The function of membrane proteins often depends on the proteins' interaction with their lipid environment, spectacularly so in the case of mechanosensitive channels, which are gated through tension mediated by the surrounding lipids. Lipid bilayer tension is distributed quite inhomogeneously, but neither the scale at which relevant variation takes place nor the effect of varying lipid composition or tension has yet been investigated in atomic detail. We calculated lateral pressure profile distributions in lipid bilayers of various composition from all-atom molecular dynamics simulations totaling 110.5 ns in length. Reproducible pressure profile features at the 1 A length scale were determined. Lipids with phosphatidylcholine headgroups were found to shift the lateral pressure out of the hydrophobic core and into the headgroup region by an amount that is independent of area per lipid. POPE bilayers simulated at areas smaller than optimal exerted dramatically higher lateral pressure in a narrow region at the start of the aliphatic chain. Stretching of POPC bilayers increased tension predominantly in the same region. A simple geometric analysis for the gating of the mechanosensitive channel MscL suggests that pressure profiles affect its gating through the second moment of the profile in a tension-independent manner.
Interpreting variants of uncertain significance (VUS) is a central challenge in medical genetics. One approach is to experimentally measure the functional consequences of VUS, but to date this approach has been post hoc and low throughput. Here we use massively parallel assays to measure the effects of nearly 2000 missense substitutions in the RING domain of BRCA1 on its E3 ubiquitin ligase activity and its binding to the BARD1 RING domain. From the resulting scores, we generate a model to predict the capacities of full-length BRCA1 variants to support homology-directed DNA repair, the essential role of BRCA1 in tumor suppression, and show that it outperforms widely used biological-effect prediction algorithms. We envision that massively parallel functional assays may facilitate the prospective interpretation of variants observed in clinical sequencing.KEYWORDS deep mutational scanning; BRCA1; variants of uncertain significance; human genetic variation; protein function I N an era of increasingly widespread genetic testing, DNA sequencing identifies many missense substitutions with unknown effects on protein function and disease risk. In the absence of genetic evidence, experimental measurement is the most reliable way to determine the functional impact of a variant of uncertain significance (VUS). However, initiating an experiment for each new variant observed in a gene is often impractical. When experiments are done, they are nearly always performed in a retrospective manner (Bouwman et al. 2013), such that the resulting data are not useful for the patient in whom the VUS was observed.By prospectively measuring, in a high-throughput fashion, the consequences of all possible missense mutations on a gene's function, we can generate a look-up table for interpreting newly observed VUS. Although functional analysis at this scale is made possible by deep mutational scanning (Fowler and Fields 2014), a central challenge is that any single assay may not recapitulate all the activities of a given protein in human disease. To address this challenge, we hypothesized that integrating the results of assays of multiple biochemical functions would strengthen estimates of the effects of mutations on disease risk (strategy outlined in Figure 1A). As a proof-ofconcept, we initiated massively parallel functional analysis of BRCA1, a protein for which there are multiple biochemical functions as well as known pathogenic and benign missense substitutions to benchmark results.BRCA1 has been subject to intense study since its implication in hereditary, early onset breast and ovarian cancer (Miki et al. 1994). All missense substitutions in BRCA1 that are known to be pathogenic occur in either the amino-terminal RING domain or the carboxy-terminal BRCT repeat (http:// brca.iarc.fr/LOVD/home.php?select_db=BRCA1). Although the RING domain represents only 5% of the BRCA1 protein, 58% of the pathogenic missense substitutions occur within this domain. Sixty-two missense substitutions in the RING domain have been observed in patient...
Molecular recognition and mechanical properties of proteins govern molecular processes in the cell
Steered molecular dynamics simulations of the mechanosensitive channel of large conductance, MscL, were used to investigate how forces arising from membrane tension induce gating of the channel. A homology model of the closed form of MscL from Escherichia coli was subjected to external forces of 35-70 pN applied to residues near the membrane-water interface. The magnitude and location of these forces corresponded to those determined from the lateral pressure profile computed from a lipid bilayer simulation. A fully expanded state was obtained on the 10-ns timescale that revealed the mechanism for transducing membrane forces into channel opening. The expanded state agrees well with proposed models of MscL gating, in that it entails an irislike expansion of the pore accompanied by tilting of the transmembrane helices. The channel was most easily opened when force was applied predominantly on the cytoplasmic side of MscL. Comparison of simulations in which gating progressed to varying degrees identified residues that pose steric hindrance to channel opening.
The mechanosensitive channel of large conductance (MscL) in prokaryotes plays a crucial role in exocytosis as well as in the response to osmotic downshock. The channel can be gated by tension in the membrane bilayer. The determination of functionally important residues in MscL, patch-clamp studies of pressure-conductance relationships, and the recently elucidated crystal structure of MscL from Mycobacterium tuberculosis have guided the search for the mechanism of MscL gating. Here, we present a molecular dynamics study of the MscL protein embedded in a fully hydrated POPC bilayer. Simulations totaling 3 ns in length were carried out under conditions of constant temperature and pressure using periodic boundary conditions and full electrostatics. The protein remained in the closed state corresponding to the crystal structure, as evidenced by its impermeability to water. Analysis of equilibrium fluctuations showed that the protein was least mobile in the narrowest part of the channel. The gating process was investigated through simulations of the bare protein under conditions of constant surface tension. Under a range of conditions, the transmembrane helices flattened as the pore widened. Implications for the gating mechanism in light of these and experimental results are discussed.
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