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The ATP-binding cassette subfamily B member 1 (ABCB1) multidrug transporter P-glycoprotein plays a central role in clearance of xenobiotics in humans and is implicated in cancer resistance to chemotherapy. We used double electron electron resonance spectroscopy to uncover the basis of stimulation of P-glycoprotein adenosine 5′-triphosphate (ATP) hydrolysis by multiple substrates and illuminate how substrates and inhibitors differentially affect its transport function. Our results reveal that substrate-induced acceleration of ATP hydrolysis correlates with stabilization of a high-energy, post-ATP hydrolysis state characterized by structurally asymmetric nucleotide-binding sites. By contrast, this state is destabilized in the substrate-free cycle and by high-affinity inhibitors in favor of structurally symmetric nucleotide binding sites. Together with previous data, our findings lead to a general model of substrate and inhibitor coupling to P-glycoprotein.

Membrane-sculpting proteins shape the morphology of cell membranes and facilitate remodeling in response to physiological and environmental cues. Complexes of the monotopic membrane protein caveolin function as essential curvature-generating components of caveolae, flask-shaped invaginations that sense and respond to plasma membrane tension. However, the structural basis for caveolin’s membrane remodeling activity is currently unknown. Here, we show that, using cryo–electron microscopy, the human caveolin-1 complex is composed of 11 protomers organized into a tightly packed disc with a flat membrane-embedded surface. The structural insights suggest a previously unrecognized mechanism for how membrane-sculpting proteins interact with membranes and reveal how key regions of caveolin-1, including its scaffolding, oligomerization, and intramembrane domains, contribute to its function.

Rationale De-differentiation of vascular smooth muscle cells (VSMC) leading to a proliferative cell phenotype significantly contributes to the development of atherosclerosis. Mitogen activated protein kinase (MAPK) phosphorylation of proteins including connexin 43 (Cx43) has been associated with VSMC proliferation in atherosclerosis. Objective To investigate whether MAPK phosphorylation of Cx43 is directly involved in VSMC proliferation. Methods and Results We show in vivo that MAPK phosphorylated Cx43 forms complexes with the cell cycle control proteins cyclin E and CDK2 in carotids of apolipoprotein-E receptor null (ApoE−/−) mice and in C57Bl/6 mice treated with platelet-derived growth factor–BB (PDGF). We tested the involvement of Cx43 MAPK phosphorylation in vitro using constructs for full length Cx43 (Cx43) or the Cx43 C-terminus (Cx43CT) and produced null phosphorylation Ser>Ala (Cx43MK4A/ Cx43CTMK4A) and phospho-mimetic Ser>Asp (Cx43MK4D/Cx43CTMK4D) mutations. Co-immunoprecipitation studies in primary VSMC isolated from Cx43 wild type (Cx43+/+) and Cx43 null (Cx43−/−) mice and analytical size exclusion studies of purified proteins identify that interactions between cyclin E and Cx43 requires Cx43 MAPK phosphorylation. We further demonstrate that Cx43 MAPK phosphorylation is required for PDGF mediated VSMC proliferation. Finally using a novel knock-in mouse containing Cx43-MK4A mutation we show in vivo that interactions between Cx43 and cyclin E are lost and VSMC proliferation does not occur following treatment of carotids with PDGF and that neointima formation is significantly reduced in carotids following injury. Conclusions We identify MAPK phosphorylated Cx43 as a novel interacting partner of cyclin E in VSMC and show that this interaction is critical for VSMC proliferation. This novel interaction may be important in the development of atherosclerotic lesions.