Summary The cytochrome bc1 complex (bc1) is the mid-segment of the cellular respiratory chain of mitochondria and many aerobic prokaryotic organisms; it is also part of the photosynthetic apparatus of non-oxygenic purple bacteria. The bc1 complex catalyzes the reaction of transferring electrons from the low potential substrate ubiquinol to high potential cytochrome c. Concomitantly, bc1 translocates protons across the membrane, contributing to the proton-motive force essential for a variety of cellular activities such as ATP synthesis. Structural investigations of bc1 have been exceedingly successful, yielding atomic resolution structures of bc1 from various organisms and trapped in different reaction intermediates. These structures have confirmed and unified results of decades of experiments and have contributed to our understanding of the mechanism of bc1 functions as well as its inactivation by respiratory inhibitors.

Edited by Norma AllewellP-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancer; it plays important roles in determining the pharmacokinetics of many drugs. Understanding the structural basis of P-gp, substrate polyspecificity has been hampered by its intrinsic flexibility, which is facilitated by a 75-residue linker that connects the two halves of P-gp. Here we constructed a mutant murine P-gp with a shortened linker to facilitate structural determination. Despite dramatic reduction in rhodamine 123 and calcein-AM transport, the linker-shortened mutant P-gp possesses basal ATPase activity and binds ATP only in its N-terminal nucleotide-binding domain. Nine independently determined structures of wild type, the linker mutant, and a methylated P-gp at up to 3.3 Å resolution display significant movements of individual transmembrane domain helices, which correlated with the opening and closing motion of the two halves of P-gp. The open-andclose motion alters the surface topology of P-gp within the drugbinding pocket, providing a mechanistic explanation for the polyspecificity of P-gp in substrate interactions.The association of multidrug resistance (MDR) 3 in cancer treatment with expression of human ABC transporters on the cell surface has raised the possibility that overcoming MDR might be achieved by inhibiting these transporters (1). One transporter in particular, the human P-glycoprotein (P-gp), has been well characterized; it is able to confer MDR by transporting numerous structurally unrelated drugs at the expense of hydrolyzing ATP (2, 3). In addition, P-gp plays important roles in drug distribution in normal physiology and is an essential component of many physiological barriers (4, 5).Long-standing efforts have been devoted to understanding the mechanism of P-gp function by various experimental approaches. Among them, structural studies of P-gp from various organisms have been reported (6 -8). In particular, a number of structures for the mouse P-gp (mP-gp) were obtained (8 -10). However, the diffraction limits of "near-native," inhibitor-bound, and nanobody-associated mP-gp crystals were relatively low, mostly near 4 Å resolution. The methylated protein gave higher resolution structures (11), but the impact of reductive methylation on the structure and function of P-gp was not addressed. The low resolution diffraction of P-gp crystals has been attributed in part to the intrinsic flexibility of the molecule (12-14).Mouse P-gp is a 1276-residue polypeptide and bears 87% sequence identity to human P-gp; it consists of two homologous halves connected by a flexible linker of ϳ75 residues. Each half is made of a transmembrane domain (TMD) implicated in drug recognition and transport and a nucleotide-binding domain (NBD). Each NBD is able to bind and hydrolyze ATP (15, 16), which is, however, dependent on the other NBD being functional (17,18). The two NBDs of P-gp are highly homologous, each featuring a consensus nucleotide-binding site with full-fledged Walker...

The cytochrome bc 1 complex (bc 1 ) is a major contributor to the proton motive force across the membrane by coupling electron transfer to proton translocation. The crystal structures of wild type and mutant bc 1 complexes from the photosynthetic purple bacterium Rhodobacter sphaeroides (Rsbc 1 ), stabilized with the quinol oxidation (Q P ) site inhibitor stigmatellin alone or in combination with the quinone reduction (Q N ) site inhibitor antimycin, were determined. The high quality electron density permitted assignments of a new metal-binding site to the cytochrome c 1 subunit and a number of lipid and detergent molecules. Structural differences between Rsbc 1 and its mitochondrial counterparts are mostly extra membranous and provide a basis for understanding the function of the predominantly longer sequences in the bacterial subunits. Functional implications for the bc 1 complex are derived from analyses of 10 independent molecules in various crystal forms and from comparisons with mitochondrial complexes.A central component of the cellular respiratory chain is the cytochrome bc 1 complex (cyt bc 1 or bc 1 ) 2 that catalyzes the electron transfer (ET) from quinol to cytochrome c (cyt c) and simultaneously pumps protons across the membrane, contributing to the electrochemical potential that drives ATP synthesis and many other cellular activities (1). In chloroplasts and cyanobacteria a related membrane protein complex, the cytochrome b 6 f (cyt b 6 f), bridges photosystem I and II, enabling oxygenic photosynthesis and conversion of light energy into a proton gradient for ATP generation (2). For non-oxygenic photosynthetic bacteria, such as R. sphaeroides (Rs), which can grow both aerobically and photosynthetically under anaerobic condition, the bc 1 complex is involved in both growth modes; however it is essential only under anaerobic conditions (3).The critical importance of bc 1 has made it a target for numerous antibiotics, fungicides, and anti-parasitic agents. As a result, resistance to these agents has been documented in a wide variety of organisms (4 -8). Disorders that are related to defects in bc 1 complex are manifest clinically as mitochondrial myopathy (9), exercise intolerance (10), and Leber's optical neuropathy (11). Mounting evidence suggests a correlation between aging and the production of reactive oxygen species from defective bc 1 complexes (12, 13). The elucidation of the molecular mechanisms underlying these phenomena requires a combination of experimental approaches and in particular, structural investigations that can provide a molecular framework for further experiments.Significant advances in elucidating architectural features of this complex have been made by crystal structure determinations of mitochondrial bc 1 (14 -17) and b 6 f from a bacterium (18) and an alga (19). In particular, crystal structures of mitochondrial bc 1 in complex with various bc 1 inhibitors provide important mechanistic insights (20 -27), leading to a significant increase in the number of experimental studies...

Emerging variants of concern for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transmit more efficiently and partially evade protective immune responses, thus necessitating continued refinement of antibody therapies and immunogen design. Here we elucidate the structural basis and mode of action for two potent SARS-CoV-2 Spike (S) neutralizing monoclonal antibodies CV3-1 and CV3-25 that remain effective against emerging variants of concern in vitro and in vivo. CV3-1 binds to the (485-GFN-487) loop within the receptor-binding domain (RBD) in the “RBD-up” position and triggers potent shedding of the S1 subunit. In contrast, CV3-25 inhibits membrane fusion by binding to an epitope in the stem helix region of the S2 subunit that is highly conserved among β-coronaviruses. Thus, vaccine immunogen designs that incorporate the conserved regions in RBD and stem helix region are candidates to elicit pan-coronavirus protective immune responses.