POSITIVE COOPERATIVITY BINDING MODEL BETWEEN LY49 NATURAL KILLER CELL RECEPTORS AND THE IMMUNOEVASIN M157: KINETIC AND THERMODYNAMIC STUDIES.

PABLO ROMASANTA; TODONE M.; SARRATEA B; ANTONOGLOU B; NOLI TRUANT S; FERNÁNDEZ LYNCH M.J.; URTASUN N; DE MARZI M; CURTO L., ; MIRANDA M.V.; DELFINO JM; FERNÁNDEZ M.M; MALCHIODI E.L.

Los Cocos, Córdoba

Congreso; LXI Reunión de la Sociedad Argentina de Inmunología (SAI); 2013

Sociedad Argentina de Inmunología (SAI)

NK cells can discriminate between infected or tumoral cells and healthy cells employing a vast panel of surface receptors, both activating and inhibitory. There are several receptor families involved in this function. Among these, homodimeric Ly49 receptors were demonstrated to be of particular importance for detection of murine cytomegalovirus (MCMV) infected cells by murine NK cells. MCMV infection has become an established model to study NK cells functions. MCMV m157 represents the only MHC-I mimic described able to recognize both activating (Ly49H) and inhibitory (Ly49I) receptors. In the present work, we conducted kinetic and thermodynamic experiments in order to elucidate a molecular model that explained the Ly49-m157 binding, employing the activating Ly49H and inhibitory Ly49I receptors. Combining Surface Plasmon Resonance (SPR), fluorescence anisotropy and circular dichroism (CD) techniques, together with an exhaustive statistical analysis, we determined that the best mechanism to describe both Ly49H-m157 and Ly49I-m157 interactions is a conformational selection model where only the closed conformation of Ly49 (Ly49*) is able to bind a first m157 ligand, followed by a bimolecular binding of a second m157 to the specie Ly49*m157. A positive cooperativity behavior is present in Ly49, being the binding of the second m157 (~108M-1) 3-fold order higher than the binding of the first m157 (~105M-1), with advantageous biological consequences. Employing far-UV CD, we found strong evidence that a conformational change is taking place during the binding process, supporting the positive cooperativity behavior. Finally, a thermodynamic analysis established that the rate-limiting step of the whole mechanism is the conformational transition from the open to the closed form. The global thermodynamic parameters from the initial state (free forms of open Ly49 and m157) to the final state (Ly49*(m157)2), is characterized by an enthalpy increase (endothermic reaction) that is compensated by an entropy increase, making the interaction spontaneous.