Structural and dynamical insights on HLA DRB1-peptide complexes which confer resistance and susceptibility to Multiple Sclerosis in Sardinia:

a Molecular Dynamics simulation study

A.Kumar1,2*, E.Cocco1, L.Atzori1, M.G.Marrosu1, E.Pieroni2
1University

of Cagliari, 2CRS4-Bioengineering group *corresponding author amit369@gmail.com

Abstract
Multiple Sclerosis is an autoimmune disease linked to inammatory and degenerative process in the central nervous system. ! Human Leukocyte Antigen (HLA) class II system has been identied as the main genetic determinant regions linked to MS [1]. ! Recent genetic studies [2], have identied and associated ve HLADRB1 alleles to Multiple Sclerosis (MS) in Sardinia. The basis of adaptive immune response has been associated with recognition of peptides bound to specic membrane glycoprotein, the Major Histocompatibility Complex (MHC) by T-cell antigen receptors (TCR)[3]. Antigen/Peptide presentation by MHC class II is critical component of the adaptive immune response to foreign pathogens. The availability of high resolution X-ray structures of the complexes in some cases have provided structural insights for antigen presentation. Our research is focused on investigation of MHC class II peptide interaction with an emphasis on identifying structural and dynamical differences between the predisposing and protective DRB1 alleles complexed with both self Myelin Basic protein (MBP) and non-self Epstein Barr Virus (EBV) peptide at a microscopic level. Our!detailed analysis conrms that a functional relation between MS predisposing genetic background and antigen presentation can be investigated by MD simulations and provide useful insights on functional mechanism at molecular level.

Results
Stability of structure during simulation

Conclusions
The binding cleft is stable only in the peptide. presence of Region D1 is narrow and rigid in presence of MBP, for the two alleles, while in presence of EBV is open and exible. On one hand Region D2 is open and rigid in presence of EBV in DR-1601 allele, while Region D3 is open and rigid in presence of MBP in DR-1501 allele. The nature of interaction of MBP with the two alleles is quite similar which is also reected in similar binding energy values -16.75, -17.52 (in kcal/mol) respectively. On the other hand for EBV the interaction picture is very different, providing an optimal binding with DR-1501 allele with an energy value of -16 kcal/mol. All together, our analysis suggest that propensity to MS in Sardinia can be linked with some specic peculiarities in the antigen presentation mechanism of the alleles.

Binding Region Analysis

Methodology
Starting X-ray structure (PDB id: 1BX2) for 1501 allele complexed with MBP (85-98) Homology model done for 1601 allele using MODELLER EBV sequence (400-413) was taken from EBNA-1 protein[4] NAMD-2.8 software Molecular Dynamics package CHARMM-27 and AMBER-99 Force Field parameters for protein and peptide, TIP3P for water molecules Periodic boundary conditions with box dimension of [78 91 82], with total number of atoms ~50.000 12 cut-off radius for both Van der Waals and electrostatic interactions along with smooth particle mesh Ewald grid [96 96 96]

Introduction
The HLA system, the MHC in humans, is controlled by genes located on chromosome 6. It encodes cell surface molecules specialized to present antigenic peptides to the TCR on T cells MHC molecules that present antigen (Ag) are divided into two main classes, i.e class I and class II Class II MHC molecules typically present peptides derived from extracellular (exogenous) Ag to CD4 TH cells; class I MHC typically present peptides derived from intracellular (endogenous) Ag (eg, viruses) to CD8 cytotoxic T cells. The activated T cell then kills the infected cell directly or through complex mechanisms.

Nature of Interactions

In Sardinia, two of the main alleles correlated with MS are DRB1*15:01 and DRB1*16:01 [2]. The binding cleft is characterized by pockets with amino acids which participate (i) in anchoring (P1, P4, P6, P9) and (ii) recognition (P4, P7) of peptide/antigen.

In the bar-plot above and on the right are shown H-bonds for the alleles DRB1:1501, DRB1:1601 with the peptide EBV and MBP, present for at least 20 % of simulation time.

Binding Energy Calculations

Binding Free Energy calculation for alleles DRB1:1501 (in blue) and DRB1:1601 (in green) in complex with MBP and EBV at different intervals of simulation time.

References
[1] The international Multiple Sclerosis Genetics Consortium. Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis. Nat Genet. 2011;476:214219. [2] Cocco E, Sardu C, Pieroni E, Valentini M, Murru R, et al. (2012) HLADRB1-DQB1 Haplotypes Confer Susceptibility and Resistance to Multiple Sclerosis in Sardinia. PLoS ONE 7(4): e33972. doi:10.1371/journal.pone. 0033972. [3] Kaas, Q.,Lefranc, M.P. (2005) T cell receptor/peptide/MHC molecular characterization and standarized pMHC contact sites in IMGT/3D structure-DB, In Silico Biology, 5, 505-528. [4]R Mechelli, J Anderson, D Vittori, G Coarelli, V Annibali, S Cannoni, F Aloisi, M Salvetti, JA James, and G Ristori EpsteinBarr virus nuclear antigen-1 B-cell epitopes in multiple sclerosis twins Mult Scler November 2011 17: 1290-1294.

In gure above is highlighted pockets identied in the binding cleft of antigen-HLA complex