native-cell-membrane-nanoparticles-system-for-membrane-protein

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Virginia Commonwealth University

Membrane proteins function in native cell membranes, but extraction into isolated particles is needed for many biochemical and structural analyses. Commonly used detergent-extraction methods destroy naturally associated lipid bilayers. Here, we devised a detergent-free method for preparing cell-membrane nanoparticles to study the multidrug exporter AcrB, by cryo-EM at 3.2-Å resolution. We discovered a remarkably well-organized lipid-bilayer structure associated with transmembrane domains of the AcrB trimer. This bilayer patch comprises 24 lipid molecules; inner leaflet chains are packed in a hexagonal array, whereas the outer leaflet has highly irregular but ordered packing. Protein side chains interact with both leaflets and participate in the hexagonal pattern. We suggest that the lipid bilayer supports and harmonizes peristaltic motions through AcrB trimers. In AcrB D407A, a putative proton-relay mutant, lipid bilayer buttresses protein interactions lost in crystal structures after detergent-solubilization. Our detergent-free system preserves lipid-protein interactions for visualization and should be broadly applicable.

Structure and activity of lipid bilayer within a membrane-protein transporter.

Mammalian pyruvate dehydrogenase (PDH) activity is tightly regulated by phosphorylation and dephosphorylation, which is catalyzed by PDH kinase isomers and PDH phosphatase isomers, respectively. PDH phosphatase isomer 1 (PDP1) is a heterodimer consisting of a catalytic subunit (PDP1c) and a regulatory subunit (PDP1r). Here, the crystal structure of bovine PDP1c determined at 2.1 Å resolution is reported. The crystals belonged to space group P321, with unit-cell parameters a = b = 75.3, c = 173.2 Å. The structure was solved by molecular-replacement methods and refined to a final R factor of 21.9% (R = 24.7%). The final model consists of 402 of a possible 467 amino-acid residues of the PDP1c monomer, two Mn ions in the active site, an additional Mn ion coordinated by His410 and His414, two MnSO ion pairs at special positions near the crystallographic twofold symmetry axis and 226 water molecules. Several new features of the PDP1c structure are revealed. The requirements are described and plausible bases are deduced for the interaction of PDP1c with PDP1r and other components of the pyruvate dehydrogenase complex.

Weihua Qiu

Department of Medicinal Chemistry,
School of Pharmacy,
Institute for Structural Biology,
Drug Discovery and Development,
Department of Medicinal Chemistry, School of Pharmacy,
Institute for Structural Biology, Drug Discovery and Development, School of Pharmacy

Virginia Commonwealth University

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Weihua Qiu

Department of Medicinal Chemistry,School of Pharmacy,Institute for Structural Biology,Drug Discovery and Development,Department of Medicinal Chemistry, School of Pharmacy,Institute for Structural Biology, Drug Discovery and Development, School of Pharmacy

Virginia Commonwealth University

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Department of Medicinal Chemistry,
School of Pharmacy,
Institute for Structural Biology,
Drug Discovery and Development,
Department of Medicinal Chemistry, School of Pharmacy,
Institute for Structural Biology, Drug Discovery and Development, School of Pharmacy