Biology subjects

De Vecchis, D.

Publications and source records attributed to De Vecchis, D..

7 recordsLinked to original sources

bioRxiv2025

Capturing transient states of heterodimeric ABC transporter TM287/288 by Time-Resolved Small-Angle X-ray Scattering

Structures of the heterodimeric ABC transporter TM287/288 have been previously determined in several states indicating large conformational changes during its reaction cycle. However, for a complete description of the cycle, transient states (such as an occluded state) are still missing, as they are difficult to capture with static structural biology techniques. Here, we investigate the kinetics and accompanying structural changes in the course of the reaction cycle of full-length TM287/288 using time-resolved small-angle X-ray scattering, initiated by stopped-flow mixing. The use of active site mutants and state-specific sybodies/nanobodies enabled us to dissect the temporal events involved in the ATP-driven conformational cycle of TM287/288 and reveal a transient occluded state of this ABC transporter. Statement of SignificanceATP-binding cassette transporters are essential molecular machines that use the power of ATP binding and hydrolysis to move substances across cellular membranes. Here, we use time-resolved small-angle X-ray scattering combined with conformation-specific nanobodies to directly capture and assign different states to the heterodimeric ABC transporter TM287/288 in solution. Our findings advance our understanding of ABC transporter and demonstrate a broadly applicable method for dissecting the different molecular states in membrane proteins on a second-to-minute time scale. TOC figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/679171v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@b83fbaorg.highwire.dtl.DTLVardef@5fec81org.highwire.dtl.DTLVardef@1a022fdorg.highwire.dtl.DTLVardef@592c92_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Coupling the role of lipids to the conformational dynamics of the ABC transporter P-gp

The ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp) is a multidrug efflux pump that is overexpressed in a variety of cancers and associated with the drug resistance phenomenon. P-gp structures were previously determined in detergent and in nanodiscs, in which different transmembrane helix conformations were found, "straight" and "kinked", respectively, indicating a possible role of the lipid environment on the P-gp structural ensemble. Here, we investigate the dynamic conformational ensembles and protein-lipid interactions of the two human P-gp inward-open conformers (straight and kinked) employing all-atom molecular dynamics simulations in asymmetric multicomponent lipid bilayers that mimic the highly specialized hepatocyte membrane in which P-gp is expressed. The two conformers are found to differ in terms of the accessibility of the substrate cavity. The MD simulations show how cholesterol and different lipid species wedge, snorkel, and partially enter within the cavity of the straight P-gp conformer solved in detergent. However, the access to the cavity of kinked P-gp conformer solved in nanodiscs is restricted. Furthermore, the volume and dynamic fluctuations of the substrate cavity largely differ between the two P-gp structures, and are modulated by the presence (or absence) of cholesterol in the membrane and/or of ATP. From the mechanistic perspective, our findings indicate that the straight conformer likely precedes the kinked conformer in the functional working cycle of P-pg, with the latter conformation representing a post substrate-bound state. The inaccessibility of the main transmembrane cavity in the kinked conformer might be crucial in preventing substrate disengagement and transport withdrawal. Remarkably, in our unbiased MD simulations, one transmembrane portal helix (TM10) of the straight conformer underwent a spontaneous conformational transition to a kinked conformation, underlining the relevance of both conformations in a native phospholipid environment and revealing structural descriptors defining the transition between two P-gp conformers.

biophysics↗

bioRxiv2023

Anionic phospholipids stimulate the proton pumping activity of the plant plasma membrane P-type H+-ATPase

The activity of membrane proteins depends strongly on the surrounding lipid environment. Here, we characterize the lipid stimulation of the plant plasma membrane H+-ATPase AHA2 upon purification and reconstitution into liposomes of defined lipid compositions. We show that the proton pumping activity of AHA2 is stimulated by anionic phospholipids, especially by phosphatidylserine. Molecular dynamics simulations revealed several preferential contact sites for anionic phospholipids in the transmembrane domain of AHA2. These contact sites are partly conserved across functionally different P-type ATPases from several organisms, suggesting a general regulation mechanism by the membrane lipid environment. Our findings highlight the fact that anionic lipids play an important role in the control of H+-ATPase activity.

biochemistry↗

bioRxiv2023

Structural basis for triacylglyceride extraction frommycobacterial inner membrane by MFS transporter Rv1410

Mycobacterium tuberculosis is protected from antibiotic therapy by a multi-layered hydrophobic cell envelope. Major facilitator superfamily (MFS) transporter Rv1410 and the periplasmic lipoprotein LprG are involved in transport of triacylglycerides (TAGs) that seal the mycomembrane. Here, we report a 2.7 [A] structure of a mycobacterial Rv1410 homologue, which adopts an outward-facing conformation and exhibits unusual transmembrane helix 11 and 12 extensions that protrude [~]20 [A] into the periplasm. A small, very hydrophobic cavity suitable for lipid transport is constricted by a unique and functionally important ion-lock likely involved in proton coupling. Combining mutational analyses and MD simulations, we propose that TAGs are extracted from the core of the inner membrane into the central cavity via lateral clefts present in the inward-facing conformation. The periplasmic helix extensions are crucial for lifting TAGs away from the membrane plane and channeling them into the lipid binding pocket of LprG.

molecular biology↗

bioRxiv2019

Molecular principles of Piezo1 activation by increased membrane tension

SUMMARY PARAGRAPHPiezo1 is a mechanosensitive channel involved in many cellular functions and responsible for sensing shear-stress and pressure forces in cells1-3. Piezo1 plays a critical role in the circulatory system and tissue development. Mutations on Piezo1 are linked to human diseases such as lymphedema2,4 or hematological disorders such as hemolytic anaemia5 and resistance to malaria6. Hypotheses for Piezo1 gating include the "force-from-lipids" principle7,8 that suggests that Piezo1 senses mechanical forces through the bilayer1,9 and a direct involvement of the cytoskeleton as well as the extracellular matrix in Piezo1 activation10,11. However, the molecular and structural changes underpinning the Piezo1 gating mechanism and how the channel senses forces in the membrane remain unknown. Here we reveal the activation mechanism of Piezo1 and the structural rearrangements that occur when Piezo1 moves from a closed to an open state when mechanical tension is applied to the cell membrane. Our results show that Piezo1s curved shape is stable in a native-like model membrane without tension creating a membrane indentation with a trilobed topology. Upon stretching Piezo1 adapts to the stretched bilayer by flattening and expansion of its blade region. In our simulations Piezo1 expands up to a planar circular area of [~]680 nm2 comparable with previous structural data and hypotheses12-14. Piezo1 flattening and expansion results in changes in the beam helix tilt angle. These movements result in the tilting and lateral movement of the pore lining TM37 and TM38 helices. This leads to the opening of the channel and to the movement of lipids that occupy Piezo1 pore region outside of this region, revealing for the first time the structural changes that happen during Piezo1 mechanical activation. The changes in the blade region are transmitted to helices TM37 and 38 via hydrophobic interactions and by interactions of neighbouring subunits via the elbow region. The flat structure of Piezo1 identified in this study exposes the C-terminal extracellular domain (CED) that in the closed state is hidden in the membrane and presumably from shear stress. Our results provide new structural data for different states of Piezo1 and suggest the molecular principles by which mechanical force opens the Piezo1 channel, thus coupling force to physiological effect via ion permeation.

biophysics↗

bioRxiv2019

Sphingomyelinase disables Piezo1 channel inactivation to enable sustained response to mechanical force

Piezo1 channels are determinants of vascular responses to fluid flow. They importantly provide sustained response to flow, but this property contrasts with the rapid inactivation that has become a hallmark of the channels in heterologous overexpression studies. Here we reveal a mechanism by which blood vessels disable inactivation to enable sustained physiological response. Creation of a molecular model of Piezo1 channel in defined lipid membranes suggested potential modulation by sphingomyelin and its product ceramide. Biological relevance was indicated by the observation that exogenous sphingomyelinase enhanced Piezo1-mediated Ca2+ entry in cultured endothelial cells. We therefore hypothesised that endogenous sphingomyelinase suppresses channel inactivation. Remarkably, in endothelium freshly-isolated from murine artery, neutral sphingomyelinase inhibitors or genetic disruption of sphingomyelin phosphodiesterase 3 (SMPD3) caused flow- and pressure-activated Piezo1 channels to become inactivating. SMPD3 retained its ability to disable inactivation in cell-free membrane patches, providing evidence for a membrane localised effect. The data suggest that inherent inactivation of Piezo1 channels is disabled by enzymatic control of lipid environment to enable physiological response to mechanical force.

physiology↗

bioRxiv2019

Computational reconstruction of the complete Piezo1 structure reveals a unique footprint and specific lipid interactions

Piezo1 is a critical mechanical sensor in many cells. It is activated by mechanical force thus allowing cells to sense the physical environment and respond to stress. Structural data have suggested that Piezo1 has a curved shape. Here, we use computational approaches to model, for the first time, the 3D structure of the full-length Piezo1 in an asymmetric membrane. A number of novel insights emerge: (i) Piezo1 creates a dome in the membrane with a trilobed topology that extends beyond the radius of the protein, (ii) Piezo1 changes the lipid environment in its vicinity via specific interactions with cholesterol and PIP2 molecules, (iii) changes in cholesterol concentration that change the membrane stiffness result in changes in the depth of the dome created by Piezo1, and iv) modelling of the N-terminal region that is missing from current structures modifies Piezo1 membrane footprint, suggesting the importance of this region in Piezo1 function.

biophysics↗