Biology subjects

Faraldo-Gomez, J.

Publications and source records attributed to Faraldo-Gomez, J..

7 recordsLinked to original sources

bioRxiv2023

Mitigation of membrane morphology defects explain stability and orientational specificity of CLC dimers

Most membrane proteins are oligomers, but the physical forces explaining the stable association of these complexes inside the lipid bilayer are not well understood. The homodimeric antiporter CLC-ec1 highlights the puzzling nature of this reaction. This complex is thermodynamically stable even though it associates via a large hydrophobic protein-protein interface that appears well adapted to interact with the membrane interior. In a previous study, however, we discovered that when CLC-ec1 is dissociated, this interface introduces a morphological defect in the surrounding membrane, leading us to hypothesize association is driven by the elimination of this defect upon dimerization. This study tests this hypothetical mechanism directly and shows it is supported by molecular and physical models. First, using coarse-grained umbrella-sampling molecular simulations, we calculated the membrane contribution to the potential-of-mean-force for dimerization in a POPC bilayer. This shows the stable association of CLC subunits prior to formation of direct protein-protein contacts, but only via the native interface that presents the membrane defect, and not others. Single-molecule photobleaching experiments show that addition of short-chain DLPC lipids, known to alleviate the membrane defect, also shifts the association equilibrium from dimers to monomers. We explain this destabilizing effect through additional umbrella-sampling and alchemical free-energy simulations, which show DLPC enrichment of the defect diminishes the membrane contribution to the association free energy, as it improves the lipid-solvation energetics of the monomer but not the dimer. In summary, this study establishes a physical model that explains the stability and orientational specificity of CLC dimers in terms of membrane-mediated forces, rather than protein-protein interactions. We posit that cells might ubiquitously leverage morphological defects in the bilayer to drive organization of membrane proteins into functional complexes, and that cellular regulation of lipid composition can modulate this organizing effect.

biophysics↗

bioRxiv2021

Structure of the Shaker Kv channel and mechanism of slow C-type inactivation

Voltage-activated potassium (Kv) channels open upon membrane depolarization and proceed to spontaneously inactivate. Inactivation controls neuronal firing rates and serves as a form of short-term memory, and is implicated in various human neurological disorders. Here, we use high-resolution cryo-electron microscopy and computer simulations to determine one of the molecular mechanisms underlying this physiologically crucial process. Structures of the activated Shaker Kv channel and of its W434F mutant in lipid bilayers demonstrate that C-type inactivation entails the dilation of the ion selectivity filter, and the repositioning of neighboring residues known to be functionally critical. Microsecond-scale molecular dynamics trajectories confirm these changes inhibit rapid ion permeation through the channel. This long-sought breakthrough establishes how eukaryotic K+ channels self-regulate their functional state through the plasticity of their selectivity filters. One-Sentence SummaryStructures of the Shaker Kv channel reveal the mechanism of slow C-type inactivation involves dilation of the selectivity filter.

biophysics↗

bioRxiv2021

Cryo-EM structure of an elusive pre-transport intermediate of the multidrug transporter BmrCD reveals modes of asymmetric drug binding

Vectorial substrate efflux by ATP binding cassette (ABC) transporters, which play a major role in multidrug resistance, entails the ATP-powered interconversion of the transporter between stable intermediates. Despite recent progress in structure elucidation of ABC transporters, a number of such intermediates have yet to be visualized and mechanistically interpreted. Here, we combine single particle cryo-EM, Double Electron Electron Resonance (DEER) spectroscopy with Molecular Dynamics simulations to profile and mechanistically frame the conformation of a hitherto unobserved intermediate in the context of BmrCD, a heterodimeric multidrug ABC exporter from Bacillus subtilis. In our cryo-EM structure, BmrCD adopts an inward-facing architecture bound to both ATP and the substrate Hoechst-33342 and is capped by an extracellular domain which undergoes ATP-dependent conformational changes. A striking feature of the structure is a symmetric arrangement of the nucleotide-binding domain (NBD) in the presence of ATP whereas binding of Hoechst at two distinct sites in an acidic pocket stabilizes an asymmetric arrangement of the transmembrane domain architecture (TMD). Mutation of residues coordinating Hoechst in the structure abrogates the cooperative stimulation of ATP hydrolysis. In conjunction with previous studies, our findings suggest a mechanistic role for symmetry mismatch between NBDs and TMDs in the conformational cycle of ABC transporters. Moreover, the resolved structures of bimodally-bound drugs are of notable importance for future rational design and optimization of molecules for targeted transport inhibition of ABC transporters. ONE SENTENCE SUMMARYCryo-EM and EPR analysis reveal cooperative substrate binding in BmrCD in an architecture primed for transport.

biophysics↗

bioRxiv2021

Weighted force analysis method for derivation of multidimensional free energy landscapes from adaptively-biased replica simulations

A methodology is proposed for the calculation of multidimensional free-energy landscapes of molecular systems, based on analysis of multiple Molecular Dynamics trajectories wherein adaptive biases have been applied to enhance the sampling of different collective variables. In this approach, which we refer to as Force Correction Analysis Method (FCAM), local averages of the total and biasing forces are evaluated post-hoc, and the latter are subtracted from the former to obtain unbiased estimates of the mean force across collective-variable space. Multidimensional free-energy surfaces and minimum free-energy pathways are then derived from integration of the mean force landscape through kinetic Monte Carlo algorithm. To evaluate the proposed method, a series of numerical tests and comparisons with existing approaches were carried out for small molecules, peptides, and proteins, based on all-atom trajectories generated with standard, concurrent and replica-exchange Metadynamics in collective-variable spaces ranging from one- to six-dimensional. The tests confirm the correctness of the FCAM formulation and demonstrate that calculated mean forces and free energies converge rapidly and accurately, outperforming other methods used to unbias this kind of simulation data. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/431654v5_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1412289org.highwire.dtl.DTLVardef@1425d53org.highwire.dtl.DTLVardef@1fcf5d0org.highwire.dtl.DTLVardef@6e6941_HPS_FORMAT_FIGEXP M_FIG TOC/Abstract Graphic C_FIG

biophysics↗

bioRxiv2020

Dimerization of a membrane transporter is driven by differential energetics of lipid solvation of dissociated and associated states

Over two-thirds of integral membrane proteins of known structure assemble into oligomers. Yet, the forces that drive the association of these proteins remain to be delineated, as the lipid bilayer is a solvent environment that is both structurally and chemically complex. In this study we reveal how the lipid solvent defines the dimerization equilibrium of the CLC-ec1 Cl-/H+ antiporter. Integrating experimental and computational approaches, we show that monomers associate to avoid a thinned-membrane defect caused by their exposed dimerization interfaces. In this defect, lipids are strongly tilted and less densely packed than in the bulk, with a larger degree of entanglement between opposing leaflets and greater water penetration into the bilayer interior. Dimerization restores the membrane to a near-native state and therefore, appears to be driven by the larger free-energy cost of lipid solvation of the dissociated protomers. Supporting this theory, we demonstrate that addition of short-chain lipids strongly shifts the dimerization equilibrium towards the monomeric state, and show that the cause of this effect is that these lipids preferentially solvate the defect. Importantly, we show that this shift requires only minimal quantities of short-chain lipids, with no measurable impact on either the macroscopic physical state of the membrane or the proteins biological function. Based on these observations, we posit that free-energy differentials for local lipid solvation define membrane-protein association equilibria. With this, we argue that preferential lipid solvation is a plausible cellular mechanism for lipid regulation of oligomerization processes, as it can occur at low concentrations and does not require global changes in membrane properties. IMPACT STATEMENTDifferences in the lipid solvation energetics of associated and dissociated states is a primary driving force for membrane protein oligomerization, presenting a molecular mechanism for lipid regulation in biology.

biophysics↗

bioRxiv2019

DHHC20 palmitoyl-transferase reshapes the membrane to foster catalysis

Cysteine palmitoylation, a form of S-acylation, is a key post-translational modification in cellular signaling. This type of reversible lipidation is catalyzed by a family of integral membrane proteins known as DHHC acyltransferases. The first step in the S-acylation process is the recognition of free acyl-CoA from the lipid bilayer. The DHHC enzyme then becomes auto-acylated, at a site defined by a conserved Asp-His-His-Cys motif. This reaction entails ionization of the catalytic Cys. Intriguingly, in known DHHC structures this catalytic Cys appears to be exposed to the hydrophobic interior of the lipid membrane, which would be highly unfavorable for a negatively charged nucleophile, thus hindering auto-acylation. Here, we use biochemical and computational methods to reconcile these seemingly contradicting facts. First, we experimentally demonstrate that human DHHC20 is active when reconstituted in POPC nanodiscs. Microsecond-long all-atom molecular dynamics simulations are then calculated for hDHHC20 and for different acyl-CoA forms, also in POPC. Strikingly, we observe that hDHHC20 induces a drastic deformation in the membrane, particularly on the cytoplasmic side where auto-acylation occurs. As a result, the catalytic Cys becomes hydrated and optimally positioned to encounter the cleavage site in acyl-CoA. In summary, we hypothesize that DHHC enzymes locally reshape the membrane to foster a morphology that is specifically adapted for acyl-CoA recognition and auto-acylation.\n\nSignificance StatementPalmitoylation, the most common form of S-acylation and the only reversible type of protein lipidation, is ubiquitous in eukaryotic cells. In humans, for example, it has been estimated that as much as [~]10% of the proteome becomes palmitoylated, i.e. thousands of proteins. Accordingly, protein palmitoylation touches every important aspect of human physiology, both in health and disease. Despite its biological and biomedical importance, little is known about the molecular mechanism of the enzymes that catalyze this post-translational modification, known as DHHC acyltransferases. Here, we leverage the recently-determined atomic-resolution structure of human DHHC20 to gain novel insights into the mechanism of this class of enzymes, using both experimental and computational approaches.

biophysics↗

bioRxiv2018

Confidence Analysis of DEER Data and its Structural Interpretation with Ensemble-Biased Metadynamics

Given its ability to measure multicomponent distance distributions between electron-spin probes, Double Electron-Electron Resonance spectroscopy (DEER) has become a leading technique to assess the structural dynamics of biomolecules. However, methodologies to evaluate the statistical error of these distributions are not standard, often hampering a rigorous interpretation of the experimental results. Distance distributions are often determined from the experimental DEER data through a mathematical method known as Tikhonov regularization, but this approach makes rigorous error estimates difficult. Here, we build upon an alternative model-based approach in which the distance probability distribution is represented as a sum of Gaussian components and use propagation of errors to calculate an associated confidence band. Our approach considers all sources of uncertainty, including the experimental noise, the uncertainty in the fitted background signal, and the limited time-span of the data collection. The resulting confidence band reveals the most and least reliable features of the probability distribution, thereby informing the structural interpretation of DEER experiments. To facilitate this interpretation, we also generalize the molecular-simulation method known as Ensemble-Biased Metadynamics. This method, originally designed to generate maximum-entropy structural ensembles consistent with one or more probability distributions, now also accounts for the uncertainty in those target distributions, exactly as dictated by their confidence bands. After careful benchmarks, we demonstrate the proposed techniques using DEER results from spin-labeled T4 lysozyme.

biophysics↗