RESUMEN
BACKGROUND: Interaction of ß2-glycoprotein I (ß2GPI) with anionic membranes is crucial in antiphospholipid syndrome (APS), implicating the role of its membrane-binding domain, domain V (DV). The mechanism of DV binding to anionic lipids is not fully understood. OBJECTIVES: This study aimed to elucidate the molecular details of ß2GPI DV binding to anionic membranes. METHODS: We utilized molecular dynamics simulations to investigate the structural basis of anionic lipid recognition by DV. To corroborate the membrane-binding mode identified in the highly mobile membrane mimetic simulations, we conducted additional simulations using a full membrane model. RESULTS: The study identified critical regions in DV, namely the lysine-rich loop and the hydrophobic loop, which are essential for membrane association via electrostatic and hydrophobic interactions, respectively. A novel lysine pair contributing to membrane binding was also discovered, providing new insights into ß2GPI's membrane interaction. Simulations revealed 2 distinct binding modes of DV to the membrane, with mode 1 characterized by the insertion of the hydrophobic loop into the lipid bilayer, suggesting a dominant mechanism for membrane association. This interaction is pivotal for the pathogenesis of APS, as it facilitates the recognition of ß2GPI by antiphospholipid antibodies. CONCLUSION: The study advances our understanding of the molecular interactions between ß2GPI's DV and anionic membranes, which are crucial for APS pathogenesis. It highlights the importance of specific regions in DV for membrane binding and reveals a predominant binding mode. These findings have significant implications for APS diagnostics and therapeutics, offering a deeper insight into the molecular basis of the syndrome.
RESUMEN
Background: Interaction of beta-2-glycoprotein I ( ß 2 GPI) with anionic membranes is crucial in antiphospholipid syndrome (APS), implicating the role of it's membrane bind-ing domain, Domain V (DV). The mechanism of DV binding to anionic lipids is not fully understood. Objectives: This study aims to elucidate the mechanism by which DV of ß 2 GPI binds to anionic membranes. Methods: We utilized molecular dynamics (MD) simulations to investigate the struc-tural basis of anionic lipid recognition by DV. To corroborate the membrane-binding mode identified in the HMMM simulations, we conducted additional simulations using a full mem-brane model. Results: The study identified critical regions in DV, namely the lysine-rich loop and the hydrophobic loop, essential for membrane association via electrostatic and hydrophobic interactions, respectively. A novel lysine pair contributing to membrane binding was also discovered, providing new insights into ß 2 GPI's membrane interaction. Simulations revealed two distinct binding modes of DV to the membrane, with mode 1 characterized by the insertion of the hydrophobic loop into the lipid bilayer, suggesting a dominant mechanism for membrane association. This interaction is pivotal for the pathogenesis of APS, as it facilitates the recognition of ß 2 GPI by antiphospholipid antibodies. Conclusion: The study advances our understanding of the molecular interactions be-tween ß 2 GPI's DV and anionic membranes, crucial for APS pathogenesis. It highlights the importance of specific regions in DV for membrane binding and reveals a predominant bind-ing mode. These findings have significant implications for APS diagnostics and therapeutics, offering a deeper insight into the molecular basis of the syndrome.
RESUMEN
The soluble flavoprotein oleate hydratase (OhyA) hydrates the 9-cis double bond of unsaturated fatty acids. OhyA substrates are embedded in membrane bilayers; OhyA must remove the fatty acid from the bilayer and enclose it in the active site. Here, we show that the positively charged helix-turn-helix motif in the carboxy terminus (CTD) is responsible for interacting with the negatively charged phosphatidylglycerol (PG) bilayer. Super-resolution microscopy of Staphylococcus aureus cells expressing green fluorescent protein fused to OhyA or the CTD sequence shows subcellular localization along the cellular boundary, indicating OhyA is membrane-associated and the CTD sequence is sufficient for membrane recruitment. Using cryo-electron microscopy, we solved the OhyA dimer structure and conducted 3D variability analysis of the reconstructions to assess CTD flexibility. Our surface plasmon resonance experiments corroborated that OhyA binds the PG bilayer with nanomolar affinity and we found the CTD sequence has intrinsic PG binding properties. We determined that the nuclear magnetic resonance structure of a peptide containing the CTD sequence resembles the OhyA crystal structure. We observed intermolecular NOE from PG liposome protons next to the phosphate group to the CTD peptide. The addition of paramagnetic MnCl2 indicated the CTD peptide binds the PG surface but does not insert into the bilayer. Molecular dynamics simulations, supported by site-directed mutagenesis experiments, identify key residues in the helix-turn-helix that drive membrane association. The data show that the OhyA CTD binds the phosphate layer of the PG surface to obtain bilayer-embedded unsaturated fatty acids.
Asunto(s)
Ácido Oléico , Péptidos , Staphylococcus aureus , Microscopía por Crioelectrón , Ácidos Grasos Insaturados , Membrana Dobles de Lípidos/metabolismo , Fosfatos , Staphylococcus aureus/enzimología , Staphylococcus aureus/genéticaRESUMEN
Here we used cryo-electron microscopy (cryo-EM), double electron-electron resonance spectroscopy (DEER), and molecular dynamics (MD) simulations, to capture and characterize ATP- and substrate-bound inward-facing (IF) and occluded (OC) conformational states of the heterodimeric ATP binding cassette (ABC) multidrug exporter BmrCD in lipid nanodiscs. Supported by DEER analysis, the structures reveal that ATP-powered isomerization entails changes in the relative symmetry of the BmrC and BmrD subunits that propagates from the transmembrane domain to the nucleotide binding domain. The structures uncover asymmetric substrate and Mg2+ binding which we hypothesize are required for triggering ATP hydrolysis preferentially in one of the nucleotide-binding sites. MD simulations demonstrate that multiple lipid molecules differentially bind the IF versus the OC conformation thus establishing that lipid interactions modulate BmrCD energy landscape. Our findings are framed in a model that highlights the role of asymmetric conformations in the ATP-coupled transport with general implications to the mechanism of ABC transporters.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Nucleótidos , Microscopía por Crioelectrón , Espectroscopía de Resonancia por Spin del Electrón , Lípidos , Adenosina TrifosfatoRESUMEN
To illuminate the structural origin of catalytic asymmetry of heterodimeric ABC transporters and how it shapes the energetics of their conformational cycles, we used cryo-electron microscopy (cryo-EM), double electron-electron resonance spectroscopy (DEER), and molecular dynamics (MD) simulations, to capture and characterize conformational states of the heterodimeric ABC multidrug exporter BmrCD in lipid nanodiscs. In addition to multiple ATP- and substrate-bound inward-facing (IF) conformations, we obtained the structure of an occluded (OC) conformation wherein the unique extracellular domain (ECD) twists to partially open the extracellular gate. In conjunction with DEER analysis of the populations of these conformations, the structures reveal that ATP-powered isomerization entails changes in the relative symmetry of the BmrC and BmrD subunits that propagates from the transmembrane domain (TMD) to the nucleotide binding domain (NBD). The structures uncover asymmetric substrate and Mg 2+ binding which we hypothesize are required for triggering ATP hydrolysis preferentially in one of the nucleotide-binding sites. MD simulations demonstrated that multiple lipid molecules, identified from the cryo-EM density maps, differentially bind the IF versus the OC conformation thus modulating their relative stability. In addition to establishing how lipid interactions with BmrCD modulate the energy landscape, our findings are framed in a distinct transport model that highlights the role of asymmetric conformations in the ATP-coupled cycle with implications to the mechanism of ABC transporters in general.