Probing the Integrin Allostery by Thermal Stability and Affinity

αXβ2 integrin is central to the migration of myeloid cells during inflammatory response. The ligand binding domain of αXβ2, called αX I-domain, has a Mg2+-binding site (MIDAS) and an allosteric α7-helix. It has been postulated that the MIDAS site crosstalks with α7-helix and this cross-talk is associated with conformational change within the fold of the αX I-domain, yet biochemical basis of this cross-talk has yet to be elucidated. Our hypothesis is that MIDAS/α7-helix crosstalk exists in a thermodynamic equilibrium between ensembles of open and closed states. Our goal is to test how the observed conformational changes alters Mg2+-affinity to MIDAS as well as the thermal stability of the αX I-domain in solution. Conclusions. Mg2+ affinity to the intact αX-I domain is 4.848 mM ± 0.133 (Fig.X1) and the αX I-domain locked in an open-alternate conformation by I314G mutation on the αI α7-helix is 1.006 mM ± 0.050 (Fig.X2). However, the high range of melting temperature in the I314G αX I-domain suggests that the transition could potentially be bi-phasic involving two different binding-phases with the dissociation constants of 0.146 mM ± 0.020 and 3.328 mM ± 0.325. This observation is an indication that a multiple ensemble of conformations could exist between the open and closed states of the αX I-domain. These observations indicate that 1) Mg2+ stabilizes the αX-I domain and 2) smaller range of melting (40-48 ºC) of the intact αX I-domain compared to that of I314G αX I-domain (43-55 ºC) shows that the close state is less dynamic than the open state.Biology and Biochemistry, Department ofHonors Colleg