Coupling X-Ray Reflectivity and In Silico Binding to Yield Dynamics of Membrane Recognition by Tim1.

The dynamic nature of lipid membranes presents significant challenges with respect to understanding the molecular basis of protein/membrane interactions. Consequently, there is relatively little known about the structural mechanisms by which membrane-binding proteins might distinguish subtle variations in lipid membrane composition and/or structure. We have previously developed a multidisciplinary approach that combines molecular dynamics simulation with interfacial x-ray scattering experiments to produce an atomistic model for phosphatidylserine recognition by the immune receptor Tim4. However, this approach requires a previously determined protein crystal structure in a membrane-bound conformation. Tim1, a Tim4 homolog with distinct differences in both immunological function and sensitivity to membrane composition, was crystalized in a closed-loop conformation that is unlikely to support membrane binding. Here we have used a previously described highly mobile membrane mimetic membrane in combination with a conventional lipid bilayer model to generate a membrane-bound configuration of Tim1 in silico. This refined structure provided a significantly improved fit of experimental x-ray reflectivity data. Moreover, the coupling of the x-ray reflectivity analysis with both highly mobile membrane mimetic membranes and conventional lipid bilayer molecular dynamics simulations yielded a dynamic model of phosphatidylserine membrane recognition by Tim1 with atomic-level detail. In addition to providing, to our knowledge, new insights into the molecular mechanisms that distinguish the various Tim receptors, these results demonstrate that in silico membrane-binding simulations can remove the requirement that the existing crystal structure be in the membrane-bound conformation for effective x-ray reflectivity analysis. Consequently, this refined methodology has the potential for much broader applicability with respect to defining the atomistic details of membrane-binding proteins.