Eur. J. Biochem. 267, 1455±1463 (2000) q FEBS 2000

Characterization of a novel type VII b-turn conformation for a bio-activetetrapeptide riginA synergy between theoretical and experimental results

Ashish, Arvinder Grover and Raghuvansh Kishore

Institute of Microbial Technology, Chandigarh, India

The conformational analysis of an immunomodulating tetrapeptide rigin (H-Gly-Gln-Pro-Arg-OH), shown to

possess diverse immunological activity, has been investigated both theoretically and experimentally for its

conformational preferences. Unrestrained molecular dynamics simulation studies in implicit dimethylsulfoxide

provide strong support for the existence of a significant population of ordered reverse turn structures for the major

trans isomer. Of the three different energy minimized families, generated from computer molecular modelling,

only one could be complemented by most of the 1D and 2D 1H NMR parameters obtained in dimethylsulfoxide-

d6. A variable temperature NMR experiment in dimethylsulfoxide-d6 revealed that the preferred conformation is

not stabilized by an intramolecular hydrogen bonding interaction. An analysis of the 2D ROESY experiment

provides evidence in favour of an uncommonly observed, rather ill-defined type VII b-turn structure. A

survey of the observed specific inter-and intra-residue NOE connectivities and their comparison with one of

the predicted low-energy conformations, demonstrates synergy between the theoretical molecular modelling

and experimentally determined NMR spectral data. The primary structure, rather than long-range

interactions, appears to be critical in determining the folding behaviour of the bio-active rigin. The

present structural attributes may be valuable in peptide drug design and development of the rigin analogs having

more effective stimulating activity.

Keywords: rigin; peptide immunomodulator; molecular dynamics simulations; 1H NMR spectroscopy; type VII

b-turn structure.

As early as in 1981, in an investigation of leukokininmolecule, Veretennikova et al. hypothesized that besidestuftsin (sequence 289±292; Thr-Lys-Pro-Arg), the tuftsin-liketetrapeptide: Gly-Gln-Pro-Arg, sequence 341±345 of IgGhuman heavy chain, may exhibit phagocytosis stimulatingactivity [1]. Later, these authors verified their prediction viachemical synthesis and bio-activity evaluations and named thisnew tetrapeptide rigin. Surprisingly, rigin not only exertedsignificant phagocytosis-stimulating activity towards rat bloodleukocytes and Staphylococcus aureus in vitro, but in additionthe observed phagocytosis index was of the same extent astuftsin [1]. Rigin was also capable of efficiently releasinginterleukin-1 and tumor necrosis factor from human monocytes[2]. In an attempt to develop more potent analogs, Rocchi et al.strategically designed and synthesized glycosylated rigin, i.e.N a-d-gluconyl-Gly-Gln-Pro-Arg, Gly-Glu(NHGlc)-Pro-Arg andGly-Gln-Pro-Arg-(NHGlc); however, these derivatives failed toexhibit any phagocytosis-stimulating activity [3]. Althoughrigin is capable of efficiently displacing [3H]tuftsin frommacrophages, it only does this at high concentrations(< 1024 m) [3]. It was suggested that the rigin molecule mayactivate macrophages through binding to its own receptor. The

peptide immunomodulators, in general, generate a physio-logical response in the target cells via their specific surfacereceptor.

Rigin may be considered similar but not identical to thetuftsin molecule. Such structurally related molecules generallyprovide excellent biological probes to elucidate biologicalprocesses for understanding their specific functions in livingsystems. Amongst the low molecular mass peptide immuno-modulators, the conformation±function relationships of theIgG-derived tetrapeptide tuftsin [4±11] and milk-derivedtripeptides Gly-Leu-Phe and Leu-Leu-Tyr [12,13] have beeninvestigated in relatively great detail. Despite the undoubtedpotential usefulness of these immunostimulating agents, nosystematic conformational analysis of rigin has been attempted[1]. To establish a probable molecular conformation of thismodulator, we employ, in the present investigation, acombination of theoretical, unrestrained molecular dynamicsimulations and experimental 1D and 2D 1H NMR spectro-scopic methods in implicit dimethylsulfoxide and in dimethyl-sulfoxide-d6, respectively, in order to make a direct comparisonof the results. One of the conformations proposed for the majortrans isomer, from molecular modelling techniques, could becomplemented by NMR-derived parameters. From thesestudies, it seems apparent that the predominant conformationat the central -Gln-Pro-residues is unique and may becritical in orienting the functional side-chains for theinteraction between rigin and its receptor. The proposedstructural information may be exploited for rational designand construction of molecules capable of exhibiting potentimmunopharmacological properties.

Correspondence to R. Kishore, Institute of Microbial Technology,

Sector 39-A, Chandigarh 160 036. India. Fax: + 91 172 690585,

E-mail: kishore@imtech.ernet.in

Abbreviation: MD, molecular dynamics.

*Note: this paper is dedicated to the memory of Prof. K. Kishore.

(Received 21 September 1999, revised 10 December 1999, accepted

12 January 2000)

1456 Ashish et al. (Eur. J. Biochem. 267) q FEBS 2000

M A T E R I A L S A N D M E T H O D S

Search for starting conformations

An extended backbone zwitterionic structure of the riginsequence, comprised of l-amino acids with standard parametersfor an all atom systems, was considered for mapping theconformational space [14]. In an exploratory protocol, asystematic grid search was performed employing atom±atompotential with force-field parameters by Momany et al. [15] anddielectric constant equal to 2, using the confd programme, asdescribed recently [16]. All the internal rotational angles: c1,f2, c2, x1

2, x22, c3, f4, x1

4 x24, x3

4 were allowed to vary with a 208resolution while f3 of the pyrrolidine ring was constrained at265.08. The systematic search of the allowed conformationalspace was performed from both N- to C- and C- to N-termini.Of the 3 779 136 (2 � 185) conformers screened, eight low-energy conformations within 6 kcal´mol21 confd energy of themost stable and having distinct backbone torsion angles wereshort listed for side-chain optimization. To find the confor-mational preferences of the side-chain torsion angles (x1

2, x22,

x14, x2

4 and x34), a grid search of 208 from both the terminals, was

again employed. After optimizing the side-chains, the 15 moststable conformations were selected for molecular dynamics(MD) simulations in implicit dimethylsulfoxide. Additionally,13 rigin sequences extracted from the Protein Databank, whosecrystal structures have been determined to a R # 2.0 AÊ

resolution, were included as starting conformations. Theaccession numbers of the pdb files that have been used are:1CAU, 1CAV, 1CAW, 3TGL, 4TGL, 1FRT, 1FCC, 1FC2,1MCO, 1FC1 (chain A and B) and 1CAX (chain B and F).Furthermore, two proteins for which three-dimensionalstructure have been reported from high resolution 1HNMR studies, 1AWE (rmsd 2.3 AÊ ), 1PMS (rmsd1.24 ^ 0.35 AÊ ), incorporating rigin sequences were alsoincluded for the analysis. A fully extended conformation ofthe rigin molecule was also used as a starting structure for theoptimization of the dynamics parameters.

Molecular dynamics studies

All the unrestrained MD simulations were performed on DECALPHA 3000 workstation with amber 4.0 package using theall-atom standard parameters with trans amide bonds [17]. Thedynamics parameters and protocols were uniformly employedwith all the 31 different starting conformations for the hightemperature dynamics calculations. A dielectric continuum,employing a representative dielectric constant of 45, was usedto influence the dimethylsulfoxide environment [10]. It hasbeen established that, `̀ while packing effects can be significant,the dominant effect on the energy surface has been shown dueto dielectric screening in polar solvents'' [10]. The advantagesof such an approach in understanding the structure±functionrelationship of low molecular mass bio-active peptides, in highpolarity media has already been described [18,19].

For quenched MD calculations, the coordinates of theeach starting conformation were first minimized and heatedgradually to 900 K over 5 ps of dynamics using theBerendsen coupling scheme [20]. To minimize the trans tocis interconversion, an additional torsional restraint of5 kcal´mol21´rad22 was added to the peptide bonds [21]. Athigh temperature, the molecular dynamics production runs werethen performed with a time step of 1 fs for a total time of100 ps employing Verlet's alogrithm [22] with Leap Frog

approximation by Hockney and Eastwood [23]. The trajectoriesfor each set were quenched (minimized by freezing) bylowering the temperature to 0 K after every 5 ps. The energyminimization procedures involved 500 steps of steepest-descentgradient with an initial step length of 0.01, followed by 1000steps of conjugate gradient, converging when the energydifference for successive steps is , 0.0001 kcal´mol21´AÊ 21.Finally, the minimized coordinates were equilibrated at 300 Kfor 5 ps and were further minimized in order to make a directcomparison with the NMR results.

Synthesis and characterization

The fully protected intermediate, Z-Gly-Gln-Pro-Arg(vNO2)-Obzl, synthesized using standard solution phaseprocedures, was purified by silica gel (60±120 mesh) columnchromatography using CHCl3-MeOH mixtures as eluants andfully characterized by 1H NMR. The final deprotection step ofhydrogenation was achieved by using 10% Pd-C in methanol at7.25 kPa The completion of the reaction was followed by thin-layer chromatography. The crude deprotected peptide waspurified to homogeneity by Waters MPLC system on a semi-preparative reversed-phase column (RP-18 LiChrosorb: HibarMerck, 2.5 � 25 cm, 7-mm particles) employing an isochraticsolvent system (water/acetonitrile mixture containing 0.01%trifluoroacetic acid) eluting at the flow rate of 5 mL´min21.Both the purity and identity of the desired peptide wasascertained by 1D and 2D 1H NMR. Further details of thesynthesis, purification and analytical data of the purified riginwill appear elsewhere.

NMR spectroscopy

The sample used for NMR study was prepared by dissolving thepurified, lyophilized peptides < 3 mg in 0.5 mL dimethylsulf-oxide-d6. All 1H NMR experiments were carried out on aBruÈker DRX-300 spectrometer at an ambient temperature of298 K, except for the variable temperature studies [24]. All thechemical shifts are reported with respect to the dimethyl-sulfoxide peak at 2.49 p.p.m. 1D 1H NMR spectra wererecorded at 32K data points, which provided direct measure-ment of 3JHN-CaH, vicinal coupling constants. The temperaturecoefficients (dd/dT) of the amide- and Arg N: protons werecalculated by analysing the chemical shifts at six differenttemperatures over the range of 298±343 K. Free inductiondecays were acquired after the temperature was stabilized foran additional four minutes. For all 2D experiments, standardpulse programs from the BruÈker software library were used.The 2D TOCSY and ROESY spectra were acquired in thephase-sensitive mode by employment of time proportionalphase increment with quadrature detection in v1 [25,26]. In theROESY experiment, the relaxation delay (D1) was 2 s. Theacquisition time for each scan was < 0.122 s and a sweep-width of 4194.6 Hz was used in both dimensions. The NOEdata reported is from a 2D ROESY experiment performed withmixing time of 200 ms, as justified by D'Ursi et al. [8] in thecase of the closely related tuftsin molecule in order to confirmthe absence of spin diffusion under similar experimentalconditions. Seventy-two scans per free induction decay wereacquired. Typically 1K data points in t2 and 256 experiments int1 were acquired. Sixteen dummy scans preceded the dataacquisition. The free induction decays were multiplied byshifted sine bell functions. Further details of the NMRexperimental data shall appear elsewhere.

q FEBS 2000 Rigin conformation, a type VII b-turn (Eur. J. Biochem. 267) 1457

R E S U LT S A N D D I S C U S S I O N

Low energy tail of the Gaussian-like distribution of energiesrepresents probable conformations in solution.

It is known that MD simulated structures exhibit bell-shapedGaussian-like distribution of energy; the low-energy tail of thedistribution represents the most probable conformers in solution[10]. Of 620 energy-minimized structures (energy ranged from218.6 to 229.2 kcal´mol21) generated for rigin by MDsimulations, 607 structures were constrained as trans Gln-Propeptide bonds and display the expected distribution of energy(Fig. 1). The 309 structures having the energy # ±23kcal´mol21 were included in the analysis. All the conformationsgenerated were plotted and verified on Ramachandran maps[27]. The points that are concentrated in the conformationallyallowed regions, energetically acceptable regions, definingrelevant extended and/or folded secondary structural featureswere included in the analysis (Fig. 2). Of these 309 low-energystructures, 222 structures satisfied the Ramachandran criteria(21708 , f , ±408, 2708 , c , ±408 and 608 , c, 1608; 508 , f , 708, 558 , c , 908). While consideringthe possibilities of various secondary structural elements,i.e. C5-structure [28,29], g-turn [30,31] and different typesof b-turn structures [32±37] that could be accessible to thisshort linear peptide, only 92 structures could be grouped intothree major families A, B and C, containing 34, 22 and 36structures, respectively. The structures having an rmsd , 0.7 AÊ

for backbone nonhydrogen atoms, including the five-memberedpyrrolidine ring of the Pro residue, were considered part of onefamily as shown in their superimpositions depicted in Fig. 3.The averaged backbone and side-chain dihedral angles: f, cand xs of each family with their deviations, characterizing themolecular conformations, are presented in Table 1.

Results from unrestrained MD simulation indicates a type VIIb-turn structure is the most probable conformation

An analysis of computationally derived low-energy confor-mations indicate that the central -Gln-Pro-segment of therigin sequence in families A and B can be classified as atype VII b-turn structure [33,38±40]. It should be emphasized

that the type VII b-turn, originally defined by Lewis et al. [40]and described by Zimmerman and Scheraga in their confor-mational energy calculations carried out on the Ac-Xaa-Pro-NHMe and Ac-Pro-Xaa-NHMe (where Xaa = proteinogenicresidue) model dipeptides, is the least understood class ofreverse turn structure [33]. This b-turn structure is character-ized as a kink in the polypeptide chain created by the f, cbackbone torsion angles: |ci+1| $ 1408 and |fi+2| # 608 or|ci+1| # 608 and |fi+2| $ 1408 and easily accommodated in theXaa-Pro sequences. Further analysis also revealed that the fixedvalue of fPro < ±65 ^ 158 severely limits the conformationalfreedom of the preceding residue and the torsion angle|ci+1| # 708 of the preceding residue is energetically disallowed[33,40]. Interestingly, the results of MD simulations of rigin arein excellent agreement with the results of conformationalenergy studies performed on -Xaa-Pro-dipeptides, i.e. thecGln < 140 ^ 108 represents the low-energy conformation inboth families. Although, the signs and the magnitudes of thebackbone torsion angles in families A and B are in good

Fig. 1. An energy histogram of the Gaussian-like distribution of the

energy minimized 607 structures for trans isomer of the rigin molecule

generated in implicit dimethylsulfoxide. The dotted line indicates the

bell-shaped regression.

Fig. 2. Ramachandran plots for the Gln and Pro residues for rigin

molecule generated by the molmol package. f, c plots for the Gln (A)

and the Pro (B) residues exhibiting a significant population of the

conformations is indeed energetically allowed.

1458 Ashish et al. (Eur. J. Biochem. 267) q FEBS 2000

agreement, the two conformations differ substantially at theirN- and C-termini. In family A, cGly is significantly extended(< ±1178), but the cArg residue is semi-folded (< ±888). Thereverse trend is observed for family B. Additionally, the ctorsion angle of the Pro residue is remarkably different in thetwo families. These critical conformational differences in theaveraged structure of the three families allowed us todistinguish the ensemble of conformations in solution by NMR.

The averaged backbone torsional angles of the structuresin family C fall in the right-handed helical region of theRamachandran map. The backbone torsion angles of theGln-Pro segment are significantly folded and characterize atypical type I (III) b-turn structure. Interestingly, the torsionangles of the terminal Gly and Arg residues adopt an extendedand a folded conformation, respectively. As the central Gln-Prosegment is highly folded in family C, the charged N- andC-terminal ends are close together in order to stabilize thebackbone conformation by electrostatic interactions.

The overall side-chain conformations of the Gln and Argresidues, in the families A and B are quite similar except the x1

of the Gln residue, which is close to a cis conformation(x1 < ±7 to 2148). Furthermore, the backbone conformationenables the maximum accessibilities of the side-chains; infamily A, they are located on the same face of the moleculewhile they are a considerable distance away from each other infamily B. However, in family C, the fully stretched side-chainsof the Gln and the Arg residues point away from the backbonein opposite directions. The pyrrolidine ring of the Pro residue isinvariably puckered and adopts a C a-endo conformation in allthe three families, i.e. the signs of the torsion angles x1, x3 andx2, x4 are positive and negative, respectively [33,36]. Therelative stability of proline puckering with respect to the natureof the imide bond (trans or cis) preceding the Pro residue, has

recently been described using an empirical potential functionECEPP/3 [41].

Resonance assignments

An extremely well resolved 1D 1H NMR spectrum of rigin, wasobtained in dimethylsulfoxide-d6. The complete resonanceassignments, using a standard approach, were straightforwardon basis of their side-chain, spin systems and characteristicchemical shifts of the constituent amino-acids in 2D TOCSYexperiment. The fully assigned, partial 1H NMR spectrum ofrigin is shown in Fig. 4 and the chemical shifts are summarizedin Table 2. The 1D spectrum of the peptide, in the amideregion, exhibits two separate sets of resonances indicating thepresence of cis±trans isomers about Gln-Pro peptide bond[40,41]. The existence of the relative abundance of the morefavoured trans isomer was established from the observedintensive NOE cross-peak between the Gln CaH and Pro CdHprotons. Only the major trans isomer (< 90%) has beenconsidered for conformational analysis.

Conformational analysis by 1H NMR spectroscopy

The structure elucidation and conformational analysis of rigin,in solution, was performed from: (a) 3JHN-CaH couplingconstants; (b) by measuring the temperature dependence ofthe chemical shifts of the amide NH and Arg N:H [42,43]; and(c) from the survey of inter- and intra-residue NOE cross-peaksusing ROESY experiments in dimethylsulfoxide-d6. In additionto an effective use of NMR spectroscopic parameters, we alsoemployed the results of MD simulations in dimethylsulfoxideboth to complement the results of NMR studies and to proposea plausible picture of the molecular conformation of rigin. Theconformation-dependent chemical shifts of the backbone Ca

Fig. 3. Energy minimized molecular structures of the three significantly

populated families: A (34 structures), B (22 structures) and C (32

structures). The rmsd values for the backbone superimposed structures,

including the Pro ring, range from 0.6 to 0.7 AÊ .

Table 1. A comparison of the averaged torsion angles (along with their

deviations) of the three MD simulated families A±C for the tetrapeptide

rigin. Angles are shown in degrees.

Parameters A B C

No. of structures 36 22 34

Energya ±25.4 ±26.1 ±24.8

Rmsd 0.66 0.69 0.56

Gly

c1 ±117.6 �^ 9.5 ±70.3 �^ 16.1 ±126.5 �^ 8.3

Gln

f2 ±91.8 �^ 13.4 ±94.3 �^ 12.8 ±62.0 �^ 5.4

c2 149.8 �^ 5.9 149.9 �^ 5.9 ±54.9 �^ 6.8

x1 ±13.6 �^ 12.9 ±7.5 �^ 11.2 ±136.1 �^ 7.9

x2 ±171.3 �^ 16.2 ±167.8 �^ 13.4 175.0 �^ 11.3

x3 142.6 �^ 23.1 ±34.2 �^ 17.4 37.8 �^ 12.2

Pro

f3 ±64.0 �^ 3.4 ±64.1 �^ 5.1 ±63.6 �^ 4.8

c3 ±11.1 �^ 8.9 105.7 �^ 9.1 ±46.3 �^ 10.5

Arg

f4 ±88.0 �^ 8.7 ±115.8 �^ 10.8 ±124.0 �^ 9.4

x1 ±151.9 �^ 11.2 ±150.5 �^ 7.3 ±141.9 �^ 11.4

x2 159.2 �^ 13.8 149.1 �^ 8.2 175.5 �^ 21.1

x3 ±179.3 �^ 23.1 ±139.3 �^ 11.9 158.7 �^ 8.9

x4 ±162.4 �^ 20.7 ±152.8 �^ 16.8 162.9 �^ 20.5

a Average amber 4.0 energy in kcal´mol21

q FEBS 2000 Rigin conformation, a type VII b-turn (Eur. J. Biochem. 267) 1459

protons of the four residues differ substantially from therandom-coil values, suggestive of the presence of the non-random structure in solution [46±48].

Vicinal coupling constants and derived dihedral angles

The preliminary information regarding the f backbone torsionangles was obtained from the 3JHN-CaH coupling constants. Anapplication of the Karplus relationship [26] permitted thedetermination of f angles of the Gln and Arg residues fromtheir observed 3J values. The results suggests that the observed3J value of 7.5 Hz may arise due to a fairly restricted backbonetorsion angle f close to either an extended, < ±1508, or a semi-extended, < ±908, conformation, keeping in mind the inherentinternal mobility associated with turn structures in solution.Here, it may be worth emphasizing that the 3J valuesbetween 6 and 8 Hz in proline-containing peptides are notnecessarily indicative of unordered/random-coil conformations[26]. Interestingly, the computed 3JHN-CaH values of the Glnand Arg residues for the averaged structure of family Acorroborates with the dihedral angles close to < ±908determined experimentally and the conformation could explainalmost all the NOEs observed. Moreover, if rigin indeedadapted a random structure in dimethylsulfoxide-d6, one wouldnot have observed a number of specific strong- and medium-range inter- and intra-residue NOE connectivities in ROESYspectra. Surprisingly, these NMR results in combination withcomputer modelling techniques permitted a detailed confor-mational analysis of the tetrapeptide in favour of an ordered,relatively ill-defined reverse-turn structure in solution.

Temperature coefficients of amide and Arg N: protons

The variable temperature study of the amidic protons and theArg N:H chemical shifts, in dimethylsulfoxide-d6 was carriedout to determine their involvement in intramolecular hydrogen-bonding interactions and/or their solvent accessibilities. Wemeasured the temperature coefficient (dd/dT) values for thepeptide backbone and the Gln side-chain amide NH and the ArgN:H by comparing spectra recorded at different temperaturesin the range 298±343 K (Table 2). In all cases, the chemicalshifts vary linearly towards upfield with temperature,indicating no major conformational changes are presentover the range studied (Fig. 5). Generally, low dd/dT values

(,20.003 p.p.m.´K21) in dimethylsulfoxide-d6 are indicativeof strongly solvent shielded and/or intramolecularly hydrogenbonded amide protons [42,43]. In rigin, all the amideprotons exhibit substantially high dd/dT values($20.0048 p.p.m.´K21) suggesting substantial exposure tothe bulk solvent, indicating that the predominant conformationdoes not seem to be stabilized by a strong intramolecularhydrogen-bonding interaction. This conclusion corroborates theresults of MD simulations as, in all the three families, theconformational forms characterized are completely devoid ofintramolecular hydrogen-bonding interactions. It is noteworthythat in marked contrast to a closely related molecule, tuftsin,the observed dd/dT of Arg amide NH under similar experi-mental conditions, is significantly higher [5,8,16]. Bearing inmind the size of the peptide in question and the nature of side-chains, the backbone conformation(s) at higher temperaturetend to display significant versatility, which in turn may have apronounced effect on the side-chain rotamer population in ahigh polarity medium. Interestingly, an interpretation of themodest dd/dT value (20.0034 p.p.m.´K21) observed for theArg N:H may not be straightforward. However, such a valuemay be indicative of a partly solvent exposed side-chain and/orweakly hydrogen-bonded conformation in equilibrium withnonbonded conformations which determines the privilegedrelative orientation having somewhat reduced conformationalmobility [49].

Synergy between experimental and theoretical results

In addition to 3JHN-CaH coupling constants and the dd/dT of theamide NH atom, we analysed a more informative ROESYspectrum to gain further insight into the type of a secondarystructure adopted by the rigin molecule. The conformation withno strong intramolecular hydrogen-bonds that satisfies most ofthe observed strong- and medium-range characteristic NOEconnectivities in dimethylsulfoxide-d6, largely consistent withthe proposed conformation, is shown in Fig. 6. A comparison ofexperimentally observed NOEs and the calculated NOEs(distances) of the three families are summarized in Table 3.The observation of an intense NOE cross-peak between Gly

Fig. 5. The temperature dependence of the amide NHs and Arg N:H

chemical shifts of the rigin molecule in dimethylsulfoxide-d6.

Fig. 4. Partial 300 MHz 1D 1H NMR spectrum of rigin in dimethyl-

sulfoxide-d6 exhibiting the NH and the C a H region. The resonance

assignments of only the major trans isomer are indicated.

1460 Ashish et al. (Eur. J. Biochem. 267) q FEBS 2000

CaH and Gln NH reflects that the terminal Gly residue does notpossess high conformational mobility. This NOE may arise dueto very short daN distance (2.2 AÊ ) expected in an extendedconformation. The existence of a diagnostic strong interresidueNOE between the Gln CaH and Pro CdH2 clearly reveals thatthe Gln-Pro imide bond of the major isomer is trans. Moreover,this NOE can be detected when the c torsion angle of the Glnresidue is restricted to either a folded (< 60 ^ 208) or anextended (< 140 ^ 208) conformation [8]. The results oftheoretical conformational analysis performed on Ac-Xaa-Pro-NHCH3 (where Xaa = Gly or Ala), which clearly indicated that|ci+1| # 708 is energetically disallowed due to nonbondedinteraction between the Pro Cd atom and the backbone NH

group of the preceding residue [33]. The lack of an amide NHin the Pro residue, however, limited further characterization ofthe molecular conformation across the -Gln-Pro-segment.Nevertheless, the potential use of the Pro CdH2 resonance inthe place of amide NH have been exploited successfully for thedetailed characterization of the molecular conformation acrossthe Xaa-Pro-Yaa segment [50,51]. The fact that a strong NOEconnectivity is present between the Gln CaH and Pro CdH2

clearly indicates that the c torsion angle of the Gln residue maybe restricted to < 140 ^ 208. This conformational behaviour isanalogous to that of a type II b-turn structure where a strongdaN (i + 1, i + 2) NOE is expected when the ci+1 (left-cornerresidue), is restricted to < 140 ^ 208 [32±37]. These con-clusions receive strong support from the molecular modelbuilding studies of the rigin sequence. As already indicated, thefive-membered pyrrolidine ring inherently restricts the ftorsion angle of the l-Pro residue to < ±65 ^ 108. The twocritical torsion angles: ci+1 and fi+2 fully satisfy therequirement of a type VII b-turn structures across the centralGln-Pro segment in rigin. Note that the conclusion about themolecular conformation presented are somewhat indirect andon basis of NOE connectivities; however, they fully satisfyalmost all the conformational features represented by family A(Table 3).

For the rigid pyrrolidine ring, the fPro restricted at about< ±65 ^ 108, there is significant flexibility for the ctorsional angle. Besides the various energetically allowedc values at < 2558 (a-helix), +1458 (poly Pro type/collagentype) and +708 (g-turn), the `bridge' region (< 0 ^ 208) isrelatively less commonly observed [37,52]. Interestingly, thethree medium range NOE connectivities, i.e. backbone±back-bone effect, Arg NH $Pro CaH and backbone±side-chaininteractions, Pro CbH2 $Arg NH $Pro CgH2, observed in theROESY spectrum can be attributed to a sharp kink in thepeptide chain, caused by the restricted cPro torsion angle closeto the `bridge' region (< 0 ^ 208). It may be presumed that theproposed conformation at the C-terminal end is not entirelyfolded and could be in dynamic equilibrium with an extendedconformation. Moreover, it should be emphasized that theobservation of a medium range daN (i, i + 1) NOE is notentirely compatible with a fully extended conformation. Theseobservations may substantiate the conclusions of the results ofMD simulations depicted in Fig. 6.

A strong dNN (i, i + 1) NOE connectivity diagnostic of thetightly folded reverse b-turn structures expected between the

Fig. 6. A comparison between an averaged low-energy conformation of

family A (upper) and the conformation derived from 1H NMR para-

meters (lower). Double headed arrows indicate diagonistic interresidue

NOE cross-peaks observed in the ROESY spectrum in dimethylsulfoxide-

d6.

Table 2. Proton chemical shifts (p.p.m.), coupling constants and NH

temperature coefficients for the tetrapeptide rigin in dimethylsulfoxide-

d6 at 25 8C.

Residue NH CH CH Others 3JHNCH dd/dT (1023)

Gly ± 3�.55 ± ± ± ±

Gln 8�.63 4�.57 1�.70 CgH2 2.14

NdH(S1) 7.28

NdH(S2) 6.84

7�.50 ±4�.82

±6.71

±6.94

Pro ± 4�.35 2�.04,

1.88 a

CgH2 1.88 a

CdH2 3.66

Arg 8�.19 4�.16 1�.53 b CgH2 1.53 b

CdH2 3.09

N:H 7.59

7�.50 ±6�.47

±3.40

a, b Indicate significant overlapping of the NMR resonances.

q FEBS 2000 Rigin conformation, a type VII b-turn (Eur. J. Biochem. 267) 1461

Pro CdH2 and Arg NH groups, considering the conformationalconstraints imposed by the l-Pro residue, is clearly absent; thismay be taken as an evidence that neither a type I (III) nora type II b-turn structure is present in solution [37,53].Moreover, a 3J value of 7.5 Hz observed for Gln and Argresidues is inconsistent with the reverse b-turn structure [5,26].Usually, in a classical b-turn structure the i + 1 residue exhibits3J values # 4 Hz [25,26]. Furthermore, the 1H NMR spectro-scopic signatures, i.e. the 3JHN-CaH values, low dd/dT andexpected NOE connectivities, required for an inverse g-turn(f < ±80 ^ 108, c < 80 ^ 108) and a C5-secondary structure(f < ±140 ^ 108, c < 140 ^ 108) are also clearly absentthereby precluding their existence in solution [36,53]. Theconclusions derived from these experimental data are inexcellent agreement with the conclusions derived from MDsimulations represented by family A (Fig. 6).

Although we detect significant overlap of the chemical shiftsof the Gln CbH2 and CgH2 protons in the 1D 1H NMRspectrum, an intense NOE cross-peak observed between GlnNH $Gln CbH2/CgH2 suggests that the preferred folded cisconformation of the x1 torsion angle of < ^108 of the Glnresidue is present, as predicted from computer molecularmodelling (Table 1). Interestingly, an observation of non-degenerate Arg CaH, CbH2/CgH2 and CdH2 proton chemicalshifts may not suggest the existence of free random rotation ofthe side-chain. The interpretation of an all-trans conformationof the methylene units of the Arg side-chain is somewhatindirect, from the ROESY spectrum as there is significantoverlap of the Arg CbH2 and CgH2 resonances. Consistentwith the proposed conformation, CaH$CbH2/CgH2, CbH2/CgH2 $N:H NOE connectivities are observed because of theirclose proximities.

Type VII b-turn in rigin is not stabilized by an intramolecular4!1 hydrogen bond

The characterization of the molecular conformation of riginincorporating a type VII b-turn structure across the -Gln-Pro-segment, inferred primarily from theoretical MD simula-tions, is in full agreement with experimental NMRparameters involving NOE connectivities in the ROESYexperiment. The experimentally estimated backbone torsionangles of the i + 1 and i + 2 residues: fGln < ±908,cGln < 140 ^ 208 and fPro < ±65 ^ 108, cPro < 0 ^ 208in fact fully characterize a type VII b-turn conformationproposed by Zimmerman and Scheraga [33] about two decadesago. The conservative Pro residue at the right-corner of the turnstructure predominantly seems to dictate the overall riginconformation. The NMR spectral parameters described here

may be the diagnostics of a type VII b-turn structureincorporating a Pro residue at the right-corner of the turnstructure. The NMR parameters presented are in excellentagreement with the statistically significant conformationalfeatures represented by family A and to a lesser extent familyB; however, are not apparent in family C. From the results, itseems apparent that in solution the tetrapeptide rigin spendsmost time in a type VII b-turn structure, which may be inequilibrium with other closely related conformers in familyB. Interestingly D'Ursi et al., while investigating tuftsinconformation from NOE restrained dynamics in dimethyl-sulfoxide, described the predominant backbone torsion anglesthat are equally compatible with type VII b-turn structure [8].

Although the topology of various types of b-turn structuresin proteins and polypeptides have been investigated extensively,observation of the conformational characteristics of a type VIIb-turn structure has been extremely rare. A systematic search ofthe highly refined X-ray structures of the globular proteins hasrevealed only seven examples [38,39]. Recently, a moredetailed investigation of the b-turn topographical featuresusing 146 examples, including type VII b-turn structures, hasprovided a characteristic conformational parameter b, a termproposed to describe various b-turns in terms of a singledihedral angle [38]. The type VII b-turn structure, devoid of anintramolecular hydrogen-bond, may explain enhanced flexi-bilities at C- and N-termini, an observation fully consistent withresults reported for proteins incorporating such structuralfeatures. Another interesting outcome of the theoretical analysisis the observation of a slightly increased distance between theGly Ca and Arg Ca, i.e. d(Ca

i ´´´´Cai+3) $ 7.5 AÊ in family A, as

compared to usual b-turn structures # 7.0 AÊ that are stabilizedby a 4!1 intramolecular hydrogen-bond [39].

C O N C L U S I O N

The present investigation, to the best of our knowledge, isthe first systematic attempt to understand the bio-activeconformation of rigin molecule that may be recognized byits receptor. It is clear that the predominant population ofthe b-turn structures are devoid of an intramolecular hydrogenbond in dimethylsulfoxide. The preferred type VII b-turnconformation of the rigin established in dimethylsulfoxide issignificantly different from the one proposed previously byVeretennikova et al. [1], i.e. the energetically favoured structureadopted a reasonably extended backbone conformation thatplaces the Gln side-chain close to the peptide backbone acrossthe Arg residue. Of special interest is whether a 4!1intramolecular hydrogen-bond is indeed a prerequisite for thestabilization of this reverse turn structure in solution; this needsfurther investigation, possibly through structural comparisonwith other designed analogs. The proposed 3D structure of riginis based on the assumption that most of the molecules are foundin the single backbone conformation and a major role is playedby the side-chain in specific receptor±ligand interactions. Itmay be noted that, as yet, no direct receptor-binding study, evenin case of tuftsin that was described almost 30 years ago, hasbeen performed. In the absence of X-ray crystallographicanalysis, a deeper insight into the NMR-deduced conformationof the peptides can be obtained simultaneously from un-restrained MD calculations; it is also possible to develop aplausible molecular model based on energy minimizations. Theapproach described here suggests that the proposed protocol forrational design of the peptide drugs by optimal use of computermodelling in combination with various spectroscopic data forthe refinement of the proposed model may be a generally useful

Table 3. A comparison of the observed 1H-1H NOEs and calculated

interproton distances (AÊ ) for the predicted three families of the rigin

molecule. s: strong; m: medium; ND: not detected.

NOE Observed Family A Family B Family C

GlyCaH2 $GlnNH s 2�.3, 3.3 2�.5, 3.6 2�.3, 3.2

GlnNH $ProCdH2 ND 4�.7, 5.5 4�.8, 5.0 2�.4, 3.1

GlnCaH $ProCdH2 s 2�.7, 3.0 2�.4, 3.3 4�.0, 4.1

ProCdH2 $Arg NH ND 3�.9, 4.2 4�.9, 5.2 2�.7, 3.9

ProCaH $ArgNH s/m 3�.1 2�.3 3�.5

ProCbH2 $ArgNH m 3�.6, 4.8 4�.1, 4.0 2�.7, 4.0

ProCgH2 $ArgNH m 3�.5, 4.4 5�.4, 4.7 2�.9, 4.0

1462 Ashish et al. (Eur. J. Biochem. 267) q FEBS 2000

protocol [54]. Finally, an understanding of the conformationalsignificance and physicochemical properties of this uncommontype of b-turn structure may provide a means of designingappropriate conformationally constrained rigin analogs capableof exhibiting more potent bio-activity.

A C K N O W L E D G E M E N T S

We acknowledge financial support from CSIR. R. K. is extremely grateful

to Dr C. M. Gupta and Dr R. Roy for help in carrying out the NMR

experiments at CDRI, Lucknow. Authors thank Prof. V. Kothekar for

valuable guidance during Grid search. This is IMTech communication

number 22/99.

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