Molecular Probing of the Microscopic Pressure at Contact Interfaces.

Obtaining insights into friction at the nanoscopic level and being able to translate these into macroscopic friction behavior in real-world systems is of paramount importance in many contexts, ranging from transportation to high-precision technology and seismology. Since friction is controlled by the local pressure at the contact it is important to be able to detect both the real contact area and the nanoscopic local pressure distribution simultaneously. In this paper, we present a method that uses planarizable molecular probes in combination with fluorescence microscopy to achieve this goal. These probes, inherently twisted in their ground states, undergo planarization under the influence of pressure, leading to bathochromic and hyperchromic shifts of their UV-vis absorption band. This allows us to map the local pressure in mechanical contact from fluorescence by exciting the emission in the long-wavelength region of the absorption band. We demonstrate a linear relationship between fluorescence intensity and (simulated) pressure at the submicron scale. This relationship enables us to experimentally depict the pressure distribution in multiasperity contacts. The method presented here offers a new way of bridging friction studies of the nanoscale model systems and practical situations for which surface roughness plays a crucial role.

Optimal reimplantation timing in two-stage exchange for periprosthetic joint infection: an observative cohort study in Asian population.

BACKGROUND: The optimal timing for reimplantation for periprosthetic joint infection (PJI) has not been established and varies from a few weeks to several months. The aim of this study was to assess the commendable time between implant removal and reimplantation in patients who underwent two-stage exchange arthroplasty for PJI. METHODS: We retrospectively reviewed 361 patients who were treated with two-stage exchange arthroplasty for hip and knee chronic PJI at our institution between January 2000 and December 2018. Patient characteristics, comorbidities, surgical variables, microbiology data, and time to reimplantation were recorded. All patients were followed for a minimum of one year. Treatment failure was defined by Delphi criteria. Logistic regression analyses were used to calculate survival rates and adjusted odds ratios (ORs) of treatment failure. RESULTS: In final analysis, 27 (7.5%) had treatment failure. Factors related to treatment failure including interim spacer exchange (OR, 3.13; confidence interval (CI), 1.04-9.09, p = 0.036), higher ESR level at reimplantation (OR, 1.85; CI, 1.05-3.57; p = 0.04), and time to reimplantation (OR, 1.00; CI, 1.003-1.005, p = 0.04). Performing revision arthroplasty surgery from 16 to 20 weeks had highest successful rate. The reimplantation over 24 weeks had a lower successful rate. However, no statistical significance in comparing each interval group. CONCLUSION: Our study emphasized the importance of timely reimplantation in achieving successful outcomes. Factors such as ESR levels, spacer exchange, and the duration of time to reimplantation influenced the likelihood of treatment failure in two-stage exchange arthroplasty for hip and knee PJI.

Structures of trehalose-6-phosphate synthase, Tps1, from the fungal pathogen Cryptococcus neoformans: a target for novel antifungals.

Invasive fungal diseases are a major threat to human health, resulting in more than 1.5 million deaths worldwide each year. Yet the arsenal of antifungal therapeutics remains limited and is in dire need of novel drugs that target additional fungal-specific biosynthetic pathways. One such pathway involves the biosynthesis of trehalose. Trehalose is a non-reducing disaccharide composed of two molecules of glucose that is required for pathogenic fungi, including Candida albicans and Cryptococcus neoformans, to survive in their human hosts. Trehalose biosynthesis is a two-step process in fungal pathogens. Trehalose-6-phosphate synthase (Tps1) converts UDP-glucose and glucose-6-phosphate to trehalose-6-phosphate (T6P). Subsequently, trehalose-6-phosphate phosphatase (Tps2) converts T6P to trehalose. The trehalose biosynthesis pathway has been identified as a top candidate for novel antifungal development based on quality, occurrence, specificity, and assay development. However, there are currently no known antifungal agents that target this pathway. As initial steps to develop Tps1 from Cryptococcus neoformans (CnTps1) as a drug target, we report the structures of full-length apo CnTps1 and CnTps1 in complex with uridine diphosphate (UDP) and glucose-6-phosphate (G6P). Both CnTps1 structures are tetramers and display D2 (222) molecular symmetry. Comparison of these two structures reveals significant movement towards the catalytic pocket by the N-terminus upon ligand binding and identifies key residues required for substrate-binding, which are conserved amongst other Tps1 enzymes, as well as residues that stabilize the tetramer. Intriguingly, an intrinsically disordered domain (IDD), encompassing residues M209 to I300, which is conserved amongst Cryptococcal species and closely related Basidiomycetes, extends from each subunit of the tetramer into the "solvent" but is not visible in the density maps. Although, activity assays revealed that the highly conserved IDD is not required for catalysis in vitro, we hypothesize that the IDD is required for C. neoformans Tps1-dependent thermotolerance and osmotic stress survival. Characterization of the substrate specificity of CnTps1 revealed that UDP-galactose, an epimer of UDP-glucose, is a very poor substrate and inhibitor of the enzyme and highlights the exquisite substrate specificity of Tps1. In toto, these studies expand our knowledge of trehalose biosynthesis in Cryptococcus and highlight the potential of developing antifungal therapeutics that disrupt the synthesis of this disaccharide or the formation of a functional tetramer and the use of cryo-EM in the structural characterization of CnTps1-ligand/drug complexes.

Ion channel chameleons: Switching ion selectivity by alternative splicing.

Voltage-gated sodium and calcium channels are distinct, evolutionarily related ion channels that achieve remarkable ion selectivity despite sharing an overall similar structure. Classical studies have shown that ion selectivity is determined by specific binding of ions to the channel pore, enabled by signature amino acid sequences within the selectivity filter (SF). By studying ancestral channels in the pond snail (Lymnaea stagnalis), Guan et al. showed in a recent JBC article that this well-established mechanism can be tuned by alternative splicing, allowing a single CaV3 gene to encode both a Ca2+-permeable and an Na+-permeable channel depending on the cellular context. These findings shed light on mechanisms that tune ion selectivity in physiology and on the evolutionary basis of ion selectivity.

Mechanism by which T7 bacteriophage protein Gp1.2 inhibits Escherichia coli dGTPase.

Levels of the cellular dNTPs, the direct precursors for DNA synthesis, are important for DNA replication fidelity, cell cycle control, and resistance against viruses. Escherichia coli encodes a dGTPase (2'-deoxyguanosine-5'-triphosphate [dGTP] triphosphohydrolase [dGTPase]; dgt gene, Dgt) that establishes the normal dGTP level required for accurate DNA replication but also plays a role in protecting E. coli against bacteriophage T7 infection by limiting the dGTP required for viral DNA replication. T7 counteracts Dgt using an inhibitor, the gene 1.2 product (Gp1.2). This interaction is a useful model system for studying the ongoing evolutionary virus/host "arms race." We determined the structure of Gp1.2 by NMR spectroscopy and solved high-resolution cryo-electron microscopy structures of the Dgt-Gp1.2 complex also including either dGTP substrate or GTP coinhibitor bound in the active site. These structures reveal the mechanism by which Gp1.2 inhibits Dgt and indicate that Gp1.2 preferentially binds the GTP-bound form of Dgt. Biochemical assays reveal that the two inhibitors use different modes of inhibition and bind to Dgt in combination to yield enhanced inhibition. We thus propose an in vivo inhibition model wherein the Dgt-Gp1.2 complex equilibrates with GTP to fully inactivate Dgt, limiting dGTP hydrolysis and preserving the dGTP pool for viral DNA replication.

A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike.

Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.

High-resolution structures of the SAMHD1 dGTPase homolog from Leeuwenhoekiella blandensis reveal a novel mechanism of allosteric activation by dATP.

Deoxynucleoside triphosphate (dNTP) triphosphohydrolases (dNTPases) are important enzymes that may perform multiple functions in the cell, including regulating the dNTP pools and contributing to innate immunity against viruses. Among the homologs that are best studied are human sterile alpha motif and HD domain-containing protein 1 (SAMHD1), a tetrameric dNTPase, and the hexameric Escherichia coli dGTPase; however, it is unclear whether these are representative of all dNTPases given their wide distribution throughout life. Here, we investigated a hexameric homolog from the marine bacterium Leeuwenhoekiella blandensis, revealing that it is a dGTPase that is subject to allosteric activation by dATP, specifically. Allosteric regulation mediated solely by dATP represents a novel regulatory feature among dNTPases that may facilitate maintenance of cellular dNTP pools in L. blandensis. We present high-resolution X-ray crystallographic structures (1.80-2.26 Å) in catalytically important conformations as well as cryo-EM structures (2.1-2.7 Å) of the enzyme bound to dGTP and dATP ligands. The structures, the highest resolution cryo-EM structures of any SAMHD1-like dNTPase to date, reveal an intact metal-binding site with the dGTP substrate coordinated to three metal ions. These structural and biochemical data yield insights into the catalytic mechanism and support a conserved catalytic mechanism for the tetrameric and hexameric dNTPase homologs. We conclude that the allosteric activation by dATP appears to rely on structural connectivity between the allosteric and active sites, as opposed to the changes in oligomeric state upon ligand binding used by SAMHD1.

Fibroblast growth factor homologous factors serve as a molecular rheostat in tuning arrhythmogenic cardiac late sodium current.

Voltage-gated sodium (Nav1.5) channels support the genesis and brisk spatial propagation of action potentials in the heart. Disruption of NaV1.5 inactivation results in a small persistent Na influx known as late Na current (I Na,L), which has emerged as a common pathogenic mechanism in both congenital and acquired cardiac arrhythmogenic syndromes. Here, using low-noise multi-channel recordings in heterologous systems, LQTS3 patient-derived iPSCs cardiomyocytes, and mouse ventricular myocytes, we demonstrate that the intracellular fibroblast growth factor homologous factors (FHF1-4) tune pathogenic I Na,L in an isoform-specific manner. This scheme suggests a complex orchestration of I Na,L in cardiomyocytes that may contribute to variable disease expressivity of NaV1.5 channelopathies. We further leverage these observations to engineer a peptide-inhibitor of I Na,L with a higher efficacy as compared to a well-established small-molecule inhibitor. Overall, these findings lend insights into molecular mechanisms underlying FHF regulation of I Na,L in pathophysiology and outline potential therapeutic avenues.

Zoledronic Acid Ameliorates the Bone Turnover Activity and Periprosthetic Bone Preservation in Cementless Total Hip Arthroplasty.

The administration of zoledronic acid (ZA) to patients who received cementless total hip arthroplasty (THA) has been reported to reduce bone turnover markers (BTMs) and increase bone mineral density (BMD). The effects of two-dose ZA versus placebo on cementless THA patients were analyzed in this five-year extension study. Alkaline phosphatase (ALP), osteocalcin (OC), procollagen 1 intact N-terminal propeptide (P1NP), serum calcium, renal function, radiological findings, and functional outcomes were compared in 49 patients, and the periprosthetic BMD of seven Gruen zones were compared in 19 patients. All the patients had normal renal function and calcium levels at their final follow-up. The mean ALP level in the ZA group was significantly lower at the fifth year, mean OC levels were significantly lower at the second and fifth year, and mean P1NP levels were significantly lower from 6 weeks to 5 years as compared with the control group. Fifth-year BMD levels were not found to be different between the ZA and control groups. The BMD Change Ratios in the ZA group were significantly increased in Gruen zone 6 at 1, 2, and 5 years. Our study results suggest that short-term ZA treatment with a subsequent 4-year drug holiday may inhibit serum BTMs and provide periprosthetic bone preservation at five years without adverse events.

A procedure to detect and identify specific chemicals of potential inhalation toxicity concern in aerosols.

OBJECTIVE: Understanding the potential inhalation toxicity of poorly characterized aerosols is challenging both because aerosols may contain numerous chemicals and because it is difficult to predict which chemicals may present significant inhalation toxicity concerns at the observed levels. We have developed a novel systematic procedure to address these challenges through non-targeted chemical analysis by two-dimensional gas chromatography-time-of-flight mass spectrometry (GC × GC-TOFMS) and assessment of the results using publicly available toxicity data to prioritize the tentatively identified detected chemicals according to potential inhalation toxicity. MATERIALS AND METHODS: The procedure involves non-targeted chemical analysis of aerosol samples utilizing GC × GC-TOFMS, which is selected because it is an effective technique for detecting chemicals in complex samples and assigning tentative identities according to the mass spectra. For data evaluation, existing toxicity data (e.g. from the U.S. Environmental Protection Agency CompTox Chemicals Dashboard) are used to calculate multiple toxicity metrics that can be compared among the tentatively identified chemicals. These metrics include hazard quotient, incremental lifetime cancer risk, and metrics analogous to hazard quotient that we designated as exposure-(toxicology endpoint) ratios. RESULTS AND DISCUSSION: We demonstrated the utility of our procedure by detecting, identifying, and prioritizing specific chemicals of potential inhalation toxicity concern in the mainstream smoke generated from the machine-smoking of marijuana blunts. CONCLUSION: By designing a systematic approach for detecting and identifying numerous chemicals in complex aerosol samples and prioritizing the chemicals in relation to different inhalation toxicology endpoints, we have developed an effective approach to elucidate the potential inhalation toxicity of aerosols.

Implant Site Development Using Titanium Mesh in the Maxilla: A Retrospective Study of 58 Mesh Procedures in 48 Patients.

This article presents a retrospective case series of implant site development using titanium mesh (Ti-mesh) in the maxilla. A total of 58 mesh procedures in combination with several different bone grafts (allograft, cellular allograft, and bovine xenograft) and biologics (including recombinant human platelet-derived growth factor, autogenous platelet-rich growth factor, and recombinant human bone morphogenetic protein-2) were performed in 48 patients. Ti-mesh guided bone regeneration procedures were performed 2 to 3 months after extraction of nonrestorable/hopeless teeth, and the implants were placed 6 to 8 months postaugmentation. The mean initial ridge width was 2.0 ± 1.0 mm, and the mean horizontal gain after Ti-mesh procedures was 4.7 ± 1.6 mm. The ridge width was first measured on the cross-sectional presurgical CBCT image and then confirmed clinically during surgical procedures. No statistical difference in the horizontal gain was found among different combinations of bone grafts and biomaterials. Ti-mesh exposure occurred 22% of the time. The middle-aged adults (odds ratio [OR] = 8.59; P = .046) and older adults (OR = 16.66; P = .02) had significantly higher chances of mesh exposure compared to young adults. While all implants were successfully placed, about 56% of the implants had < 2 mm of bone to the facial aspect of the osteotomy and received additional contour augmentation when placed in a prosthetically appropriate position for a screw-retained restoration. This study demonstrates that although Ti-mesh procedures result in significant bone regeneration in narrow alveolar ridges to predictably allow implant placement, the age-related mesh exposure rate and frequency of need for additional contour grafting should be discussed with patients.

A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike.

Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.

Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies.

Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geographically diverse HIV-1 strains using a unique heavy-chain (VH) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization. Here, we describe HIV-1 Env Fab-dimerized glycan (FDG)-reactive bnAbs without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques. FDG Abs also recognized cell-surface glycans on diverse pathogens, including yeast and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike. FDG precursors were expanded by glycan-bearing immunogens in macaques and were abundant in HIV-1-naive humans. Moreover, FDG precursors were predominately mutated IgM+IgD+CD27+, thus suggesting that they originated from a pool of antigen-experienced IgM+ or marginal zone B cells.

Cryo-EM structures of the SARS-CoV-2 endoribonuclease Nsp15 reveal insight into nuclease specificity and dynamics.

Nsp15, a uridine specific endoribonuclease conserved across coronaviruses, processes viral RNA to evade detection by host defense systems. Crystal structures of Nsp15 from different coronaviruses have shown a common hexameric assembly, yet how the enzyme recognizes and processes RNA remains poorly understood. Here we report a series of cryo-EM reconstructions of SARS-CoV-2 Nsp15, in both apo and UTP-bound states. The cryo-EM reconstructions, combined with biochemistry, mass spectrometry, and molecular dynamics, expose molecular details of how critical active site residues recognize uridine and facilitate catalysis of the phosphodiester bond. Mass spectrometry revealed the accumulation of cyclic phosphate cleavage products, while analysis of the apo and UTP-bound datasets revealed conformational dynamics not observed by crystal structures that are likely important to facilitate substrate recognition and regulate nuclease activity. Collectively, these findings advance understanding of how Nsp15 processes viral RNA and provide a structural framework for the development of new therapeutics.

Structural Basis for Virulence Activation of Francisella tularensis.

The bacterium Francisella tularensis (Ft) is one of the most infectious agents known. Ft virulence is controlled by a unique combination of transcription regulators: the MglA-SspA heterodimer, PigR, and the stress signal, ppGpp. MglA-SspA assembles with the σ70-associated RNAP holoenzyme (RNAPσ70), forming a virulence-specialized polymerase. These factors activate Francisella pathogenicity island (FPI) gene expression, which is required for virulence, but the mechanism is unknown. Here we report FtRNAPσ70-promoter-DNA, FtRNAPσ70-(MglA-SspA)-promoter DNA, and FtRNAPσ70-(MglA-SspA)-ppGpp-PigR-promoter DNA cryo-EM structures. Structural and genetic analyses show MglA-SspA facilitates σ70 binding to DNA to regulate virulence and virulence-enhancing genes. Our Escherichia coli RNAPσ70-homodimeric EcSspA structure suggests this is a general SspA-transcription regulation mechanism. Strikingly, our FtRNAPσ70-(MglA-SspA)-ppGpp-PigR-DNA structure reveals ppGpp binding to MglA-SspA tethers PigR to promoters. PigR in turn recruits FtRNAP αCTDs to DNA UP elements. Thus, these studies unveil a unique mechanism for Ft pathogenesis involving a virulence-specialized RNAP that employs two (MglA-SspA)-based strategies to activate virulence genes.

Structural impact of K63 ubiquitin on yeast translocating ribosomes under oxidative stress.

Subpopulations of ribosomes are responsible for fine tuning the control of protein synthesis in dynamic environments. K63 ubiquitination of ribosomes has emerged as a new posttranslational modification that regulates protein synthesis during cellular response to oxidative stress. K63 ubiquitin, a type of ubiquitin chain that functions independently of the proteasome, modifies several sites at the surface of the ribosome, however, we lack a molecular understanding on how this modification affects ribosome structure and function. Using cryoelectron microscopy (cryo-EM), we resolved the first three-dimensional (3D) structures of K63 ubiquitinated ribosomes from oxidatively stressed yeast cells at 3.5-3.2 Å resolution. We found that K63 ubiquitinated ribosomes are also present in a polysome arrangement, similar to that observed in yeast polysomes, which we determined using cryoelectron tomography (cryo-ET). We further showed that K63 ubiquitinated ribosomes are captured uniquely at the rotated pretranslocation stage of translation elongation. In contrast, cryo-EM structures of ribosomes from mutant cells lacking K63 ubiquitin resolved at 4.4-2.7 Å showed 80S ribosomes represented in multiple states of translation, suggesting that K63 ubiquitin regulates protein synthesis at a selective stage of elongation. Among the observed structural changes, ubiquitin mediates the destabilization of proteins in the 60S P-stalk and in the 40S beak, two binding regions of the eukaryotic elongation factor eEF2. These changes would impact eEF2 function, thus, inhibiting translocation. Our findings help uncover the molecular effects of K63 ubiquitination on ribosomes, providing a model of translation control during oxidative stress, which supports elongation halt at pretranslocation.

Cryo-EM Structures of the SARS-CoV-2 Endoribonuclease Nsp15.

New therapeutics are urgently needed to inhibit SARS-CoV-2, the virus responsible for the on-going Covid-19 pandemic. Nsp15, a uridine-specific endoribonuclease found in all coronaviruses, processes viral RNA to evade detection by RNA-activated host defense systems, making it a promising drug target. Previous work with SARS-CoV-1 established that Nsp15 is active as a hexamer, yet how Nsp15 recognizes and processes viral RNA remains unknown. Here we report a series of cryo-EM reconstructions of SARS-CoV-2 Nsp15. The UTP-bound cryo-EM reconstruction at 3.36 Å resolution provides molecular details into how critical residues within the Nsp15 active site recognize uridine and facilitate catalysis of the phosphodiester bond, whereas the apo-states reveal active site conformational heterogeneity. We further demonstrate the specificity and mechanism of nuclease activity by analyzing Nsp15 products using mass spectrometry. Collectively, these findings advance understanding of how Nsp15 processes viral RNA and provide a structural framework for the development of new therapeutics.

Controlling the SARS-CoV-2 spike glycoprotein conformation.

The coronavirus (CoV) spike (S) protein, involved in viral-host cell fusion, is the primary immunogenic target for virus neutralization and the current focus of many vaccine design efforts. The highly flexible S-protein, with its mobile domains, presents a moving target to the immune system. Here, to better understand S-protein mobility, we implemented a structure-based vector analysis of available ß-CoV S-protein structures. Despite an overall similarity in domain organization, we found that S-proteins from different ß-CoVs display distinct configurations. Based on this analysis, we developed two soluble ectodomain constructs for the SARS-CoV-2 S-protein, in which the highly immunogenic and mobile receptor binding domain (RBD) is either locked in the all-RBDs 'down' position or adopts 'up' state conformations more readily than the wild-type S-protein. These results demonstrate that the conformation of the S-protein can be controlled via rational design and can provide a framework for the development of engineered CoV S-proteins for vaccine applications.

Controlling the SARS-CoV-2 Spike Glycoprotein Conformation.

The coronavirus (CoV) viral host cell fusion spike (S) protein is the primary immunogenic target for virus neutralization and the current focus of many vaccine design efforts. The highly flexible S-protein, with its mobile domains, presents a moving target to the immune system. Here, to better understand S-protein mobility, we implemented a structure-based vector analysis of available ß-CoV S-protein structures. We found that despite overall similarity in domain organization, different ß-CoV strains display distinct S-protein configurations. Based on this analysis, we developed two soluble ectodomain constructs in which the highly immunogenic and mobile receptor binding domain (RBD) is locked in either the all-RBDs 'down' position or is induced to display a previously unobserved in SARS-CoV-2 2-RBDs 'up' configuration. These results demonstrate that the conformation of the S-protein can be controlled via rational design and provide a framework for the development of engineered coronavirus spike proteins for vaccine applications.

Structures of AAA protein translocase Bcs1 suggest translocation mechanism of a folded protein.

The mitochondrial membrane-bound AAA protein Bcs1 translocate substrates across the mitochondrial inner membrane without previous unfolding. One substrate of Bcs1 is the iron-sulfur protein (ISP), a subunit of the respiratory Complex III. How Bcs1 translocates ISP across the membrane is unknown. Here we report structures of mouse Bcs1 in two different conformations, representing three nucleotide states. The apo and ADP-bound structures reveal a homo-heptamer and show a large putative substrate-binding cavity accessible to the matrix space. ATP binding drives a contraction of the cavity by concerted motion of the ATPase domains, which could push substrate across the membrane. Our findings shed light on the potential mechanism of translocating folded proteins across a membrane, offer insights into the assembly process of Complex III and allow mapping of human disease-associated mutations onto the Bcs1 structure.