No abstract

Spontaneous transitions between the native and non-native protein conformations are normally rare events that hardly take place in typical unbiased molecular dynamics simulations. It was recently demonstrated that such transitions can be well described by a reaction coordinate, Q, that represents the collective fraction of the native contacts between the protein atoms. Here we attempt to use this reaction coordinate to enhance the conformational sampling. We perform umbrella sampling simulations with biasing potentials on Q for two model proteins, Trp-Cage and BBA, using the CHARMM force field. Hamiltonian replica exchange is implemented in these simulations to further facilitate the sampling. The simulations appear to have reached satisfactory convergence, resulting in unbiased free energies as a function of Q. In addition to the native structure, multiple folded conformations are identified in the reconstructed equilibrium ensemble. Some conformations without any native contacts nonetheless have rather compact geometries and are stabilized by hydrogen bonds not present in the native

HIV-1 maturation inhibitors (MIs), Bevirimat (BVM) and its analogs interfere with the catalytic cleavage of spacer peptide 1 (SP1) from the capsid protein C-terminal domain (CACTD), by binding to and stabilizing the CACTD-SP1 region. MIs are under development as alternative drugs to augment current antiretroviral therapies. Although promising, their mechanism of action and associated virus resistance pathways remain poorly understood at the molecular, biochemical, and structural levels. We report atomic-resolution magic-angle-spinning NMR structures of microcrystalline assemblies of CACTD-SP1 complexed with BVM and/or the assembly cofactor inositol hexakisphosphate (IP6). Our results reveal a mechanism by which BVM disrupts maturation, tightening the 6-helix bundle pore and quenching the motions of SP1 and the simultaneously bound IP6. In addition, BVM-resistant SP1-A1V and SP1-V7A variants exhibit distinct conformational and binding characteristics. Taken together, our study provides a structural explanation for BVM resistance as well as guidance for the design of new MIs.

It has been challenging to obtain reliable free energies for protein conformational changes from all-atom molecular dynamics simulations, despite the availability of many enhanced sampling techniques. To alleviate the difficulties associated with the enormous complexity of the conformational space, here we propose a few practical strategies for such calculations, including (1) a stringent method to examine convergence by comparing independent simulations starting from different initial coordinates, (2) adoption of multistep schemes in which the complete conformational change consists of multiple transition steps, each sampled using a distinct reaction coordinate, and (3) application of boundary restraints to simplify the conformational space. We demonstrate these strategies on the conformational changes between the outward-facing and outward-occluded states of the Mhp1 membrane transporter, obtaining the equilibrium thermodynamics of the relevant metastable states, the kinetic rates between these states, and the reactive trajectories that reveal the atomic details of spontaneous transitions. Our approaches thus promise convergent and reliable calculations to examine intuition-based hypotheses and to eventually elucidate the underlying molecular mechanisms of reversible conformational changes in complex protein systems.

Protein sequence matching does not properly account for some well-known features of protein structures: surface residues being more variable than core residues, the high packing densities in globular proteins, and does not yield good matches of sequences of many proteins known to be close structural relatives. There are now abundant protein sequences and structures to enable major improvements to sequence matching. Here, we utilize structural frameworks to mount the observed correlated sequences to identify the most important correlated parts. The rationale is that protein structures provide the important physical framework for improving sequence matching. Combining the sequence and structure data in this way enables the incorporation of allosteric information into sequence matching and transforms it effectively from a 1-D to a 3-D procedure. Our results show that there are major gains in the specificity of sequence matching across diverse proteins. Specifically, all known cases where protein structures match but sequences do not match well are resolved.