Human plasminogen activator inhibitor-1 (PAI-1) is the fast-acting inhibitor of tissue plasminogen activator and urokinase and is a member of the serpin family of protease inhibitors. Serpins normally form complexes with their target proteases that dissociate very slowly as cleaved species and then fold into a highly stable inactive state in which the residues that flank the scissile bond (P1 and P1';) are separated by about 70 A. PAI-1 also spontaneously folds into a stable inactive state without cleavage; this state is termed 'latent' because inhibitory activity can be restored through denaturation and renaturation. Here we report the structure of intact latent PAI-1 determined by single-crystal X-ray diffraction to 2.6 A resolution. The three-dimensional structure reveals that residues on the N-terminal side of the primary recognition site are inserted as a central strand of the largest beta sheet, in positions similar to the corresponding residues in the cleaved form of the serpin alpha 1-proteinase inhibitor (alpha 1-PI). Residues C-terminal to the recognition site occupy positions on the surface of the molecule distinct from those of the corresponding residues in cleaved serpins or in the intact inactive serpin homologue, ovalbumin, and its cleavage product, plakalbumin. The structure of latent PAI-1 is similar to one formed after cleavage in other serpins, and the stability of both latent PAI-1 and cleaved serpins may be derived from the same structural features.

Multidrug transporters belonging to the multidrug and toxic compound extrusion (MATE) family expel dissimilar lipophilic and cationic drugs across cell membranes by dissipating a preexisting Na + or H + gradient. Despite its clinical relevance, the transport mechanism of MATE proteins remains poorly understood, largely owing to a lack of structural information on the substrate-bound transporter. Here we report crystal structures of a Na + -coupled MATE transporter NorM from Neisseria gonorrheae in complexes with three distinct translocation substrates (ethidium, rhodamine 6G, and tetraphenylphosphonium), as well as Cs + (a Na + congener), all captured in extracellular-facing and drug-bound states. The structures revealed a multidrug-binding cavity festooned with four negatively charged amino acids and surprisingly limited hydrophobic moieties, in stark contrast to the general belief that aromatic amino acids play a prominent role in multidrug recognition. Furthermore, we discovered an uncommon cation-π interaction in the Na + -binding site located outside the drug-binding cavity and validated the biological relevance of both the substrate-and cationbinding sites by conducting drug resistance and transport assays. Additionally, we uncovered potential rearrangement of at least two transmembrane helices upon Na + -induced drug export. Based on our structural and functional analyses, we suggest that Na + triggers multidrug extrusion by inducing protein conformational changes rather than by directly competing for the substrate-binding amino acids. This scenario is distinct from the canonical antiport mechanism, in which both substrate and counterion compete for a shared binding site in the transporter. Collectively, our findings provide an important step toward a detailed and mechanistic understanding of multidrug transport.cation coordination | substrate recognition | membrane protein | multidrug resistance | monobody

Mitochondrial ATP synthase comprises a membrane embedded Fo motor that rotates to drive ATP synthesis in the F1 subunit. We used single-particle cryo-EM to obtain structures of the full complex in a lipid bilayer in the absence or presence of the inhibitor oligomycin, at 3.6 Å and 3.8 Å resolution, respectively. To limit conformational heterogeneity, we locked the rotor in a single conformation by fusing the F6 subunit of the stator with the δ-subunit of the rotor. Assembly of the enzyme with the F6-δ fusion caused a twisting of the rotor and a 9° rotation of the Fo c10-ring in the direction of ATP synthesis, relative to the structure of isolated Fo. Our cryo-EM structures show how F1 and Fo are coupled, give insight into the proton translocation pathway and show how oligomycin blocks ATP synthesis.

We report the high-resolution (1.9 Å) crystal structure of oligomycin bound to the subunit c 10 ring of the yeast mitochondrial ATP synthase. Oligomycin binds to the surface of the c 10 ring making contact with two neighboring molecules at a position that explains the inhibitory effect on ATP synthesis. The carboxyl side chain of Glu59, which is essential for proton translocation, forms an H-bond with oligomycin via a bridging water molecule but is otherwise shielded from the aqueous environment. The remaining contacts between oligomycin and subunit c are primarily hydrophobic. The amino acid residues that form the oligomycin-binding site are 100% conserved between human and yeast but are widely different from those in bacterial homologs, thus explaining the differential sensitivity to oligomycin. Prior genetics studies suggest that the oligomycin-binding site overlaps with the binding site of other antibiotics, including those effective against Mycobacterium tuberculosis, and thereby frames a common "drug-binding site." We anticipate that this drug-binding site will serve as an effective target for new antibiotics developed by rational design. (1). Studies in the 1960s from the laboratory of Efraim Racker demonstrated that the mitochondrial ATP synthase can be separated into two parts, coupling factor 1, or F 1 , which contains the catalytic site for ATP synthesis, and coupling factor o, or F o , which is able to confer sensitivity to oligomycin (2-4). Despite more than 50 y of studies on mitochondrial F 1 F o ATP synthase, the binding site of oligomycin on F o has been elusive. Here we report the oligomycin-binding site on subunit-c of the F o portion of the ATP synthase.Subunit-c of the ATP synthase is an integral membrane protein consisting of two helices, 1 and 2, which span the inner mitochondrial membrane (Fig. 1). Subunit-c assembles as a homomeric ring consisting of 10 subunits in the yeast ATP synthase and eight subunits in the bovine ATP synthase (5, 6). The c-ring forms an essential component of the proton turbine of the ATP synthase, which spins coupled to the movement of protons down a potential gradient. The essential carboxylate of Glu59 in helix 2 of the yeast subunit-c is postulated to participate directly in the movement of protons from the cytosol to the mitochondrial matrix during ATP synthesis. The side-chain carboxyl of Glu59 is nearly in the middle of helix 2, positioning it in the lipid bilayer in the protonated, "closed" conformation (7). Subunit-a is postulated to form two aqueous half-channels that allow protons to gain access to the carboxylate of Glu59 in the "open" conformation, allowing protonation and deprotonation reaction (7).Based on the results presented here, we propose that in the intact ATP synthase, oligomycin binds at the c-ring positioned at the proton channel and blocks proton translocation by blocking access to the essential carboxyl. Furthermore, we propose that the binding site framed out by oligomycin is a common drug-binding site for inhibitors that bind to the ba...

Multidrug and toxic compound extrusion (MATE) transporters contribute to multidrug resistance by coupling the efflux of drugs to the influx of Na+ or H+. Known structures of Na+-coupled, extracellular-facing MATE transporters from the NorM subfamily revealed twelve membrane-spanning segments related by a quasi-twofold rotational symmetry and a multidrug-binding cavity situated near the membrane surface. Here we report the crystal structure of an H+-coupled MATE transporter from Bacillus halodurans and the DinF subfamily at 3.2 Å-resolution, unveiling a surprisingly asymmetric arrangement of twelve transmembrane helices. We also identified a membrane-embedded substrate-binding chamber by combining crystallographic and biochemical analyses. Our studies further suggested a direct competition between H+ and substrate during DinF-mediated transport, and how a MATE transporter alternates between its extracellular- and intracellular-facing conformations to propel multidrug extrusion. Collectively, our results demonstrated hitherto unrecognized mechanistic diversity among MATE transporters.