Magnitude of structural changes of the T-cell receptor binding regions determine the strength of T-cell antagonism: molecular dynamics simulations of HLA-DR4 (DRB1*0405) complexed with analogue peptide

Hidehiro Toh1, Nobuhiro Kamikawaji2, Takeshi Tana2, Shigeru Muta1, Takehiko Sasazuki2 and Satoru Kuhara1,3

1 Graduate School of Genetic Resources Technology, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-8581 and 2 Department of Genetics, Medical Institute of Bioregulation, Kyushu University, Maidashi,Higashi-ku, Fukuoka 812-8582, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In our model system, we generated T cell clones specific for the HLA-DR4 (DRB1*0405)–index peptide (YWALEAAAD) complex. Based on response patterns of the T cell clones, analogue peptides containing single amino acid substitutions of the index peptide were classified into three types, agonists, antagonists or null peptides (non-agonistic and non-antagonistic peptides). Subtle structural changes induced by the antagonists in the T-cell receptor (TCR) binding regions have already been explained using the root mean square (r.m.s.) deviations from the DR4–index peptide complex in the molecular dynamics (MD) trajectory. In this work, we performed additional MD simulations at 300 K with explicit solvent molecules to reveal the structural character of the HLA-DR4 complexed with the analogue peptides. We examined the r.m.s. deviations of the TCR-binding sites and the exposed areas of the bound peptides. Remarkable differences of the r.m.s. deviations among the DR4–antagonist complexes, together with our previous data, suggest that the magnitude of structural changes of TCR-binding regions would determine the strength of TCR antagonism. The simulations also indicate that TCR could discriminate null peptides from other ligands mainly through the changes of exposed side chains of the bound peptide, rather than the conformational changes of TCR-binding surfaces on HLA molecule.

Keywords: analogue peptide/HLA class II molecule/molecular dynamics simulation/recognition/T cell receptor


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Human CD4+ Th cells recognize antigenic peptides in the context of human major histocompatibility complex (MHC) class II molecules by T cell receptor (TCR) and proliferate to exert effector functions through various biological activities of secreted lymphokines. Recent studies have indicated that variant synthetic peptides with amino acid substitutions can induce qualitative changes in T cell responses such as T cell anergy, TCR antagonism, lymphokine production or cytolysis in the absence of proliferation (De Magistris et al., 1992Go; Sloan-Lancaster et al., 1993Go; Chen et al., 1996Go; Ikegawa et al., 1996Go; Kersh and Allen, 1996aGo,bGo; Matsuoka et al., 1996Go; Dittel et al., 1997Go). The crystal structures of HLA class I molecule complexed with altered peptide have been solved (Reid et al., 1996Go; Ding et al., 1999Go), but it has remained less clear whether there is any significant potential for peptide-induced variation in the MHC molecule to contribute directly to the antigenic nature of a specific MHC–peptide complex.

As a model system to examine the molecular mechanisms of TCR antagonistic activities, we used the human class II HLA-DR4, peptide YWALEAAAD (Y = position 1, p1) and a T cell clone which recognizes an index peptide (YWALEAAAD) in the context of DRB1*0405 (Tana et al., 1998Go; Toh et al., 1998Go). HLA-DRB1*0405 is one of the DR4-associated subtypes and strongly associated with rheumatoid arthritis (Kinouchi et al., 1994Go; Matsushita et al., 1994Go). To determine whether the analogue peptides which induced no proliferative responses of the T cell clone functioned as TCR antagonism, proliferation of the T cell clone for the index peptide bound to HLA-DR4 was tested in the presence of various concentrations of the analogue peptides. Based on the results of this investigation, we classified the analogue peptides into three groups, agonists, antagonists or null peptides (non-agonistic and non-antagonistic peptides) (Table IGo). Analogue peptides carrying single residue substitutions at p3, p4, p5, p6, p7 or p8 in the index peptide were used.


View this table:
[in this window]
[in a new window]
 
Table I. Activation of T cells by analogue peptides with substitutions in the index peptide, YWALEAAAD
 
According to crystallographic analysis of peptides associated with HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4 (DRB1*0401), I-Ad and I-Ek (a murine class II MHC molecule) (Stern et al., 1994Go; Ghosh et al., 1995Go; Fremont et al., 1996Go; Jardetzky et al., 1996Go; Dessen et al., 1997Go; Murthy and Stern, 1997Go; Scott et al., 1998Go; Smith et al., 1998Go), MHC class II molecules may generally constrain bound peptides to a regular polyproline II-like conformation. Figure 1AGo shows the direction of the side chains of the bound peptide. The side chains at p5 and p8 are completely exposed to solvent. The p7 side chain is at least partially buried in a shallow pocket and the p3 side chain is exposed to solvent on a shelf-like pocket. The p4 and p6 side chains, defined as an anchor residue, are buried in a deep pocket within the peptide binding groove.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 1. (A) The direction of the side chains of the peptide bound to HLA-DR molecule: side view of the peptide with the {alpha}1 helix to the left and the ß1 helix to the right. The side chains at p1 and p4 are shown. The arrows represent the direction of the side chains at p3, p5, p6, p7 and p8. (B) Putative TCR-binding regions on the DR4 molecule: white balls, C{alpha} atoms of selected residues as the putative TCR-contacting sites; black balls, C{alpha} atoms of p3, p4, p5, p6, p7 and p8. The DR4 molecule and antigenic peptide are indicated by the C{alpha} trace: {alpha}-chain, thin line; ß-chain, bold line; peptide, wide bold line. The circles represent each pocket in the peptide-binding groove. The TCR-contact sites were defined as follows: {alpha}55 Glu, {alpha}57 Gln, {alpha}61 Ala, {alpha}62 Asn, {alpha}64 Ala, {alpha}65 Val, {alpha}67 Lys, {alpha}68 Ala, ß63 Ser, ß64 Gln, ß66 Asp, ß70 Gln, ß73 Ala, ß77 Thr, ß81 His and ß85 Val. This figure was generated using the program MOLSCRIPT (Kraulis, 1991Go).

 
Knowing the structure of a TCR complexed with a class I MHC molecule bound to a peptide (Garboczi et al., 1996Go; Garcia et al., 1996Go, 1998Go; Ding et al., 1998Go; Teng et al., 1998Go), questions about orientation of TCR on the MHC–peptide ligand can be generalized to all TCR interactions with class I molecules and perhaps also with class II MHC molecules (Jorgensen et al., 1992Go; Sant'Angelo et al.,1996Go; Chang et al., 1997Go). The TCR interacts with limited regions of the long {alpha}-helix in the diagonal configuration (Bjorkman, 1997Go; Smith and Lutz, 1997Go). Thus, helical regions ({alpha}55, {alpha}57, {alpha}61, {alpha}62, {alpha}64, {alpha}65, {alpha}67, {alpha}68, ß63, ß64, ß66, ß70, ß73, ß77, ß81 and ß85) of {alpha} and ß chains were previously defined as the putative TCR-binding regions (Toh et al., 1998Go) (Figure 1BGo).

The molecular dynamics (MD) simulation technique has the attractive feature that motions on the atomic level can be monitored and analyzed in atomic detail. To study the role of water molecules in the binding of peptides to MHC (Rognan et al., 1992aGo,bGo; Meng et al., 1997Go), to predict the binding epitope sequence (Rognan et al., 1994Go; Lim et al., 1996Go), to design the class I MHC ligands (Rognan et al., 1995Go, Krebs et al., 1998Go) and to explain the existing MHC-binding motifs (Hadida et al., 1995Go; Rognan et al., 1997Go; Steele et al., 1998Go), the MD simulations methods have been used for MHC–peptide complexes.

In our previous work, we performed MD simulation of the DR4–index peptide (YWALEAAAD) and DR4–analogue peptide (agonists and antagonists) complexes. The root mean square (r.m.s.) deviation between two structures is a measure of their differences. The MD simulations showed that the r.m.s. deviations of the DR4–antagonist complexes in the TCR-binding regions from the DR4–index peptide complex were larger than those of the DR4–agonist complexes. These results indicated that subtle changes of the TCR-binding regions on HLA molecule by the antagonist peptides could induce TCR antagonistic activities (Toh et al., 1998Go). We report here the results of additional MD simulations of the DR4–analogue peptide (including null peptide) complexes. To analyze the principal determining factor in discriminating between the strong and the weak antagonists, we calculated the r.m.s. deviations from the index complex and the exposed areas of the side chains of the bound peptide. The results show that the weak antagonistic peptides could cause larger main chain shifts of the TCR-binding regions than do the strong antagonistic peptides. The simulations also indicate that the null peptides would be treated as completely different ligands mainly through the changes of the exposed side chains at p3, p5 or p7, rather than the conformational changes of the TCR-binding surfaces on the DR4 molecule.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Preparation of the input coordinates

All simulations were performed using the InsightII/Discover software package (Molecular Simulations, San Diego, CA). The starting structure for all simulations of the DR4 complexes was the X-ray crystal structure of HLA–DRB1*0401 complex with a peptide derived from human collagen II (Protein Data Bank entry 2SEB; Dessen et al., 1997). Hydrogen atoms were added to this model using the InsightII program. To save computational time, only antigen-binding sites ({alpha}3–{alpha}84, ß3–ß94) were taken into account in the study. This approximation was shown not to alter the accuracy of molecular dynamics simulations because only limited interactions exist between the {alpha}1ß1 and {alpha}2ß2 domains that do not significantly influence the shape of the peptide-binding groove (Rognan et al., 1992aGo). DRB1*0405–collagen peptide complex was constructed by replacement of two DRß residues (DRß57 and DRß71) that differ between DRB1*0401 and DRB1*0405. A peptide (NH2–AAYWALEAAADAA–COOH) and all analogue peptides bound to DRB1*0405 molecule were similarly generated from the backbone of the bound collagen peptide (residues 1168–1180), QYMRADQAAGGLR, co-crystallized with DRB1*0401 by substituting by corresponding residues.

Molecular mechanics and dynamics simulation protocol

Calculations were performed using the Discover 95.0 program. The parameter used was the consistent valence force field (CVFF). No cross-terms were used in the energy expression and a simple harmonic valence potential was used to model the valence bond stretching term. The minimized structures were solvated by a 10.0 Å layer of water molecules using the SOAK option of InsightII. For the water molecule the SPC model was used (Hermans et al., 1984Go; Berendesen et al., 1987Go). During the molecular dynamics and minimization, a dielectric constant of 1.0 was used. Before the MD simulations were started, we performed energy minimizations in a four-step procedure: (1) the coordinates of all the protein atoms were fixed and solvent molecules were minimized using the steepest descent method (in all constrained minimizations for up to 1000 iterations); (2) the heavy chain atoms were tethered loosely (10 kcal/mol.Å2) while the hydrogen atoms were adjusted; (3) the backbone atoms were tethered loosely (10 kcal/mol.Å2) while the side chain was adjusted; (4) finally, the conjugated gradients minimizations were performed with all atoms free to move until the maximum derivative was <0.5 kcal/mol.Å. The minimized coordinates were used as a starting point for NVT (constant volume and temperature) molecular dynamics at 300 K to generate possible stable conformations. The system was warmed to 300 K. Bond lengths were constrained to equilibrium values during the simulations using the RATTLE method with a tolerance of 10–5 (Andersen, 1983Go). For the treatment of the non-bond interactions, the cell multipole method (CMM) was used. The CMM is more rigorous and efficient than the application of cut-offs (Ding et al., 1992Go; Mathiowetz et al., 1994Go; Sugita and Kitao, 1998Go). After a 10 ps equilibration stage, the simulations were continued at 300 K for 590 ps using a 2.0 fs time step. The simulations were carried out using the Verlet velocity algorithm (Swope and Anderson, 1982Go) and the structures were stored in the computer every 1.0 ps. Acquired data were analyzed during the last 300 ps of the 600 ps simulation. Initially, we defined an average atomic conformation of the DR4–index peptide (YWALEAAAD) complex during the last 300 ps of the simulation as an `index complex'.

Solvent-accessible surface areas

Solvent-accessible surface areas were computed for the structures during the last 300 ps of the simulations. The Connolly algorithm with a solvent probe radius of 1.4 Å was used (Connolly, 1983Go).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Time course of the simulations

To study the basis for the observed phenomena, molecular dynamics simulations of DR4 (DRB1*0405)–analogue peptide complexes were carried out for 600 ps. A standard way to evaluate the quality of an MD simulation is to monitor the r.m.s. deviations of protein atoms from the starting structure. Figure 2Go shows the r.m.s. positional deviations of the C{alpha} atoms of DR4–peptide complexes from each starting structure. Slight fluctuations were observed, but the r.m.s. deviations were stable around a constant value over the last 300 ps of the trajectory. Acquired data were analyzed during the last 300 ps of the 600 ps simulation.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 2. R.m.s. deviations of DRB1*0405 ({alpha}3–{alpha}84, ß3–ß94) C{alpha} atoms from the starting structure before the MD simulation as a function of time: index complex, bold line; p7–Phe complex, open circles; p7–Pro complex, closed circles; p7–Trp complex, thin line.

 
Structural differences between the DR4–strong and DR4–weak antagonist complexes

In the proposed TCR-binding regions on the DR4 molecule, the r.m.s. deviations for C{alpha} traces between the index complex and DR4–analogue peptides (p3, p4, p5, p6, p7 and p8) complexes were computed (Figure 3Go). The r.m.s. deviations of the antagonist complexes were larger than those of the agonists complexes, as reported previously (Toh et al., 1998Go).



View larger version (53K):
[in this window]
[in a new window]
 
Fig. 3. The C{alpha} r.m.s. deviations of the DR4–agonist and DR4–antagonist complexes from those of the time-averaged conformation of the DR4–index peptide (YWALEAAAD) complexes in the putative TCR-binding sites as a function of time: agonists, bold lines; strong antagonists, open circles and squares; weak antagonists, closed circles and squares. (A) p3–variants; (B) p4–variants; (C) p6–variants; (D) p7–variants; (E) p8–variants. Average ± standard deviations (SD) of the r.m.s. deviations are shown.

 
Furthermore, we analyzed the structural difference between the strong antagonist and the weak antagonist using the r.m.s. deviations and the exposed surface areas. In the antagonist complexes, the r.m.s. deviations of the weak antagonist complexes were relatively larger than those of the strong antagonist complexes (Figure 3Go). The effects of the analogue peptides on the conformational changes were different among the variants at each position.

The p3 side chain contacts with open pocket in the binding site. The effects of the substitutions at p3 on the ß1 {alpha}-helix were hardly found. In the simulations of the p3–Leu (a weak antagonist) complex, the calculated r.m.s. deviations were higher than those of the p3–Val (a strong antagonist) complex (Figure 3AGo). The p3–Leu side chain was more buried than other p3 side chains, indicating the buried portion at pocket 3 induces the structural changes of the TCR binding region (Figure 4Go). In the p3–Phe (a weak antagonist) complex, the r.m.s. deviations were comparable to those of the p3–Gly (an agonist) complex. The p3–Phe side chain, however, was more exposed to solvent (Figure 4Go). In the p3–agonist and p3–antagonist complexes, ligands with more exposed side chain at p3 tended to be the weak antagonists (Figure 4Go). A large portion of the p4 side chain (about 20% residue exposure) is buried in a hydrophobic pocket. In the simulation of the p4–variants, the r.m.s. deviations of the p4–Tyr (a weak antagonist) were higher than those of other p4–variants (Figure 3BGo). The p4–Tyr side chain was too large to fit into pocket 4. Thus, the ß1 {alpha}-helix between ß53 and ß73 shifted. The p6 side chain is buried in small pocket. In the p6–variants, the r.m.s. deviations of the p6–Val (a weak antagonist) were higher than those of other p6–variants (Figure 3CGo) and the {alpha}1 {alpha}-helix near pocket 6 fluctuated. In the solvent-accessible areas of the p6 side chains in the p6–variants, there were no remarkable differences (Figure 4Go). The p7 side chain is partly buried in a shallow pocket 7. The p7 side chains in both the index and the p7–agonist complexes were much less exposed in pocket 7 (Figure 4Go). In the p7–weak antagonist complexes, the r.m.s. deviations were much higher than those of the other p7–variants (Figure 3DGo). A wider range of C{alpha} deviations of the p7–antagonist complexes was observed throughout the simulation. The r.m.s. deviations of the p7–variants were higher than those of the p3–, p4–, p6– and p8–variants, indicating that substitutions at p7 have a relatively larger influence to alter conformations of the TCR-binding sites than those at other positions (Figure 3Go). The ß-chain helical region around pocket 7 which exhibits high temperature factors in all class II MHC structures was flexible. This flexible regions around pocket 7 induced the larger effects at p7 (Figure 5Go). The p8 side chain projects into solvent. In the p8–variants, a smaller range of C{alpha} deviations was observed throughout the simulation than the other variants (Figure 3Go). The calculated r.m.s. deviations of the p8–Asp (a weak antagonist) were much higher than those of the other p8–variants. In the p8–antagonist complexes, small shifts occurred in the location of the {alpha}1 {alpha}-helix between {alpha}53 and {alpha}61, but the ß1 {alpha}-helix did not move. In the solvent-accessible areas of the p8 side chains of the p8–variants, there was no relationship between the agonists and the antagonists (Figure 4Go).



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 4. Mean and SD values of the surface-exposed areas of the side chain substituted at each position in the analogue peptides. All values were computed during the last 300 ps of the MD simulations.

 


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5. The structural differences between the p7–Ala (index) and the p7–Gln (weak antagonist) complexes. The p7 side chains of the bound peptides are shown. In the TCR-binding regions on the DR4 molecule, the two complexes are superimposed: white balls, C{alpha} atoms of the putative TCR-contacting sites; p7–Ala (index) complex, thin line; p7–Gln (weak antagonist), bold line. This figure was generated using the program MOLSCRIPT (Kraulis, 1991Go).

 
Remarkable features of the DR4–null peptide complexes

We also investigated the factors discriminating the null peptides from other ligands (especially antagonist). In the null peptide (p3, p5 and p7) complexes, the calculated deviations for C{alpha} traces from the index complex in the TCR-binding regions were relatively larger than those of the agonist complexes (Figure 6Go). The r.m.s. deviations of the p3–null peptide complexes were comparable to those of the p3–antagonist complexes (Figures 3A and 6AGoGo). There were no differences in the r.m.s. deviations in the TCR-binding regions between the p3–null peptide and the p3–antagonist complexes. In the p3–null peptide complexes, the p3 side chains were more exposed to solvent than in the p3–antagonist complexes (Figure 4Go). In the p5–null peptide complexes, the r.m.s. deviations were comparable to those of the p5-Gly (an agonist) (Figure 6BGo). In the p5–null peptides, a smaller range of C{alpha} deviations was observed throughout the simulation than in the p3– and p7–null peptide complexes (Figure 6Go). In the p5–variant complexes, a correlation between the solvent-accessible areas of the p5 side chains and the groups of the analogue peptide was not found (Figure 4Go). In the p7–null peptide variants, the r.m.s. deviations from the index complex in the TCR-binding regions were lower than those of the p7–weak antagonist complexes (Figures 3D and 6CGoGo). In the p7–Ile and p7–Met complexes, the {alpha}61, {alpha}62 and ß63–ß73 region, situated at the center in the groove, shifted. The surface-exposed areas of the p7 side chain were relatively larger than those of the index, agonist and antagonist complexes (Figure 4Go).



View larger version (45K):
[in this window]
[in a new window]
 
Fig. 6. The C{alpha} r.m.s. deviations of the DR4–null peptide complexes from those of the time-averaged conformation of the DR4–index peptide (YWALEAAAD) complexes in the putative TCR-binding sites as a function of time: agonists, bold lines; null peptides, circles and squares. (A) p3–variants; (B) p5–variants; (C) p7–variants. Average ± SD of the r.m.s. deviations are shown.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have performed the MD simulations of the DR4–analogue peptide substituted at p3, p4, p5, p6, p7 or p8 (agonists, antagonists and null peptides) complexes in a model system developed for analyzing the specific features of the T cell clone that recognizes the index peptide (YWALEAAAD) presented by the DR4 (DRB1*0405) molecule. Our previous work showed that subtle structural changes in TCR-contact regions on HLA molecule could induce TCR antagonism (Toh et al., 1998Go). The present studies demonstrate the differences between the strong and the weak antagonists and the characters of the null peptide complexes.

About two thirds of the total energy for the TCR–ligand (peptide–MHC) interaction is directed toward the MHC (Manning et al., 1998Go). Our simulations, together with our previous study, showed that the r.m.s. deviations of the weak antagonists were relatively larger than those of the strong antagonists at each position (Figure 3Go). We see that the weak antagonistic peptides could cause larger structural changes in the TCR-binding sites on HLA molecule than do the strong antagonistic peptides. Thus, these subtle changes of the TCR-binding regions on HLA molecule would reduce affinity for TCR. The affinity (Kd) of the antagonist peptide complexes for the TCR was lower than that of the agonist peptide complexes (Alam et al., 1996Go). This would result in a reduction in the time that TCR remain engaged with its ligand (Matsui et al., 1994Go). A shorter interaction time between the TCR and its ligand then results in a qualitatively different signal through the TCR (McKeithan, 1995Go; Lyons et al., 1996Go; Rabinowitz et al., 1996Go). Thus, the magnitude of the conformational changes of the HLA scaffold would be relevant to the strength of the TCR antagonism.

The effects of the antagonists on the conformational changes are different among the variants at each peptide position. In the p3–variants, peptides with more exposed side chain at p3 tended to be the weak antagonists except the p3–Leu (Figure 4Go). These data suggest that the p3–antagonists are distinguished by both the structural changes on the TCR-binding region and the exposed p3 side chain. The TCR could not recognize directly the p4 and p6 side chains which are buried in a pocket as a second anchor residue. The simulations of the p4– and p6–variants indicate that changes of the anchor residues have potential to cause the structural alterations of the TCR-recognition surface on the HLA molecule. While the changes at p4 situated under the ß1 {alpha}-helix induced the ß1 {alpha}-helix, those at p6 situated under the {alpha}1 {alpha}-helix induced the {alpha}1 {alpha}-helix. Substitutions of p7 induce shifts of both the {alpha}1 and the ß1 {alpha}-helix (Figure 5Go) and have a greater potential to cause the structural changes of the TCR-binding sites than those at p3, p4, p5, p6 and p8 (Figures 3 and 6GoGo). In the p8–variants, no correlation between the solvent-accessible areas of the p8 side chains and the types of the analogue peptides is found (Figure 4Go). No p8–variants are classified in the null peptides (Table IGo). Therefore, although the p8 side chain projects into solvent like the p5 side chain, the side chain at p8 could not be recognized directly by the TCR. A smaller range of C{alpha} deviations was observed throughout the simulation than the other variant complexes (Figure 3Go). The r.m.s. fluctuations of the p8–variant complexes were hardly affected by substitutions at p8 (data not shown). These data indicated that the p8–variants are distinguished through the structural changes at the TCR-binding sites.

We also analyzed the structural features of the null peptide complexes. The r.m.s. deviations of the p3–null peptide complexes were comparable to those of the p3–antagonists (Figures 3 and 6GoGo). It was difficult to explain from the r.m.s. deviations in the TCR-binding regions the differences between the null peptides and the antagonists. The p3 side chains of the p3–null peptides were more exposed to solvent than the p3–antagonists (Figure 4Go). The p3–null peptides would be discriminated by changes of the surfaces on the bound peptide components, rather than the conformational change of the TCR-binding surfaces on the HLA molecule. The simulations showed that the r.m.s. deviations of the p5–null peptide complexes were lower than those of the p3– and p7–null peptides and comparable to the p5–Gly (an agonist) (Figure 6Go). These data suggested that the changes at p5 hardly affected the structure of the TCR-binding sites. In our model system, the TCR is very sensitive to minor changes in charge and shape at p5 such as the Glu to Asp exchange (Table IGo). The p5 side chain projects completely into solvent at the center of the peptide-binding groove. These results indicate that the p5–null peptides would be recognized by TCR as completely different ligands mainly through the change of the exposed p5 side chain in the bound peptide. The simulations of the p7–null peptide complexes showed that the r.m.s. deviations were smaller than those of the p7–weak antagonists (Figures 3 and 6GoGo), suggesting that the structural changes of the TCR-binding sites are not the main factor to be null ligands with regard to p7. The r.m.s. deviations of the p7–Trp and p7–Tyr complexes were comparable to those of the p7–agonists (Figure 6Go). The large p7–Trp and p7–Tyr side chains were not able to fit into shallow pocket 7 and were exposed (Figure 4Go). Thus, the p7–Trp and p7–Tyr complexes were discriminated by the exposed p7 side chains. The p7–Ile and p7–Met side chains were partly buried, but induced the shifts at the center of the peptide-binding groove unlike the p7–antagonists. Thus, the bulky p7 side chain, which is partly buried, affects the center surface of the HLA–peptide complex. The p7–null peptides would be discriminated by changes of the surfaces on peptide–HLA complexes, especially the center of the peptide-binding groove.

Our previous simulations showed that the TCR antagonists could cause larger structural changes in TCR contact regions on the HLA molecule than do the agonists (Toh et al., 1998Go). In addition, the present MD simulations show that the weak antagonists could induce relatively larger structural changes in TCR-binding regions than do the strong antagonists. Thus, the magnitude of the structural changes of the TCR-binding regions would determine the strength of T-cell antagonism. The simulations also indicate that the changes of the exposed side chain in the bound peptide are the factors in treating the null peptides as completely different ligands. Subtypes of HLA–DR4 are strongly associated with autoimmune diseases including rheumatoid arthritis (Todd et al., 1988Go). Our observations may be useful for targeted vaccine design or immunotherapy with analogue peptides.


    Notes
 
3 To whom correspondence should be addressed Back


    Acknowledgments
 
This work was partially supported by a Grant-in-Aid for Scientific Research on Priority Areas, `Genomic Informatics', from the Ministry of Education, Science, Sports and Culture of Japan and by the Research for the Future Program of the Japan Society for the Promotion of Science. Computation time was provided by the Supercomputer Laboratory, Institute for Chemical Research, Kyoto University and by the Super Computer System, Human Genome Center, Institute of Medical Science, University of Tokyo.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Alam,S.M., Travers,P.J., Wung,J.L., Nasholds,W., Redpath,S., Jameson,S.C. and Gascoigne,N.R.J. (1996) Nature, 381, 616–620.[ISI][Medline]

Andersen,H.C. (1983) J. Comput. Phys., 52, 24–34.[ISI]

Berendesen,H.J.C., Grigera,J.R. and Straatsma,T.P. (1987) J. Phys. Chem., 91, 6269–6271.[ISI]

Bjorkman,P.J. (1997) Cell, 89, 167–170.[ISI][Medline]

Chang,H.-C., Smolyar,A., Spoerl,R., Witte,T., Yao,Y., Goyarts,E.C., Nathenson,S.G. and Reinherz,E.L. (1997) J. Mol. Biol., 271, 278–293.[ISI][Medline]

Chen,Y.-Z., Mastushita,S. and Nishimura,Y. (1996) J. Immunol., 157, 3783–3790.[Abstract]

Connolly,M.L. (1983) Science, 221, 709–713.[ISI][Medline]

De Magistris,M.T., Alexander,J., Coggeshall,M., Altman,A., Gaeta,F.C., Grey,H.M. and Sette,A. (1992) Cell, 68, 625–634.[ISI][Medline]

Dessen,A., Lawrence,C.M., Cupo,S., Zaller,D.M. and Wiley,D.C. (1997) Immunity, 7, 473–481.[ISI][Medline]

Ding,H.-Q., Karasawa,N. and Goddard,W.A.,III (1992) J. Chem. Phys., 97, 4309–4315.[ISI]

Ding,Y.-H., Smith,K.J., Garboczi,D.N., Utz,U., Biddison,W.E. and Wiley,D.C. (1998) Immunity, 8, 403–411.[ISI][Medline]

Ding,Y.-H., Baker,B.M., Garboczi,D.N., Biddison,W.E. and Wiley,D.C. (1999) Immunity, 11, 45–56.[ISI][Medline]

Dittel,B.N., Sant'Angelo,D.B. and Janeway,C.A.,Jr (1997) J. Immunol., 158, 4065–4073.[Abstract]

Fremont,D.H., Henderrickson,W.A., Marrack,P. and Kappler,J. (1996) Science, 272, 1001–1004.[Abstract]

Garboczi,D.N., Ghosh,P., Utz,U., Fan,Q.R., Biddison,W.E. and Wiley,D.C. (1996) Nature, 384, 134–141.[ISI][Medline]

Garcia,K.C., Degano,M., Stanfield,R.L., Brunmark,A., Jackson,M.R., Peterson,P.A., Teyton,L. and Wilson,I.A. (1996) Science, 274, 209–219.[Abstract/Free Full Text]

Garcia,K.C., Degano,M., Pease,L.R., Huang,M., Peterson,P.A., Teyton,L. and Wilson,I.A. (1998) Science, 279, 1166–1172.[Abstract/Free Full Text]

Ghosh,P., Amaya,M., Mellins,E. and Wiley,D.C. (1995) Nature, 378, 457–462.[ISI][Medline]

Hadida,F., Haas,G., Zimmermann,N., Hosmalin,A., Spohn,R., Samri,A., Jung,G., Debre,P. and Autran,B. (1995) J. Immunol., 154, 4174–4186.[Abstract/Free Full Text]

Hermans,J., Berendsen,H.J.C. and Postma,J.P.M. (1984) Biopolymers, 23, 1513–1518.[ISI]

Ikegawa,S., Matsushita,S., Chen,Y.-Z., Ishikawa,T. and Nishimura,Y. (1996) J. Allergy Clin. Immunol., 97, 53–64.[Medline]

Jardetzky,T.S., Brown,J.H., Gorga,J.C., Stern,L.J., Urban,R.G., Strominger,J.L. and Wiley,D.C. (1996) Proc. Natl Acad. Sci. USA, 93, 734–738.[Abstract/Free Full Text]

Jorgensen,J., Esser,U., Fazekas de St. Groth,B., Reay,P.A. and Davis,M.M. (1992) Nature, 355, 224–230.[ISI][Medline]

Kersh,G.J. and Allen,P.M. (1996a) Nature, 380, 495–498.[ISI][Medline]

Kersh,G.J. and Allen,P.M. (1996b) J. Exp. Med., 184, 1259–1268.[Abstract]

Kinouchi,R., Kobayashi,H., Sato,K., Kimura,S. and Katagiri,M. (1994) Immunogenetics, 40, 376–378.[ISI][Medline]

Kraulis,P.J. (1991) J. Appl. Crystallogr., 24, 946–950.[ISI]

Krebs,S., Lamas,J.R., Poenaru,S., Folkers,G., Lopez de Castro,J.A. and Rognan,D. (1998) J. Biol. Chem., 273, 19072–19079.[Abstract/Free Full Text]

Lim,J.S., Kim,S., Lee,H.G., Lee,K.Y., Kwon,T.J. and Kim,K. (1996) Mol. Immunol., 33, 221–230.[ISI][Medline]

Lyons,D.S., Lieberman,S.A., Hampl,J., Boniface,J.J., Chien,Y.-h., Berg,L.J. and Davis,M.M. (1996) Immunity, 5, 53–61.[ISI][Medline]

Manning,T.C., Schlueter,C.J., Brodnicki,T.C., Parke,E.A., Speir,J.A., Garcia,K.C., Teyton,L., Wilson,I.A. and Kranz,D.M. (1998) Immunity, 8, 413–425.[ISI][Medline]

Mathiowetz,A.M., Jain,A., Karasawa,N. and Goddard,W.A.,III (1994) Proteins: Struct. Funct. Genet., 20, 227–247.[ISI][Medline]

Matsui,K., Boniface,J.J., Steffner,P., Reay,P.A. and Davis,M.M. (1994) Proc. Natl Acad. Sci. USA, 91, 12862–12866.[Abstract/Free Full Text]

Matsuoka,T., Kohrogi,H., Ando,M., Nishimura,Y. and Matsushita,S. (1996) J. Immunol., 157, 4837–4843.[Abstract]

Matsushita,S., Takahashi,K., Motoki,M., Komiyama,K., Ikagawa,S. and Nishimura,Y. (1994) J. Exp. Med., 180, 873–883.[Abstract]

McKeithan,T.W. (1995) Proc. Natl Acad. Sci. USA, 92, 5042–5046.[Abstract]

Meng,W.S., Grafensen,H. and Haworth,I.S. (1997) Int. Immunol., 9, 1339–1346.[Abstract]

Murthy,V.L. and Stern,L.J. (1997) Structure, 5, 1385–1396.[ISI][Medline]

Rabinowitz,J.D., Beeson,C., Lyons,D.S., Davis,M.M. and McConnell,H.M. (1996) Proc. Natl Acad. Sci. USA, 93, 1401–1405.[Abstract/Free Full Text]

Reid,S.W. et al. (1996) J. Exp. Med., 184, 2270–2286.

Rognan,D., Reddehase,M.J., Koszinowski,U.H. and Folkers,G. (1992a) Proteins: Struct. Funct. Genet., 13, 70–85.[ISI][Medline]

Rognan,D., Zimmermann,N., Jung,G. and Folkers,G. (1992b) Eur. J. Biochem., 208, 101–113.[Abstract]

Rognan,D., Scapozza,L., Folkers,G. and Daser,A. (1994) Biochemistry, 33, 11476–11485.[ISI][Medline]

Rognan,D., Scapozza,L., Folkers,G. and Daser,A. (1995) Proc. Natl Acad. Sci. USA, 92, 753–757.[Abstract]

Rognan,D., Krebs,S., Kuonen,O., Lamas,J.R., Lopez de Castro,J.A. and Folkers,G. (1997) J. Comput.-Aided Mol. Des., 11, 463–478.

Sant'Angelo,D.B., Waterbury,G., Preston-Hurlburt,P., Voon,S.T., Medzhitov,R., Hong,S.-C. and Janeway,C.A.,Jr (1996) Immunity, 4, 367–376.[ISI][Medline]

Scott,C.A., Peterson,P.A., Teyron,L. and Wilson,I.A. (1998) Immunity, 8, 319–329.[ISI][Medline]

Sloan-Lancaster,J., Evavold,B.D. and Allen,P.M. (1993) Nature, 363, 156–159.[ISI][Medline]

Smith,K.D. and Lutz,C.T. (1997) J. Immunol., 158, 2805–2812.[Abstract]

Smith,K.J., Pyrdol,J., Gauthier,L., Wiley,D.C. and Wucherpfenning,K.W. (1998) J. Exp. Med., 188, 1511–1520.[Abstract/Free Full Text]

Steele,J.C., Young,S.P., Goodall,J.C. and Gallimore,P.H. (1998) J. Immunol., 161, 4745–4752.[Abstract/Free Full Text]

Stern,L.J., Brown,J.H., Jardetzky,T.S., Gorga,J.C., Urban,R.G., Stominger,J.L. and Wiley,D.C. (1994) Nature, 368, 215–221.[ISI][Medline]

Sugita,Y and Kitao,A. (1998) Proteins: Struct. Funct. Genet., 30, 388–400.[Medline]

Swope,W.C. and Anderson,H.C. (1982) J. Chem. Phys., 76, 637–649.[ISI]

Tana,T., Kamikawaji,N., Savoie,C.J., Sudo,T., Kinoshita,Y. and Sasazuki,T. (1998) J. Hum. Genet., 43, 14–21.[ISI][Medline]

Teng,M.-K., Smolyar,A., Tse,A.G.D., Liu,J.-H., Liu,J., Hussey,R.E., Nathenson,S.G., Chang,H.-C., Reinherz,E.L. and Wang,J. (1998) Curr. Biol., 8, 409–412.[ISI][Medline]

Todd,J.A. et al. (1988) Science, 240, 1003–1009.[ISI][Medline]

Toh,H., Kamikawaji,N., Tana,T., Sasazuki,T. and Kuhara,S. (1998) Protein Eng., 11, 1027–1032.[Abstract]

Received December 2, 1999; revised March 2, 2000; accepted April 7, 2000.





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (7)
Request Permissions
Google Scholar
Articles by Toh, H.
Articles by Kuhara, S.
PubMed
PubMed Citation
Articles by Toh, H.
Articles by Kuhara, S.