crystallographic studies of two proteases: the aminopeptidase from vibrio proteolyticus (aap) and...
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Crystallographic Studies of Two Proteases: the Aminopeptidase from Vibrio proteolyticus (AAP)
and γ-chymotrypsin.
Aaron G. MoulinPetsko/Ringe LabJanuary 15, 2007
Outline
1. Background on dinuclear metallohydrolases and AAP
2. E151 mutants of AAP
3. Synthesis, kinetics and structure of a Tris-based inhibitor of AAP
Bridged Dinuclear Metalloenzyme
Dinuclear Metalloenzymes
• Large class of enzymes with various functions—amidohydrolases, phosphatases, nucleases.
• Involved in many cellular processes—cell-cycle control, protein degradation, carcinogenesis, tissue repair.
• Implicated in many diseases—neural disorders, bacterial and HIV infections, cancers.
Dinuclear metalloenzymes Enzyme Metal Reference
Aeromonas aminopeptidase Zn2+
Structure, (1994), 2, 283-291
Streptomyces griceus aminopeptidase Zn2+ J. Mol. Bio.,(1997), 265, 620-636
Bovine Lens aminopeptidase Zn2+ J. Mol. Bio., (1992), 224, 113-140
Proline aminopeptidase Mn2+ Proc. Nat. Acad. Sci., (1998), 95, 3472
Arginase Mn2+ Nature, (1996), 383, 554
DNA Polymerase Zn2+
Mg2+
EMBO J., (1991), 10, 25-33
Alkaline Phosphatase Zn2+ J. Mol. Bio., (1991), 218, 449-464
HIV-1 Reverse Transcriptase Mn2+ Science, (1991), 252, 88-95
P1 Nuclease Zn2+ EMBO J., (1991), 10, 1607-1619
Glutamine Synthetase Mn2+ J. Mol. Bio., (1989), 264, 17681
Phospholipase C Ca2+ Nature, (1989), 338, 357-360
Manganese Catalase Mn2+ JACS, (1992), 114, 5869-5870
Aminopeptidase from Vibrio proteolyticus
• Dizinc metalloprotease.• Bridging water species.• Isolated from organism Vibrio
proteolyticus.• N-terminal exo-peptidase.• Small (291 a.a., 32 kDa),
nonspecific.• Easily crystallized, diffracts to
atomic resolution.• Recently cloned, mutants
available and are easily purified.
O
Glu152
O
N
N
His256
NN
His97
OO
Asp117
O
Asp179
O
Zn2 Zn1
OH(H)
O
Glu151
O
The Active Site of AAP
Zn2 Zn1O
HNO
NH2
I
OH
Tyr225
3.7
2.41.8 2.1
3.0
Zn2 Zn1O
H2N
HN
O
O
O-
2.3 2.3
3.6
1.8 2.2
OH
NH2HO
OH
Zn1Zn2
CH2
OH
Tyr225
3.45
2.132.211.95 2.21
2.70
Inhibitor Structures of AAP
• p-iodo-D-phenylalanine hydroxamate Ki = 0.4 μM
• L-leucine phosphonic acid Ki = 6.7 μM
• Bestatin Ki = 18 nM• Tris > 20 mM
Zn2 Zn1
O
P
H2N
2.2
O
O
3.9
1.9 2.3
2.1
Chevrier et al. Eur. Jour. Biochem. (1996) 237(2):393-8. Stamper et al. Biochemistry 40, 7035 Stamper et al. Biochemistry. 43(30), pp. 9620-8 Desmarais et al. Structure 10, pp. 1063-1072
O
Glu152
O
N
N
His256
NN
His97
OO
Asp117
O
Asp179
O
Zn2 Zn1
OH(H)
O
Glu151
O
The Active Site of AAP
The Role of E151
• The active sites of several dinuclear metallopeptidases have a putative glutamate base.
• Kinetics studies have demonstrated that replacement of E151 in AAP results in kinetically retarded enzymes.
• Data are consistent with E151 being the catalytic general acid-base.
Roles of various active site residues in AAP, CPG2, SAP and
GCP II
AAP CPG2 SAP GCP II Proposed Function
H97 H112 H85 H377 Zn2+ binding
D99 D114 D87 D379 Hydrogen bonding
D117 D141 D97 D387 Zn2+ binding
E151 E175 E131 E424 Catalytic base
E152 E176 E132 E425 Zn2+ binding
D179 E200 D160 D453 Zn2+ binding
H256 H385 H247 H553 Zn2+ binding
Speno et al. Mol. Pharmacol. (1999) 55(1), pp. 179-185
rAAP E151D-AAP E151H-AAP E151Q-AAP E151A-AAP
kcat (min-1) 4280 2.24 2.00 0.17 ND
Km (μM) 16.4 22.9 25.5 18.5 ---
Bzymek & Holz Jour. Biol. Chem. (2004) 279(30), pp. 31018-25Bzymek et al. Biochemistry (2005) 44(36), pp. 12030-40
Stamper, C. et al. Biochem., 40, p. 7035
Proposed Mechanism1
O
Glu152
O
N
N
His256
NN
His97
OO
Asp117
O
Asp179
O
Zn2 Zn1
OH(H)
O
Glu151
O(H)
Leu-Phe
2Zn2 Zn1
OH(H)
NH
O COO-
H2NO
Glu151
O(H)
3
HN
-OOC
H2N
Zn2
O
Zn1
HO
O
Glu151
O(H)
4
HN
-OOC
H2N
Zn2
O
Zn1
O
H
Glu151
O(H)
O
5H2N O
Zn1Zn2
OH
HN
-OOC
Glu151
O(H)
O
Phe
6
OH2N
Zn2
O
Zn1
H2OLeu
Still Some Questions…
• Which way does the substrate bind?
• What are the reasons for the kinetic retardation of E151 mutants?
• What are the roles of the two zinc ions?
Outline
1. Background on dinuclear metallohydrolases and AAP
2. E151 mutants of AAP
3. Synthesis, kinetics and structure of a Tris-based inhibitor of AAP
E151 Recap
• Mutants E151D- and E151H-AAP are functional, while mutant E151A-AAP is inactive.
• Are there any gross structural changes?
• Can any subtleties in the structures explain rate differences, particularly in the active site?
rAAP E151D-AAP E151H-AAP E151Q-AAP E151A-AAP
kcat (min-1) 4280 2.24 2.00 0.17 ND
Km (μM) 16.4 22.9 25.5 18.5 ---
Crystallization of E151 Mutants
• All mutants crystallized from 4.5 M NaCl, 100 mM KSCN, 100 mM HEPES pH 7.2.
• Crystals grow in 3-4 days for E151D and E151H, in 2-4 weeks for E151A.
• All crystals have hexagonal symmetry.
Data StatisticsData collection and refinement statistics.
E151D E151A E151H
Crystal Data
Space Group P6122 P6122 P6122
Unit Cell Parameters (Å) a= 109.23 109.6 109.1
b= 109.23 109.6 109.1
c= 91.42 91.4 98.4
Data Processing
No. reflections, observed 2,102,450 534,226 457,444
Cutoff (I/) 2.0 2.0 2.8
Rmerge (overall) (%) 8.4 9.3 10.5
Completeness, overall (%) 99.2 99.4 98.4
Model Refinement
Resolution range (Å) 50.0-1.14 50.0-1.75 50.0-1.9
R-factor (%) 13.2 17.8 18.0
Rfree (for 10% of reflections; %) 14.3 20.1 20.5
The Structures Agree Well with the Wild-Type Structure
• The global fold is the same for all mutants.
• There are no gross structural changes anywhere in any mutant.
• The main-chain overlap is good.
E151D-AAP E151A-AAP E151H-AAP
RMSD (Å) 0.21 0.24 0.20
Representative Density
Superimpositions of the Active Sites of E151 Mutants with Wild-Type AAP
Images of the Active Site
• E151H-AAP
• E151D-AAP
• E151A-AAP
E151H-AAP Active Site
Chemical Models and Distances
Zn2 Zn1O
H2N O
2.4
3.4
2.1 1.82.8
Zn2 Zn1O
H2N O
2.5
3.4
2.2 1.93.1
Zn2 Zn1O
H2N O
2.21
3.51
2.10 1.982.88
E151D-AAP
E151A-AAP
Zn2 Zn1O
H2N
HN
O
O
O-
2.3 2.3
3.6
1.8 2.2Bestatin
Zn2 Zn1
O
P
H2N
2.2
O
O
3.9
1.9 2.3
2.1
6
OH2N
Zn2
O
Zn1
Observations
• E151D and E151A crystallize in a product complex whereas E151H does not.
• Product complex looks much like predicted from inhibitor complexes and EPR data.
• Bridging oxygen species seems to be negatively charged.
OH2N
Zn2
O
Zn1
Glu151
OH
O
Zn2 Zn1
HO(H)
H2O
H3NO O
Lower the pH• E151D-AAP crystals were
grown and transferred to pH 4.5.
O O
H3N
Zn1 Zn2
O O
Asp117
OH
A Revised Mechanism for AAP1
O
Glu152
O
N
N
His256
NN
His97
OO
Asp117
O
Asp179
O
Zn2 Zn1
OH(H)
O
Glu151
O(H)
3
HN
-OOC
H2N
Zn2
O
Zn1
HO
O
Glu151
O(H)
Leu-Phe
2Zn2 Zn1
OH(H)
NH
O COO-
H2NO
Glu151
O(H)
4
HN
-OOC
H2N
Zn2
O
Zn1
O
H
Glu151
O(H)
O
6
OH2N
Zn2
O
Zn1
Glu151
OH
O
Phe
Leu3a
HN
-OOC
H2N
Zn2
O
Zn1
OH
O
Glu151
O(H)
5
H2N O
Zn1Zn2
OH
HN
-OOC
Glu151
O(H)
O
OH3N
Zn2
O
Zn1
H2O
OH(H)
7
Conclusions
• The E151 mutants are kinetically retarded due to distance/orientation. • The bridging oxygen species of the product complex carries negative
charge.• The product (and likely the substrate as well) binds with the amine
coordinating to Zn2.• Zn2 seems to serve to coordinate the amine, as predicted.• Zn1 is still likely to active the carbonyl.• A terminal water species is observed at Zn2, the opposite of
prediction.• E151 is apparently responsible for a number of proton shuttling
events, including the protonation of the amine of the product.
Outline
• Background on dinuclear metallohydrolases and AAP
• E151 mutants of AAP
• Synthesis, kinetics and structure of a Tris-based inhibitor of AAP
Tris Can Inhibit AAP
• Tris inhibits AAP with Ki > 20 mM
• Inhibits competitively
• Binds to the active site in “backwards” fashion
Desmarais et al.Structure (2002) 10(8):1063-72
Can Tris Be Used as a Scaffold to Design New Inhibitors?
• Substitution of one hydroxyl for a gain in binding energy
• Synthesis should be general
• Should be possible with commercially available starting materials
Desmarais et al.Structure (2002) 10(8):1063-72
The Synthesis Scheme for Tris-Based Inhibitor “Benzyl-Tris”
“Benzyl-Tris” Inhibits AAP Competitively
• Inhibits with calculated Ki = 180 μM.
Crystal Growth and Diffraction Statistics
• Wild-type crystals grown from 4.5 M NaCl, 100 mM KSCN, 100 mM HEPES pH 7.2
• Soaked with “benzyl-tris” at 300 mM
Data collection and refinement statistics.
Crystal Data
Space Group P6122
Unit Cell Parameters (Å) a=110.11b=110.11c=91.23
Data Processing
No. reflections, observed 2340130
Rmerge (overall) (%) 10.1
Completeness, overall (%) 98.9
Model Refinement
Resolution range (Å) 50-1.14
R-factor (%) 12.1
Rfree (for 2053 reflections; %) 14.2
The Active Site Density
Comparison of the Tris and “Benzyl-Tris” Structures
Anisotropic Displacement Paramters for Tris and “Benzyl-Tris”
Conclusions
• Tris can be used as scaffold to build inhibitors for AAP, and possibly other dinuclear metalloenzymes.
• A general synthesis has been developed and implemented for several compounds.
• The “benzyl-Tris” inhibitor is competitive with AAP, with a Ki = 180 μM.
• The structure reveals a binding mode analogous to Tris, though one metal-coordinating ligand has been lost.
• Analysis of anisotropic displacement parameters shows that Tris ligand loss had highest thermal motion and likely lower binding energy.
• Loss of the metal-coordinating ligand is compensated by gain of hydrophobic interactions in the binding pocket.
Acknowledgements• Greg• Dagmar
• Tim• Mark• Todd
• Alejandro• Cheryl• Bryan• Gabe• Ed• Wally• Jose• Dali• Nilou• Quyen• Shulin• Rest of the PR Lab• Alex Milshteyn
• Rick Holz• Krzysztof Bzymek
• Rex Pratt• Jason Bell
• BioCARS• SSRL• GM/CA CAT• X25A• X6A
• Mom• Dad• Karen• George & Vera• Dorothy
Fenn T.D. et al. (2003) POVScript. J. Appl. Cryst. 36(2), pp. 944-947.