In recent years, atomistic molecular simulations have become a method of choice for studying the interaction of small molecules, peptides, and proteins with biological membranes. Here, we critically examine the statistical convergence of equilibrium properties in molecular simulations of two amino acid side-chain analogs, leucine and arginine, in the presence of a hydrated phospholipid bilayer. To this end, the convergence of the standard binding free energy for the reversible insertion of the solutes in the bilayer is systematically assessed by evaluating dozens of separate sets of umbrella sampling calculations for a total simulation time exceeding 400 μs. We identify rare and abrupt transitions in bilayer structure as a function of solute insertion depth. These transitions correspond to the slow reorganization of ionic interactions involving zwitterionic phospholipid headgroups when the solutes penetrate the lipid-water interface and when arginine is forced through the bilayer center. These rare events are shown to constitute hidden sampling barriers that limit the rate of convergence of equilibrium properties and result in systematic sampling errors. Our analysis demonstrates that the difficulty of attaining convergence for lipid bilayer-embedded solutes has, in general, been drastically underestimated. This information will assist future studies in improving accuracy by selecting a more appropriate reaction coordinate or by focusing computational resources on those regions of the reaction coordinate that exhibit slow convergence of equilibrium properties.

The first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we present crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures reveal that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutations at the KRAS-CRD interface result in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.

ENSEMBLE is a program implemented in C and embedded in a Perl wrapper. It is supported on main Linux distributions. Source codes and installation files, including a detailed example, can be freely downloaded at http://abragam.med.utoronto.ca/∼JFKlab.