Ind ian Journal of Biochemi stry & Biophysics Vo l. 39, Oc tober 2002. pp. 325 -33 1

Conformational preferences of two peptides DY ASL and DY A from haemagglutinin of influenza virus and their possible role in the initiation of

protein folding

C Raina Prabha

Departme nt of Bioche mistry. Fac ulty of Sc ience, M S University of Baroda. Vadodara 390 002. India

Received 8 March 2002; revised 26 JUli e 2002; accepted 19 July 2002

T he conformational pre fere nces of two pepti des DY ASL and DY A fro m haemagg lutin in of influenza virus were stud ied using PClLO programme. T his was done to understand the poss ibl e role of DY AS in the in iti ation of prote in fo ld ing and to understand the co ntribution of the fo urth residue serine in the fo rmati on of turn . O ur resul ts indicate that thi s sequence shows an inherent pre ference fo r tu rn confo rmation , w ith a stab ili z ing Asx turn . DY A with NH group in the C­term inal pro tect ion models a type I p tum more c losely than DY AS, because serine has a weak potenti al fo r turn conformation.

~-Turns are the smalles t e lements of secondary structure in a protein I . They reverse the direc tio n of a peptide backbone and are oft en referred to as ' reverse turns ' . Amino acid substitutions in certain reverse turns have been reported to confer stability to the proteins2

,J Certain short peptides have been observed to adopt turn structures in solution4

-8. Some of them

were found to be hi ghly immunogenic . Short peptides with secondary structure and immunogeni c potentia l can act as initiation sites of protein fo lding in an unfo lded prote in9.

The sequence 98 YPYDVPDY A 106 forms an

immunodo minant part of a highly immunogeni c 36-residue peptide of HA 1 chain of haemagglutinin (HA) of influenza virus. Thi s peptide is located in the trimeric interface of the nati ve prote in9

.10

, Under ac idic conditions, the conformation of the prote in is alte red di ssociating the prote in into monomers, exposing the sequence on the surface. Antibodies raised against the 36-res idue peptide (residues 75-1 IO of HA 1 chain) could recogni ze the cognate sequence in the prote in. T he soluti on structure of the peptide YPYDVPDY A was studi ed us ing NMR spectroscopy and the C- terminal portion was fo und to show extended structure 10 .

X-ray crys tallographic studies have shown th at the peptide looYDVPDY ASL I08 in complex with antibody di splayed ex tended confo rmati on up to YDV and adopted a type I ~ turn in the C-te rmina l region constituted by DY AS II . Thi s structure is simil ar to

E-mail : chi vuku la_r@yahoo.com

that exhibited by the sequence in intact HA 10 . Si nce the peptide retains its nati ve conformatio n, it could be a potenti al initi ation site for prote in fo lding . Further, turns have been reported to initiate prote in fo lding in many proteins I2

, 13. The peptide DY ASL serves as an excellent example for corre lating theoretical studies and experimental observations with a view to predict the in iti ation sites for prote in fo lding. Earli er, another turn formed by YDVP sequence adj acent to DYASL had been studied in great detail to understand the sequence requirements for the formatio n of a turn5

, 14.

Turn potenti als of amino ac ids at various pos itions in ~ turns have been evaluated using stati stica l methods l 5

.

In the present study conformational energy calcul ations were perfo rmed using a quantum mechani cal method, Perturbati ve Configurati on Interac tion over Localized Orbita ls (PCI LO) developed by Pullman and Pullman 15 . PCILO has been used previo usly to model the confo rmation sampled by peptides I 7

.19

, nucle ic ac ids2o.22 and

d n~5 Th f . rugs ' . e mtnlmum energy con onnatlons arri ved at often re fl ect the tendency of the peptides in h . . 17- 19 t e lr conti guous seq uence .

Another interes ting aspect of this problem is to understand the ro le of the fo urth res idue serine in the formation of turn , whi ch could be contributing to the di fference in the observatio ns mentioned above using NMR spec troscopy and X-ray crysta llography. In an attempt to interpret the cont ributio n o f serine, the minimum energy conformations of DY A and DYAS L are co mpared here.

326 INDIAN J. 1310CHEM. 1310PIIYS., VOL. 39, OCTOBER 2002

Methodology of Computation The conformational energy calculations on the

peptides DY ASL and DY A were performed using PClLO method 16. This method evalu ates the energy associated with the different atomic configurations of the system and thereby , predicts the preferred molecular conformation. The calculations were carri ed out with the peptides using three different methods namely, Sr, Allex and Simin, where the starting conformations di ffer in their side chain tors ion angles . The Nand C terminals of the peptides have been capped with CH1-CO- and - NH-CH} respectively, in all the protocols . This end protection simulates a situation where the peptide is part of a protein.

Modeling lIIillillllllll el/ ergy cOl/forlllatiol/ willi DY;\SL and DYA

The calculations were carried out with three different starting confo rmati ons namely Sr, Allex and Simin . [n Sr, in the start ing conformation , the side chain torsion angles of the amino acid residues were fi xed at the va lues, obtai ned for single am ino acid residues after energy minimization. [n the case of st<1ning: conformation of All ex, the side chain torsion angles of air the amino ac id residues were extended or kept close to 180° wi th no Van der Waals colli sions. Whil e for conforll1;.)tion Simin, the side chains of the starting conformation were minimized on the extended backbone before starting the calcu lations.

The details of the methodology adopted for arri vi ng at minimum energy conformations with Sr, Allex and Sim in were as follows. To start with , the main chain or the pept ide was kept ex tended, that is <P and \jf

va lues along the length were fixed at 180° and the X va lues of the side chains were fixed at va lues specifi c for that particular starti ng conrormat ion. The <p, \jf

va lues of the first residue or the <PI, \jf l were rotated by 30° interva ls up to - 180° and the energy for each conforma ti on was calculated. A mong the <PI, \j/ l pai r or torsion angl es for the first residue, <PI was selected from the comb in ation with minimum energy and the <PI of the peptide backbone was fixed at thi s val ue. Aga in the \VI of first residue and <P2 of the second res idue were rotated by 30° interval s up to -1 80° and the energy for different conformations was ca lculated. The \jf I of fi rst residue was selected from the conformation with minimum energy and the backbone \jfl was fixed at this va lue. Once again the cycle was repeated by varying the <P2, \Ih. The process was

continued to the C-terminal end of the peptide. Then the X values of the side chains were rotated by the same interval, taking two at a time, start ing wi th the first residue. This was continued to the last res idue. At thi s point to check whether the minimum energy conformation was reached or not once aga in the <P, \jf

values of the first res idue were rotated and energy was calculated. If the resultant <P, \jf va lues were same as the original values then the minImum energy conformation is reached. Otherwise, the above steps were repeated. In all the calculations the Nand C terminal protections were kept ex tended .

The starting conformations and the methods used for arriving at minimum energy conformat ions Sr, Allex and Simin wi th DY A were simi lar to that of DYASL.

Results

Millimum ellergy cOllformations of D Y ASL with starting cOllformatiolls Sr, Allex alld Simill display tum-like cOllformations

The <jl, \jf and X of the starting and final conformations of DY ASL with the above th ree methods are given in Table I. [n an ideal type [ ~ turn the <jl, \jf . va lues of (n+ l) and (n+2) are - 60, -30 and -90, 0, where n is the first residue. The <p, \jf va lues observed with the minimum energy conformat ions are different from the ideal values. The \jf of Y and <jl, \[I

of A are either identical or within the allowed limits of deviation of 30° of the ideal va lues . But, the <jl value of Y shows a large difference int erferi ng with the formation of ideal turn in the min imum encrgy co nformati ons. The Ca ( lI ) -Ca ( 11 + 3 ) distance is 6.34, 6.3 and 5.8A in conformati ons Sr, All ex and Simin , respectively. [f the Co. (II ) -Clf. ( 11 + 3) distance is < 7 A,

I . f · I 2(, those secondary structures are c assl I CC as turns . Based on thi s criterion all the three minimum energy conformations can be judged as open turns.

Conformati on Sr seems to be stabilized by the fo llowing non-native interac ti ons: i), the side chain carboxy l of D participates in hydrogen bonding with backbone N-H protons of Y, A and S. The hydrogen bonding observed between the side chain of D and the main chain NH of A is call ed the '·Asx turn", where Asx stands for either asparti c acid or asparagine27

.

The Asx turns are integra l to most type I turns , rendering the latter more stable; ii ), hydroxy l of S interacts with backbone N-H of L.

PRABHA: CONFORM ATIONAL PREFERENCES OF PEPTIDES DYASL AND DYA 327

Table I-\b, wand X va lu c:s of the peptide DY ASL in the start ing and fin al conformations using the methods Sr, Allex and Simin

Torsion ang les

All ex

<P \jl

XI X2

X .1. 1

X 3.2

Simin q'>

IJf XI X2

X.1 .1

D

180 ISO lRO

- 150

180 180 180

-150

180 180 -60

60

Starting conformati on

Y

180 180 -60 90

IRO 180

- 150 180

180 180 - 60 90

A

180 180 60

180 lXO 180

180 180 60

S

180 180 180 180

ISO 180 ISO 180

180 180 -90 60

L

180 180 60 180 60

- 60

ISO 180

- 120 180 - 60 -60

180 180 60 150 - 60

- 60

D

-90 180 ISO

- ISO

- 90 180

-150 150

120 -60 60

-60

Y

180 30

-60 90

- ISO - 30 - 60 -30

60 o

60 - 90

Fin al conformat ion

A

180 30 60

180 60 60

- 120 o

±60

S

150 120 150 -30

180 180 60 -30

ISO 120 60 30

L

- 90 60 -60 180 -60 -60

- 90 60

- 60 180 -60 -60

90 - 60 - 60 180 -60 - 60

Table: 2-Energic:s of starting and fin al conformat ions o f peptides DY AS L and DY A and dec rc:ase in energy after energy mini miza ti on lIsing the methods Sr. Allex and Simin

Peptide Conformation Encrgy of start ing Energy of fina l Energy eli fferc nce co nformation conformation (kca l/mol)

Sr

DYASL Allex

()YA

Si ll1in

Sr

Allcx

Si min

(kcallll1o l)

-30'+90.+.'+6

- 30.+894.5 1

-304907.75

-20737.+. 86

- 20736 1.80

- 207375.58

I n con formati on A lIex , the interacti on between side chain carboxy l of' D w ith backbone N-H proton s of Y , A , S, L and C- terminal end protection (CE) seems to stabili ze the final structure. Hyd roxy l of S does not interact w ith any other group.

The backbone -H protons of Y , Sand C- terminal end protect ion appear to participate in H-bonding with side chain carboxy l of D in the conformation Si min . Side chain of S does not interact with any other group.

There is a large lowering in energy in all three cases, compared to starting conformations (Tab le 2). Conformati on A llex is largely different from a type I o turn . The energy lowering in thi s case is much less

(kca l/mol)

-304953. 14 4X .68

- 30'+938.25 43 .74

-30'+954. 12 46.37

-207398. 14 23 .28

- 207396.26 34.46

- 2074 15.78 40.20

compared to Sr and Simin. A mong the three con formations, con formati on S i ll1i n approaches th e type I turn better, from two criteria , i .e . the C,,- ( 11 ) -

CIj. (ll e3) di stances and <», \If values . The images of the final modeled conformati ons of DY ASL drawn wi th the help of Capm are presented in Fig. I (a)-(c) .

Millimum ellergy cOllformatiolls 51', Allex alld Simill ofDYA are similar to those ofDYASL

In the final conformati ons of DYA obtained with Sr, A llex and Simin the <», \j1 values of Y and A are

di fferent from the ideal <», \If va lues of a type I 0 turn (Tab le 3). But, in th e case of Simin the <», \If va lues are

328 INDIAN 1. BIOCHEM. BIOPHYS., VOL. 39, OCTOBER 2002

(0) ( b) (c)

Fig. I- -Minimum energy conformations of DYAS L obtained with starting conformati on of (a), Sr; (b), Allex ; and (c ), Simin

Table 3----<jJ, \jI and X va lues of the peptide DY A in the starting and final conformation using the methods Sr, Allex and Simin

Torsion Starting conformation Fi nal conformati on angles

D Y A D Y A Sr

<Il 180 180 180 -90 180 180

\jI 180 180 180 180 30 -30

Xl 180 - 60 60 180 -60 60

Xz - ISO 90 -150 90

Allcx

<Il 180 180 180 -60 180 180

\jI 180 180 180 180 30 -30

Xl - ISO - 150 180 180 -90 60

Xz 180 180 -150 90

Si min

<Il 180 180 180 120 180 -90

\jI 180 180 180 -90 - 30 -30

Xl -60 - 60 60 60 -60 ±60

Xz 60 90 - 60 90

close to type I ~ tum except for <j> of Y. Interestingly, in the final conformations of Sr, Allex and Simin the Ca (11 ) - Cc;t. (n; 3) di stances are 6.00, 6.00 and 6.18 A, respectively and can be classified as turns. The three conformations seem to be stabilized by the non-native interaction between the side chain carboxyl of D with main chain N-H protons of Y, A and C-terminal end protection (CE) forming hydrogen bonds.

There is considerably large lowering in energy even with DY A in all the three cases, compared to starting conformations . Conformation Simin approaches the type I ~ tum better, from the two criteria of distances and <j> , \jf values. Thi s conformation also gives the maximum lowering in energy. The three final

(0) ( b)

--0t + "'.... " V' D

/

Fig. 2-Minimum energy conformat ions of DYA obtained with starting conformation of (a) , Sr; and (b), Allex

conformations can be described as open turn-like structures. The images of these modeled minimum energy conformations Sr and Allex of DY A are shown in Fig. 2 (a) and (b) (the image of Simin is not presented here). These results also show that in theoretical modeling the starting conformation plays an important role in determining the final conformation. Although it is difficult to generalize which starting conformation models the experimentally observed conformation better. It may be possible by trying the same methods with many peptides.

Discussion Justification for the use of peILO as a method for theoretical modeling of the millimum energy conformations for the peptides DYA and DYASL

The PCILO method has been found to be reliable for conformational work28 and for conformational study of biological molecules29

• It has also been observed that PCILO calculations are in agreement

PR AB HA: CON FORM ATIONA L PREFERENCES OF PEPTIDES DY ASL AN D DYA 329

with ab inilio STO-3G calcul ati ons in some model systems30

. It has been reported that the calculati ons using PCILO on alanine res idue l 6 are in agreement with the <», \jf values for allowed and di sallowed regions of Ramachandran plot for alanine residue. The results obta ined on peptides with PCILO were correlated to and fo und to be in agreement with experimental observati ons deri ved from NMR and CD I7 1 ~.3 1 In view of the above observations PCILO has been chosen as a method for modeling the confo rmati ons of the peptides DY A and DY ASL and so was used in the present study.

Importance of startillg cOllformatioll ill theoretical modeling using PCILO method

The fin al <», \jf values in the minimal energy confo rmati ons in Sr, Allex and Simin of both DY A and DY AS L are not identi cal pointing to the signi ficance of side chain torsion angles in the starting confo rmat ion. It can be rati onali zed th at some torsion angles of the side chains in the starting confo rmati on raise the energy of the intermedi ate confo rmati on at certain torsion angles of the backbone. Minimum energy being the criterion fo r selection of intermedi ate at each step of calculation, an intermedi ate which is chosen for furth er rounds of calculation with one protocol could be eliminated with other protocol. The di ffe rence in the energy of startin g confo rmati ons supports thi s observation. If fo lding of pept ide is compared to downhill journey of a ball , thi s situati on is ak in to the ball starting at di fferent points In mountain ranges, fo ll owing diffe rent tracks and ending up at di ffe rent spots. It should be born in mind that in so me cases the tracks can converge and lead to one fin al point.

Tum potential of residues Y and A in second and third positions of type I p tum in DYA SL and DYA

The tu rn forming potenti als of amino acids have been analyzed stat istica ll y and rati onali zed on the basis of fo rmat ion of hydrogell bonds and the prefe rences of the ami no ac ids to adopt the <», \jf

values of the particu lar turn l5. Based on thi s analys is,

in the seq uence DY AS, the residue D is the most preferred with hi ghest potenti al in the first pos ition in a type I ~ turn . Being a hyd rogen bond acceptor, the side chain of D fo rms a hydrogen bond with the bac kbone nitrogen of third res idue, fo rming an 'Asx turn ,2h. S in the fo urth position shows only a moderate potential. In most turn s thi s position is occupi ed by a glyc ine fac ilitating the reversal or backbone direc ti on.

This explains why DY A can model a type I ~ turn better than DY AS. In the above three final conformations of the peptides DY A and DY ASL, the <», \jf values of Y and A are di fferent from the ideal <», \jf values of a type I turn . It is known from earlier studies that these two residues have very low turn potenti al in their respec ti ve positi ons IS. The <» of Y is far from the acceptable va lue, although all the other torsion angles adopt ideal va lues or dev iate by 30°, that is with in the limits of normal cut off.

Comparison of the minimum energy conformations of D Y ASL with antibody bound Ilollapeptide

The X-ray crystallographic structure of antibody bound nonapeptide YDVPDY ASL revealed that in the C-terminal of the peptide the res idues DY AS adapt a type I turn II, which has been fo und to be similar to the structure of conti guous sequence in haemagglutinin . In addition, the peptide antibody could recognize the seq uence in haemagglut i ni n ~. There are number of interactions between the side chains of peptide and those of antibody. The side chain of D in the peptide fo rms salt bridge with arginine in the antibody. The side chain of Y is sandwiched between arginine and glutamate of antibody. Further, the hydroxy l of Y forms hydrogen bonds with so me residues in the antibody. The backbone carbonyl of A is in volved in a hydrogen bond with an asparag ine of the antibody. No such interac ti ons are poss ible in iso lated peptides in the cal cul ati ons, giving scope fo r non-nati ve in terac ti ons di storting the fin al structure.

NMR spectra of YPYDVPDYA showed that DYA remaills extended

The NMR spectra of YPYD VPDY A revealed that DY A is in extended conformati on in the aq ueous environment lo

. The diffe rences between experimental observa ti on and theoreti ca l modeling can be due to fo ll owing reasons . In the peptide YPYDY A, the fo urth resid ue of the turn S is mi ss ing. In theoreti cal modeling of DY A the NH grou p of CE substitutes fo r NH of S. Additionall y, PCI LO calculati ons are carri ed out in vacuum conditi ons. Although these conditi ons are diffe rent from aqueous environment , where proteins fo ld, the role played by water in determi ning the final co nfo rmation can be interpreted the following way. Water leads to clustering of hydrophobic mOieti es, competes for hydrogen bondi ng with other groups and solvates the charges interfe ring with the sa lt bri dge formation. DY A and

330 INDI AN J. 1310C HEM. 13 10PHYS. , VOL. 39, OCT OBER 2002

DY AS being small peptides, there cannot be any possibility for hydrophobic collapse. But some of the hydrogen bonding interactions observed in these calcul ati ons may not be present in aqueous environment. These interactions are playing a major role in distorting the structure and preventing the peptides from adopting an idea l type I turn in the modeled conformations. As a result, the conformations stabili zed in these calculations can show only the inherent preferences, but do not model the exact structure observed in the native protein . Moreover, 0104 and YIOS are known to be involved in various interactions with other residues of the protei n. These interactions may enforce the ideal type I turn conformation in the protein.

Possible role of the peptide to act as an initiation site for protein folding

In the initiation of protein folding the protein is completely unfolded. At this stage local interactions playa deterministic role in the formati on of initiation si tes . These initiation sites need not be very stable32

.35

.

According to diffusion-collision model of protein folding these initi ation sites or microdomains, which may be marginally stable, diffuse and form the native structure34

,35. In the present study, the minimum energy conformations arrived at the end of the calcul ations adopt open turn strucure as the di stance between Ca (n) -Ca (n+3) is less than 7 A. Such an open turn conformation was originally suggested with the peptide sequence TGAA by Sundarlingam and Sekharudu36

. The formation of open turn in the peptide TGAA was later established experimentally' 7.37 . Although the open turn conformations obtained with DY A and DY ASL do not model classical ~ turn, the Asx turn di splayed by these structures, is a stabilizing feature observed in most type I ~ turns. The open tum confonnation together with Asx turn and energy difference between the starting and final conformations, point at the propensity of the peptides for attaining turn-like conformation. The immunological evidence in literature" along wi th the present results suggest a strong possibility for the peptides to act as initiation sites for protein folding in the contiguous sequence.

Conclusions From the above studies it can be concluded that the

peptides DY A with C-terminal end protection (CE) and DY ASL displ ay a propensity for attaining turn­like conformati ons. This resu lt is in good agreement

with the experimental observation in X-ray studies, where DY AS forms a type I turn, suggesting strong possibility for the sequence to act as an initi ati on si te for protein folding in the HA 1 chain of haemagglutinin . The deviation from ideal turn in the above models could be attributed to the following reasons: firstly , very short peptides were chosen for calculat ions, eliminating any scope for tertiary interacti ons for side chains. This situation is different from the conti guous sequence in the protein or the peptide is in complex with the antibody . The second reason could be due to the calcu lation cond itions being set in vacuum. Under these conditi ons, the side chains involve in many non-native inrerac ti ons with the backbone amides, which may not be observed in the presence of water. Although, the modeled conformations are not ideal type I turns, they can be broadly classified as open turns, stabili zed by Asx turns. These structures reflect the overall conformational preferences and consequent potential of the sequence to act as initiation site for protein folding.

Acknowledgement The author thanks Dr Y U Sasidhar, Department of

Chemistry, lIT, Mumbai for the many di scussions and the advice given and lIT, Mumbai for the computational facilities.

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