Modeling the bound conformation of Pemphigus Vulgaris-associated peptides to MHC Class II DR and DQ Alleles

Allele comparisons – HLA DR4 PV

The sequence identity between the DR4 alleles (excluding DRB1*0401) with their corresponding templates ranges from 97.9 to 99.0%, and the sequence similarity (representing identical and conservatively substituted residues) was between 98.4 and 99.5% (Table 1). All five important peptide-binding pockets 1, 4, 6, 7 and 9 show extremely high structural conservation at the Cα positions, suggesting that any peptide discrimination leading to epitope selection between the alleles is mainly due to the size and nature of the side chains of the pocket residues. In order to further isolate the true disease-relevant allele within a haplotype, we compared specific residues in the polymorphic pockets regarded as important in conferring specificity for antigen presentation (Figure 1). Pocket 1, characterized by a Val/Gly β86 dimorphism, is the deepest cavity and thus, the most important anchor for peptide binding [29]. In addition, the functional specificity of DR4 molecules is also affected by polymorphisms at position β70, β71, β74, which contribute to pocket 4. Two negatively charged residues at position β70 and β71 that were previously suggested to influence peptide selectivity in PV patients [30] could be found in DRB1*0402 (Asp β70 and Glu β71) but a positively charge Arg/Lys β71 was found in DRB1*0404, *0406 and *0401. Amino acid polymorphism can also be observed at position β11 of pocket 6, β71 of pocket 7 and β37 of pocket 9 respectively.

Allele	Template	Sequence Identity	Positives	Cα RMSD (Å)
				α & β chains	Pockets
					P1	P4	P6	P7	P9
DRB1*0402	1D5Z	97.9%	99.0%	0.35	0.12	0.06	0.07	0.10	0.09
DRB1*0404	1D5Z	99.0%	99.5%	0.31	0.15	0.10	0.06	0.07	0.18
DRB1*0406	1D5Z	97.9%	98.4%	0.32	0.11	0.15	0.07	0.11	0.22
DRB1*1401	1D5Z	94.1%	97.3%	0.25	0.11	0.09	0.02	0.09	0.18
DRB1*1404	1D5Z	85.8%	89.5%	0.29	0.12	0.10	0.02	0.06	0.22
DRB1*1405	1D5Z	81.0%	83.2%	0.24	0.11	0.07	0.02	0.08	0.07
DQB1*0202	1S9V	98.0%	99.0%	0.57	0.16	0.09	0.04	0.15	0.05
DQB1*0503	1UVQ	93.0%	96.0%	0.39	0.03	0.07	0.01	0.10	0.06

Allele comparisons – HLA DR6 PV

Study of individual allele frequencies in DR6 PV patients revealed that the relevant disease susceptibility allele is DQB1*0503 instead of DR6 alleles [Sinha et al., manuscript in preparation]. DQB1*0503 and the DR6 PV non-associated alleles investigated in this study show a significant degree of overlap in alignment, with 14 amino acid differences in areas of the binding cleft that could affect peptide binding. Clear differences in the amino acid sequences are observed at residue β86 of pocket 1, residues β13, β70, β71, β74, β78 of pocket 4, residue β11 of pocket 6, residues β28, β30, β67, β71 of pocket 7 and residues β9, β37, β57, β60 of pocket 9. Similar to the DR4 alleles, all five important peptide-binding pockets 1, 4, 6, 7 and 9 in DBQ1*0503 and DR6 alleles demonstrate exceptionally high structural conservation at the Cα positions. A significant difference is that DQB1*0503 contains a negatively charged Asp β57 that differs from the uncharged Ala β57 found in non-PV associated DRB1*1401 and *1404. Also, at positions β70 and β71, DQB1*0503 does not contain negatively charged residues identified in DRB1*0402 that are critical for binding of self-antigens in DR4 PV patients. Instead, these positions were replaced by two small neutral hydrophobic residues (Gly β70 and Ala β71), suggesting that DRB1*0402 and DQB1*0503 may recognize different sets of PV epitopes under the influence of a different balance of intermolecular forces. Positions β70 and β74 show charge reversal, in the non-PV associated DRB1*1401, *1404 and *1405 alleles while the negative charge at β71 alone is conserved, compared to DRB1*0402, making pocket 4 the single dominant factor discriminating between PV non-association and susceptibility.

Allele comparisons – PV protective and susceptible alleles

Differences in the amino acid sequences are observed at residue β86 of pocket 1, residue β70, β71 of pocket 4, residues β28, β30, β47, β71 of pocket 7 and residues β37, β57 of pocket 9. Both protective alleles (DQB1*0201 and DQB1*0202) do not contain negatively charged residues at position β70 (pocket 4) and β71 (pocket 7). Instead, these positions were replaced by two large and positively charged amino acids (Arg β70 and Lys β71). The functional specificities of PV protective and susceptible alleles are also affected by clear structural differences in the Cα positions of both α and β chains (Cα RMSD > 0.57Å) indicating that any differences in peptide discrimination between the alleles is due to a combination of both the backbone conformation as well as the size and nature of the side chains of the pocket residues.

Epitope comparisons – HLA DR4 PV

Eight previously identified stimulatory Dsg3 epitopes (Dsg3 191–205, Dsg3 206–220, Dsg3 252–266, Dsg3 342–356, Dsg3 380–394, Dsg3 763–777, Dsg3 810–824 and Dsg3 963–977) for DRB1*0402 were docked into the binding groove of all DR4 (DRB1*0401, *0402, *0404, *0406) alleles investigated in this study. Analysis of these Dsg3 peptide-bound alleles revealed that only one peptide conformation can fit perfectly into the binding cleft of DRB1*0402, and atomic clashes of these Dsg3 peptides are obtained for all other DR4 subtypes investigated in this study. Notably, two epitopes (Dsg3 342–356 and Dsg3 810–824) have small residues (Ser/Cys) in pocket 1, suggesting that large anchor residues may play a critical role for high affinity binding in DR4 PV molecules, an observation previously documented for influenza-associated I-A^d allele of mice [31]. This finding provides support to the evidence that DRB1*0402 is associated with PV whereas other DR4 subtypes are non-associated, with the exception of DRB1*0406 that is reported to be associated in the Japanese population [32]. As such, there is a possibility of the existence of other peptides relevant in the Japanese populations that bind to *0406 but are yet to be determined.

Epitope comparisons – HLA DR6 PV

Dsg3 96–112, a recently identified epitope in DR6 PV patients [28], fits perfectly into the binding groove of DQB1*0503 with two identified core sequences at residues 101–109 and residues 102–110. The identified 101–109 core has four intermolecular hydrogen bonds compared to seven intermolecular hydrogen bonds in the core of 102–110. Perfect fitting of Dsg3 206–220, Dsg3 252–266, Dsg3 342–356, Dsg3 810–824 and Dsg3 963–977 into the binding groove of DQB1*0503 is also obtained. Atomic clashes are obtained for Dsg3 191–205, Dsg3 380–394 and Dsg3 763–777 as well as all DR6 alleles investigated in this study. The proportion of DRB1*1401, *1404 and *1405 has been reported to be increased in PV probably due to linkage disequilibrium. The lack of binding of all stimulatory peptides investigated in this study to these alleles indicates that the HLA association in DR6 PV patients is more likely at the DQB1 locus (DQB1*0503 allele) and not the linked DRB1 loci (DRB1*1401, *1404 and *1405). Our data supports the notion that the reported associations of this disease with DRB1*1401, *1404, *1405 are due to linkage disequilibrium with the true disease associated allele (DQB1*0503).

Epitope comparisons – PV susceptibility Alleles

Epitope comparisons – PV protective alleles

Our simulation results indicate that DQB1*0201 and DQB1*0202 can bind to multiple core sequences for the majority of PV epitopes investigated in this study. DQB1*0201 can bind one epitope (Dsg3 963–977) at two core regions, one epitope (Dsg3 206–220) at three core regions, three epitopes (Dsg3 191–205, 252–266 and 342–356) at four core regions, and two epitopes (Dsg3 96–112 and 810–824) at five core regions. DQB1*0202 can bind two epitopes (Dsg3 96–112 and 963–977) at three core regions, two epitopes (Dsg3 342–356 and 810–824) at four core regions and one epitope (Dsg3 252–266) at five core regions. In contrast, the majority of PV epitopes (with the exception of Dsg3 96–112 and 252–266) can bind to PV susceptible alleles DRB1*0402 and DQB1*0503 at a single core. This finding lends support to the hypothesis that the protective alleles DQB1*0201, *0202 may be capable of binding to most peptides with greater affinity than PV susceptible alleles, allowing for efficient deletion of autoreactive T cells [33].

Role of flanking residues in peptide selection

Our data demonstrates that the conformations of flanking peptide residues that extend beyond the binding groove are critical to peptide selection in MHC class II alleles. The core sequences of Dsg3 963–977 fit perfectly within the binding grooves of non-associated alleles DRB1*0401, *0404, and *1404 but poor contacts to the respective alleles at Phe α50 are obtained when the conformation of the N-terminal flanking residue Ile4 is taken into account. These results suggest that binding is determined by both the core and flanking segments while considering the overall interactions between each peptide and the respective alleles.

Sequence motifs

Peptide VII (Dsg3 763–777) agrees well with the motifs from Veldman et al. [28] and Sinha et al. (unpublished results), while all other peptides show low to moderate compliance. Of the four positions compared, peptide IV (Dsg3 252–266) shows agreement only at position p6. For Dsg3 342–356 peptide, the core nonamer identified by our models is 346–354, which is register-shifted by one residue from the core of 347–355 reported by Veldman et al. [28], and 345–353 identified by Sinha et al. (unpublished results), for the binding groove of *0402. This shift is critical as residues p1 and p4 identified by us do not fit well into both binding motifs. Our modeling studies suggest that peptide position p4 need not be positively charged as indicated by Veldman et al. [28], supporting the existence of a more degenerate motif by Sinha et al. (unpublished results) at this position. In addition, p1 also appears to be more degenerate than previously suggested [28], showing a preference for hydrophobic and large residues but can accommodate residues of other sizes as well. Hence for generating sequence patterns to design peptides for vaccine design, structural information is important [34] and the exact peptide in the binding groove identified by our docking protocol will be most useful here.

Disease progression in PV

T cell response to a number of epitopes among PV patients has been reported in several studies [2, 8, 26, 29, 30]. There may be disease heterogeneity, meaning that clinically similar but distinct phenotypes could operate by alternate pathways, each with a different initial immunodominant epitope(s). The differential T cell reactivities among individual patients to individual peptides may also be a function of the disease stage or severity and correlate with mechanisms of disease progression. While there may be a limited set of epitopes present in patients in the early stages of the disease, epitope spreading can occur during disease progression, resulting in reactivity to previously innocuous epitopes. In addition, reactivities to multiple epitopes within individual patients were detected in two cases (Dsg3 191–205 and 342–356 for PV107; Dsg3 191–205, 810–824 and 963–977 for PV117). Autoantibodies against desmoglein 1 have also been reported in severe disease [35]. One other incidence of multiple T cell reactivities within a PV patient has been previously reported [29]. These findings, together with our simulation results, lend further credence to the hypothesis that no single epitope is responsible for both disease initiation and propagation and are consistent with the expected and observed ability to generate multiple pMHC complexes from a single target autoantigen.

Template search

In this study, ten PV associated, closely related non-associated and protective MHC class II alleles DRB1*0401, *0402, *0404, *0406, *1401, *1404, *1405, DQB1*0201, *0202, and *0503 were selected for analysis. MHC sequence data were obtained from IMGT-HLA http://www.ebi.ac.uk/imgt/hla/[36] database. The α chain of all DR alleles investigated in this study is the DRA1*0101 sequence, with the β chain from the allele sub-type. To identify potential structural templates available in the Protein Data Bank (PDB) [37] for model building, a sequence similarity search was performed using PSI-BLAST [38] running on the servers at NCBI http://www.ncbi.nlm.nih.gov/blast/ and the highest quality templates were selected among the returned results. Among these, the crystal structures of HLA-DR4 (PDB code 1D5Z) and HLA-DQ2 (PDB code 1S9V) were adopted as the structures of DRB1*0401 and DQB1*0201 respectively (100% sequence identity). The crystal structures of DRB1*0401 (PDB code 1D5Z), DQB1*0602 (PDB code 1UVQ) and DQB1*0201 (PDB code 1S9V) were selected as templates for all other DR subtypes, DQB1*0503 and DQB1*0202 respectively (Table 1).

Model building

The program MODELLER [39] was employed for comparative modeling of both DRB1 (*0402, *0404, *0406, *1401, *1404, *1405) and DQB1 (*0202, *0503) subtypes. The models are constructed by optimally satisfying spatial constraints obtained from the alignment of the template structure with the target sequence and from the CHARMM-22 force field [40]. The initial model was refined by assigning the rotameric states of essential side chains according to the corresponding crystal structure, followed by a short energy minimization [41] from the program Internal Coordinates Mechanics (ICM; Molsoft LLC, San Diego, CA) [42].

Patient recruitment and groupings

Peptide set

Nine previously identified epitopes Dsg3 96–112, 191–205, Dsg3 206–220, Dsg3 252–266, Dsg3 342–356, Dsg3 380–394, Dsg3 763–777, Dsg3 810–824 and Dsg3 963–977 that elicited primary proliferative T cell response in PV patients [2, 8, 26, 29, 30] were selected for modeling studies. T cell response to eight of these peptides (Dsg3 191–205, Dsg3 206–220, Dsg3 252–266, Dsg3 342–356, Dsg3 380–394, Dsg3 763–777, Dsg3 810–824 and Dsg3 963–977) has been reported in patients carrying DRB1*0402. Dsg3 96–112 has been reported to elicit T cell response in patients with DQB1*0503 but lacking DRB1*0402 [28]. Of these Dsg3 191–205, Dsg3 342–356, Dsg3 810–824 and Dsg3 963–977 were shown to directly bind to the DRB1*0402 receptor by competitive binding assays (Sinha et al., unpublished results). Briefly, soluble HLA DRA1*0101/DRB1*0402 were purified by DR-specific affinity chromatography and incubated with different concentrations of experimental peptides (0–40 μM) in the presence of biotinylated class II-associated invariant-chain peptide (CLIP) (1 μM) for 2 hours. The MHC-peptide complexes were then captured on a 96-well plate coated with anti-HLA-DR (L243) (BD Pharmingen, San Diego, CA). The CLIP bound to the MHC molecules was directly assayed using Europium (Eu)-labeled streptavidin (Perkin Elmer, Boston, MA). The relative binding of peptides was subsequently determined by measuring the displacement of the CLIP at different peptide concentrations.

Peptide docking

Analysis of binding motifs http://www.syfpeithi.de[43] and available crystal structures suggested a core region of nine amino acids as essential for binding. Based on this observation, a sliding window input of size nine (Figure 2) is applied to generate all combinations of nonameric core peptide residues to be modeled into the binding groove of each allele. Each core peptide fragment is docked into the binding groove in a three-step protocol described in an earlier study [27]. In brief, docking of core peptide residues is performed as follows: (i) core peptide fragments at the ends of the binding groove is docked using ICM biased Monte Carlo procedure, followed by (ii) loop closure of peptide core residues by satisfaction of spatial constraints, and finally (iii) refinement of the backbone and side-chains of peptide core residues as well as atomic clash regions at receptor. Next, flanking residues are extended from the core peptide residues using ICM biased Monte Carlo procedure. The adopted empirical scoring function [44, 45] takes into account continuum and discrete electrostatics, and hydrophobic and entropy loss [45, 46]. This methodology allows rapid and accurate docking of peptides with an average computing time of approximately 18 minutes for the complete modeling of each peptide on a 4-CPU SGI Origin 3200 workstation. For each ligand, the best solution is obtained based on the following criteria: pattern of hydrogen bonding to the MHC molecule, pattern of hydrophobic burial of peptide side chains, and the absence of atomic clashes or repulsive contacts.

Definition of contact residues

In this study, MHC-peptide residues were considered to be in contact if at least one pair of their non-hydrogen ("heavy") atoms was found to be within 4.00Å radius [47]. Intra-peptide interactions and intra-MHC interactions were not considered as they have minor influence on peptide/protein backbone structure. Any atom in the peptide and any atom in the MHC were considered to be experiencing atomic clash if their separation is below 2.00Å [48] for non-hydrogen atoms and below 1.60Å for atoms participating in hydrogen bonds [49, 50].

Definition of binding pocket for MHC Class II alleles

Interactions between side-chains of bound peptide ligands and polymorphic cavities (or anchor "pockets") in the binding site of MHC class II alleles are important in determining the peptide binding affinity and sequence specificity of MHC molecules and are defined according to the work of Stern et al. [51, 52].