onchorinchus mykiss. Identity ranges from 70% with CA2Z and 77% with CAHZ of Danio rerio to 79% of gill CA and 77% of CAII from Onchorinchus mykiss.

Figure 6: Comparison between the structural models of the reaction centers of CAice (blue) and CAIIh (red) obtained in the MXAN analysis. Zinc atom (violet) and coordinated waters are shown in ball and stick mode, while Thr199 and coordinated histidines are shown in wire frame mode. In addition to the three coodinating histidine residues on the left side of the figure, Thr199 is also shown .

The comparison of the ice fish sequence with alpha carbonic anhydrases present in the PDB structural database indicates a larger similarity with mammalian CAII than other mammalian isoforms for the amino acids within 10 Å (Figure 1) from the catalytic zinc atom, which are more likely to play a key role in the reaction mechanism. Some further considerations are due on the residues that define the completely conserved local chemical environment of the zinc atom. The first group of conserved amino acids are involved in proton shuttling (G63, H64, S65/A65, and F66, using a numbering that is valid for all mammal CAIIs), where serine is always present in position 65 except in the human enzyme where an Ala65 is present. The presence of a relatively small aa (i.e., Ala or Ser) in proximity of the proton shuttle His65 does not affect the mobility of the latter and, consequentially, the kinetic parameters of the enzyme. The second group of conserved amino acids are those that define the hydrophobic pocket (V121, Val 143,W209, and Leu198) that are conserved in CAice and in all mammalian CAIIs. A third class of extremely conserved amino acids in all CAs from vertebrates are those in the second coordination shell of the metal ion in the active site. These are the residues that form a hydrogen bond to the non-coordinating nitrogen of the zinc ligand histidines (Q92 with H94, E117 with H119, and N244 with H96) and to the fourth metal ligand, the exchangeable water molecule (Thr 199).

26

However, some amino acids in positions close to the zinc atom are different in icefish CA compared to human CA. These are positions 144 and 146, where Leu and Ile are present in CA2h, and valines are present for icefish in both 144 and 146. It should be mentioned that these positions are not particularly conserved among CAs from vertebrates, although the variability is confined to Leu, Ile and Val. For example, CAs from Bos taurus and Ovis aries show the same amino acids in this region as icefish (which is also the more represented in fish CA with Val144 and Val146), while Danio rerio CAII has a Val144 and Ile146, suggesting that these positions are not specific for icefish. The last relevant difference among CA2h and icefish CA refers to position 245 that is always a Trp in mammalian and a Tyr among fish. This position refers to a difference between fishes and mammals and not to a peculiarity of ice fish. In conclusion, the comparative analysis of the amino acids placed within 10 Å from the zinc atom allows the identification of key residues for the reaction mechanism that represent the criteria in the diversification of the various CA isoforms. However, no indication emerges for a unique sequence of ice fish CA that can rationalize the low temperature adaptation, simply be referring to the amino acids close to the zinc. The aa conservation, as expected, decreases moving away from the zinc atom. Consequentially, it becomes difficult to assign relevance to any of the observed difference between ice fish CA and other CAs in the database. On these premises, the most plausible structural template to be used as starting point for the MXAN analysis is the human carbonic anhydrase II (PDB files 2cba). As mentioned above, the simulation of the k-edge absorption spectra with MXAN allows for the definition of a structural model of the active site where the position of the individual atoms of the protein is defined up to about 6 Å from the zinc atom. The experimental strategy implied the measurement of a known sample of human carbonic anhydrase II to be used as a validation test for the analysis of the structurally unknown ice fish CA. The structural metric of the active site of Human CA coming from the MXAN analysis is substantially in agreement with the crystallographic data. The limit in this comparison is caused by the resolution of the crystal structure that permits errors in the atomic positions larger than the observed differences. On the other hand, the accuracy of the MXAN analysis allows discrimination between the human and the ice fish proteins, highlighting the structural differences of the active sites. The Zn-coordinated water molecule is an essential player in the reaction mechanism of CA; consequentially, the definition of the bond length was of key importance. As observed in figure 5A, the proposed difference between CA2h and CAice is substantiated by the presence of two distinct minima when the χ2 value is plotted against bond length. A further difference between CA2h and CAice is the angle of the Zn-H20 bond with the plane, defined by the three coordinating nitrogen atoms of the histidine ligands. As observed in figure 5B, the effect is less pronounced than that observed for the bond length, but a minimum in χ2 value is still observed. A structural feature that relates to those mentioned above is the position of the δO of Thr199, an atom in the crystal structures that is always reported as hydrogen bonded to the Zn-coordinated water molecule. In the χ2 minimization procedure, the movement of this atom, placed at 3.9 Å from the zinc atom, is correlated with the movement of the Zn-coordinated water molecule even when these two atoms are allowed to move independently. Consequentially, a shorter Zn-δOThr199 distance corresponds also to the shorter Zn-H2O bond length of the CA2h . It is tempting to propose that all of these structural differences concur to the modulation of the pK value of the coordinated water molecule in the two proteins, but no reliable correlation has been defined between these structural features and the actual pK values that are also likely to depend on other properties, such as the local dielectric constant. No simple correlation can be made between the observed structural difference in the active site and the aa composition for the region around the metal center. The aa conservation is complete between human CAII and CAice for the amino acids within a sphere of radius 6 Å centered on the zinc atom.

27

The features that define the observed differences in structure and kinetic properties between these two proteins must be related to something other than what is usually described as governing the molecular mechanism of CA, i.e., Thr199, the hydrophobic pocket and the region around the proton shuttle. The final step in the structural analysis presented here was the generation of a molecular model of CAice by homology modeling of the template provided by the PDB file of CA2h. For both the generated model of CAice and the crystallographic structure of CA2h, the electrostatic potential was calculated. A striking difference emerged from the comparison: an overall negative potential for CAice (-0,2 mV) and a significant positive potential for CA2h (+0.7 mV). This difference can be easily visualized when the two proteins are presented with a color coding that refers to the surface potential (figure 2 A,E). The surface potential distribution is substantially different, with a difference in the region of the substrate entrance to the active site but more pronounced in the back side of the protein. To evaluate the observed differences in surface potential to a larger set of Cas, the molecular modeling was extended to other fish CAs from erythrocytes. Among these, Danio rerio (CA2Z, sequence identity with CA2h 63%) and Onchorynchus mykiss (CA2oncho, sequence identity with CA2h 61%) and to cytosolic isoforms of Oncorhynchus mykiss (CA1oncho, sequence identity with CA2h 63%). For all of the molecular models, the surface potentials were calculated with the results reported in figure 2. The two CAs from erythrocytes show an electrostatic potential distribution similar to CA2h with an overall positive potential. On the contrary, the cytosolic CA from O. mykiss shows an overall negative electrostatic potential distribution very similar to CAice. The latter result suggests a diversification of fish CAs based more on cell type rather than on species. In this regard, in a recent study on the CA of a green algae Dunaliella salina (33), a relation has been proposed between the halo-tolerance of this organism and the observed electrostatic potential distribution that is reported figure 2 F for comparison. The authors emphasize the relation between the overall very negative electrostatic potential and the interaction with anions (particularly halides). More precisely, the low potential in the reaction center associated to the surface potential could rationalize the modulation in the affinity constant of the zinc atom in the active site toward halide binding, together with the other unusual properties of solubility and stability of the algae CA. On these bases, the predicted difference in the electrostatic potential between CA2h and CAice represents the best candidate to justify the measured structural differences between these two proteins. The modulation of the interaction between the catalytic water molecule and the zinc atom could, as demonstrated for an analogous interaction of halides, depend on the effect of the electrostatic potential distribution. It should be mentioned, however, that the electrostatic potential distribution itself, being substantially comparable in the “only” two cytosolic fish CA considered, may be not sufficient to account for the cold adaptation of this molecule calling for future studies on other cold-adapted species.

1 The abbreviations used are: CA, carbonic anhydrase; XANES, X-ray Absorption Near Edge Spectroscopy; MXAN, Minuit Xanes; aa, amino acid; FPLC, Fast Protein Liquid chromatography; XAS, X-ray Absorption Spectroscopy; rbc, red blood cells Acknowledgements

This work is in the framework of the Italian National Programme for Antarctic Research. The authors also wish to thank Dr. Isabella Ascone for the excellent technical support at the LURE facility. We wish to thank Prof. Federico Fogolari for the helpful discussions on the manuscript.

29

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31

CHAPTER 3

MOLECULAR MECHANICS INVESTIGATION INTO S. antibioticus

TYROSINASE ACTIVITY TOWARD CHLOROPHENOLS

AND NAPHTHOLS

Abstract

The activity of the type 3 copper enzyme Tyrosinase toward the three isomers of mono-

chlorophenols (MCPs) 2-, 3-, and 4-chlorophenols and the two isomers of naphthol was

studied using a combined kinetic and molecular mechanics computational approach.

3-, 4-chlorophenol and 2-naphtol react with Tyrosinase to give products that then

proceed to a non enzymatic polymerization process. 2-chlorophenol is not reactive and

acts as a competitive inhibitor in the enzymatic oxidation of 3,4-dihydroxyphenylalanine

(L-dopa). 1-naphtol resulted to be neither a substrate nor a detectable inhibitor.

Docking calculations on the Oxy form of the enzyme address the issue of the

binding mode for MCPs and naphthols to the active site of bacterial Tyrosinase. A good

agreement was observed between simulations and the kinetic experimental data . The

results show that substrates point to one of the copper atoms of the binuclear centre

(copper B) and that reactive species interact preferentially with one of the two

coordinated oxygen atoms (called O1, maintaining the labelling of the crystallographic

PDB structure with 1wx2 code) . The reactive approach of the substrate is favoured

first by pi-stacking interaction with one of the copper coordinated histidine (H194) and

then by H-bond interaction with O1 oxygen. Before coordination to copper B, the

substrates have to be deprotonated by a base protruding into the active site cleft. The

following step is a decrease in the strength of the interactions with O1 and with H194.

In the proposed reaction scheme the weakening of these two interactions anticipate the

redirection of the substrate with its anionic oxygen that approach the copper B atoms at

a distances compatible with coordination. The not reactive 1-naphthol show low affinity

for the enzymatic cleft and point toward O2: its best pose of pre-coordination is not

consistent with reactivity. The inhibitor 2-CP can easily enter the cleft but cannot

approach the copper atoms at a distance consistent with coordination. The reactive

substrates are instead predicted to be reactive with favourable energies in a way

consistent with kinetic data. Calculations predicted a specific orto-hydroxylation at the

C-6 position of the 3-CP molecule: H-NMR experiments confirmed the formation of only

one specie of stereo-specific quinone (4-Cl-1,2-o-benzoquinone).

32

INTRODUCTION

Tyrosinases (Ty) (E.C. 1.14.18.1) are copper-containing enzymes which are ubiquitously distributed in nature (2,4,7)). They are involved in the early steps of melanin formation (7) and in several other functions. Which are, in plants, sponges and many invertebrates, the process of wound healing and the primary immune response(1, 3,). In arthropods the sclerotization of the cuticle after molting or injury. In mammals tyrosinases are found in melanocytes of the retina and skin [4]. Moreover, the tyrosinases of bacterial origin secreted into the soil have been found recently to be involved in the formation of humus through random coupling of different aromatic compounds. These findings have attracted interest in the activity of Ty as a potential detoxifying agent for xenobiotic compounds with phenolic structure (6,7). All tyrosinases use molecular oxygen to catalyse two different enzymatic reactions, the ortho-hydroxylation of monophenols to o-diphenols (monophenolase, cresolase acticity) and the oxidation of o-diphenols to o-quinones (diphenolase, catecholase activity). The reactive quinones in turn polymerize non-enzymatically to the macromolecular melanins. On the basis of spectral comparison with a corresponding model complex from Kitajima and Moro-oka,[8] it was further concluded that tyrosinases in their oxy form should bind dioxygen as peroxide in the unique side-on bridging (µ-η2 :η2) manner, as was recently confirmed by the crystal structures of Streptomyces castaneoglobisporus Ty (10). In spite of new structural informations available after the publication of the crystallographic structure, as well as the wide spectroscopic literature regarding type-III copper proteins, cfr 11,15,16), details of the intermediate steps in the reaction mechanism remain substantially unclear, with different hypothesis that still wait for direct experimental prove. Among these hypothesis the DFT theoretical detailed description proposed by Siegbahn (17) in which a novel oxygen coordination is proposed, with a hydroperoxide structure for the reaction centre. This structure has not been observed spectroscopically and can be hardly reconciled with the crystallographic structure , where the structure of the peroxo is side-on bridging (µ-η2 :η2). Another recent hypothesis of mechanism, solely concocted on the observation of the recently released crystal structure, has been proposed by Decker et al (14), which implies coordination to CuA (the copper are classified according to the numbering of the Motaba model (PDB, 1wx2) as CuA coordinated with H38, H54, H63 and CuB coordinated to H190, H194, H216) and a reorientation of the peroxo bridge before reaction with the aromatic ring. In this study to gain further insight into the enzymatic mechanism of Tyrosinases, a series of substrates was chosen and investigated on the basis of their different stereochemistry and on their relevance from an industrial/technological point of view. Two distinct group of isomers were used, mono-chloro-phenols (2CP, 3CP,4CP) and naphthols (1-naphthol and 2-naphthol), differentiating greatly both from a chemical point of view (i.e functional group and aromatic weight) and from a physical point of view (i.e. dimension, relative position of the functional group against the principal axe of the molecule). These molecules can provide important information on Ty reactivity, related to both the structural features that govern the affinity for the enzymatic cleft and to the differences between the energetically favoured poses of the substrates near the reaction centre as well .

33

MATERIALS AND METHODS

Computational Methodologies. All modeling studies were carried out on a 10 CPU (PIV-3.0GHZ and AMD64) linux cluster running under openMosix architecture. Modelling of S. antibioticus Tyrosinase: Starting from the X-ray structure of the oxy-form of the S. castaneoglobisporus Ty (8 ; 1wx2 pdb code), a theoretical model for S.antibioticus Ty was built using MOE2006 comparative modelling modules (27). Validation of the protein model was carried out using MOE protein report and Savs server (http://nihserver.mbi.ucla.edu/SAVS) Hydrogen atoms were added using standard geometries to the protein structure with the MOE program. To minimize clashes between hydrogen atoms, the structures were subjected to energy minimization (Amber99 force field, 28,29) until the gradient rms was

34

The rationale of imposing restrains stem from the necessity to explore only configurations consistent with Cu coordination of the substrate without involving unjustified changes in reaction centre organization. Protein purification. The enzyme was prepared from the growth medium liquid cultures of S. antibioticus, harbouring the pIJ703 expression plasmid, and purified from the according to published procedures (13). Protein concentrations were determined optically using a value of 82 mM-1 cm-1 for the extinction coefficient at 280 nm (22). Purity was checked by SDS-PAGE and western blot. The protein was stored at -20°C at different concentration in 100 mM Phosphate buffer at pH 6.8 containing 20% glycerol as a cryoprotectant. Prior to experiments, glycerol was removed from the storage buffer by dialysis (cut off 10-14 KDa) in 100 mM Phosphate buffer at pH 6.8 Kinetic calculation. : Kinetic studies on the catalytic oxidation of CP's were performed with 0.9 µM Ty in 100 mM phosphate buffer at pH 6.8 at 25°C, using a thermostatted cell with 1 cm optical path length. In the inhibition studies , the effect of o-Cp on the kinetic of L-DOPA oxidation was evaluated at five different concentration values. Formation of the chlorphenols derived quinone was monitored through the development of their characteristic optical band at 420nm (ε ≈ 2000 M-1cm-1) , while The 1,2-naphthoquinone production was follow at 345 nm (ε:4724 M-1cm-1,24) . Uv/Vis. Analysis. Uv/vis analysis was performed using Diode Array spectrophotomer , with quartz cuvette characterized by 10 mm optical length and 100 µl reaction volume, while for kinetic constants calculation an Aglilent 8453 UV-visibile Spectroscopy System was used. For naphthols absorbation coffecient was obtain from literature: 2-naphthol have a ε328 of 1700 M

-

1cm-1 (25), while 1-naphthol have ε322 of 2200 M-1cm-1 (Dongfang 2001).

Determination of quinine moieties in 4-CP and 3CP Ty mediated degradation was monitored through the development of their characteristic optical band at 420nm (ε ≈ 2000 M-1cm-1,12). Constants calculation. The kinetic convention constants was obtain by Lineawearin-Burk plot. Concentration of 2-naphthol varied in the range 0.2 -2 mM. The temperature controller was set at 25 °C. Tyrosinase 1µM was add together with stechiometric L-DOPA as activating agent. Kinetic studies on the catalytic oxidation of CP's were performed with 0.9 µM Ty in 100 mM phosphate buffer at pH 6.8 at 25°C. Concentrations of the substrates was varied between 0.1-20 mM for o-CP, 1-20 mM for m-CP, 0.25 - 5mM p-CP. In inhibition studies , the concentrations of o-Cp was kept fixed (at each one of the value between 0.1-20mM) and that of L-DOPA was varied between 1-15mM. Determination of Ki for o-CP was made with the Dixon plot method NMR experimens. NMR spectrum was recorded on a Bruker Avance DMX600 spectrometer equipped with a gradient triple resonance probe.

Sample was obtained by incubating 1 mM 3-Clorophenol with 1 µM S. antibioticus Tyrosinase in 100 mM phosphate buffer, pH 6.8, in the presence of 5 mM ascorbic acid. After a reaction time of 120 min, the product was separated from the 3-Clorophenol by reverse phase high-performance liquid chromatography using a Zorbax Eclipse XDB-C18 column with linear gradient (30% in 10 min) of 95% acetonitrile and water containing 0.1% trifluoroacetic acid. The experiment, consisting of 512 scans and 16K data points, was carried out at 298 K. Water suppression was achieved using WATERGATE sequence (30) while the acetonitrile signal was suppressed with presaturation during the relaxation time. The spectral width was set to 7183.91 Hz and the frequency offset to 2813.92 Hz Prior to Fourier transformation, the time domain data were multiplied by exponential function and zero filling to 32K points was employed to increase the digital resolution. Sodium 3-(trimethylsilyl)proprionate-2,2,3,3-d4 (TSP) was used as an internal chemical shift reference.

35

RESULTS

Kinetic results. Time dependent measurements of the Ty activity toward the reactive Chlorphenols m-CP, p-CP and 2-naphthol were carried out respectively at 420 nm and 345 nm as a function of substrate concentration. The absorbance versus time curves showed an induction period (lag phase) that decreases with the increase of substrate concentration. When the kinetic analysis was performed in the presence of quantities of L-dopa equimolar to the enzyme, the observed behaviour changed completely. The lag phase previously observed in the enzymatic reactions without L-dopa was signific