Characterization of the putative binding site of Staphylococcal Enterotoxin R (SER) to the murine TCR Vb8.2.

CHIAPPINI S; ROMASANTA P; DE MARZI M; FERNÁNDEZ M; MALCHIODI E

Buenos Aires

Congreso; I Congreso Argentino –Francés de Inmunología y LVIII Reunión Anual de la SAI; 2010

SAI

Superantigens (SAgs) are bacterial or viral protein characterized by simultaneously binding to MCH-II molecules and TCRs. As a consequence of this interaction they activate a large number of T-cells through interaction with Vb domain of the TCR, triggering the massive release of cytokines that may cause Toxic Shock Syndrome. In addition, SAgs are tightly involved in autoimmune processes such as diabetes mellitus, rheumatoid arthritis and multiple sclerosis. The potential use of SAgs and TCRs as therapeutics agents for these pathologies makes the biophysical and structural studies of importance. The latest incorporations to SEB subfamily or Group II were the staphylococcal enterotoxins G (SEG) and R (SER), which have amino acid sequence similarity. The Group II is characterized by interacting with the murine Vb8.2 TCR, the principal receptor involved in multiple sclerosis mouse model. Previously we have reported that SER and SEG present a differential behavior with Vb8.2, compared with other members of the group. The aim of the present work was to determine the putative binding site of the complex SER-Vb8.2. On that purpose, a model of SER structure was carried out with the Swiss Model server using SEG as a search model. The refined model was superimposed onto SEG-Vb8.2 structure using the Pymol computational program. Based on structural analysis, we designed SER mutants with point mutations in the Vβ8.2 putative binding site. The mutants were expressed in E. coli and purified by exclusion molecular chromatography. The affinity for Vb8.2 was determinated by surface plasmon resonance showing mutants K19A (53 uM) and G20A (34 uM) lower affinity than the wild type (8 uM); or N24D, N24A and F204A mutants no interaction at all. The determined affinities allow us to characterize SER-Vb8.2 binding site and identify N24 and F204 as the residues that make the greatest energetic contribution to stabilizing the interaction.