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Enzymes in the respiratory chain are increasingly seen as potential targets against multi-drug resistance of human pathogens and cancerous cells. However, a detailed understanding of the mechanism and specificity determinants of known inhibitors is still lacking. Oligomycin, for example, has been known to be an inhibitor of the membrane motor of the mitochondrial ATP synthase for over five decades, and yet little is known about its mode of action at the molecular level. In a recent breakthrough, a crystal structure of the S. cerevisiae c-subunit ring with bound oligomycin revealed the inhibitor docked on the outer face of the proton-binding sites, deep into the transmembrane region. However, the structure of the complex was obtained in an organic solvent rather than detergent or a lipid bilayer, and therefore it has been unclear whether this mode of recognition is physiologically relevant. Here, we use molecular dynamics simulations to address this question and gain insights into the mechanism of oligomycin inhibition. Our findings lead us to propose that oligomycin naturally partitions into the lipid/water interface, and that in this environment the inhibitor can indeed bind to any of the c-ring proton-carrying sites that are exposed to the membrane, thereby becoming an integral component of the proton-coordinating network. As the c-ring rotates within the membrane, driven either by downhill proton permeation or ATP hydrolysis, one of the protonated, oligomycin-bound sites eventually reaches the subunit-a interface and halts the rotary mechanism of the enzyme. [ABSTRACT FROM AUTHOR]

ZAP-70 and Syk are essential tyrosine kinases in intracellular immunological signaling. Both contain an inhibitory SH2 domain tandem, which assembles onto the catalytic domain. Upon binding to doubly phosphorylated ITAM motifs on activated antigen receptors, the arrangement of the SH2 domains changes. From available structures, this event is not obviously conducive to dissociation of the autoinhibited complex, yet it ultimately translates into kinase activation through a mechanism not yet understood. We present a comprehensive theoretical study of this molecular mechanism, using atomic resolution simulations and free-energy calculations, totaling >10 μs of simulation time. Through these, we dissect the microscopic mechanism coupling stepwise ITAM engagement and SH2 tandem structural change and reveal key differences between ZAP-70 and Syk. Importantly, we show that a subtle conformational bias in the inter-SH2 connector causes ITAM to bind preferentially to kinase-dissociated tandems. We thus propose that phosphorylated antigen receptors selectively recruit kinases that are uninhibited and that the resulting population shift in the membrane vicinity sustains signal transduction. [ABSTRACT FROM AUTHOR]

The molecular mechanisms of membrane proteins that are activated either by ions or by ATP are just beginning to come into focus, as long-awaited structural data are revealed. This information is being leveraged and supplemented to great effect by molecular modeling and computer simulation studies. Important examples include the homology modeling of eukaryotic protein structures based on distantly related templates, as well as the use of internal structural symmetry for modeling different states in conformational cycles. Molecular simulation studies have elucidated the location and coordination structure of ion binding sites, and explained their selectivity, while also providing tantalizing insights into the mechanisms that couple conformational change to ion translocation or ATP hydrolysis. [ABSTRACT FROM AUTHOR]

Autophagy is a catabolic process involving capture of cytoplasmic materials into double-membraned autophagosomes that subsequently fuse with lysosomes for degradation of the materials by lysosomal hydrolases. One of the least understood components of the autophagy machinery is the transmembrane protein ATG9. Here, we report a cryoelectron microscopy structure of the human ATG9A isoform at 2.9-Å resolution. The structure reveals a fold with a homotrimeric domain-swapped architecture, multiple membrane spans, and a network of branched cavities, consistent with ATG9A being a membrane transporter. Mutational analyses support a role for the cavities in the function of ATG9A. In addition, structure-guided molecular simulations predict that ATG9A causes membrane bending, explaining the localization of this protein to small vesicles and highly curved edges of growing autophagosomes. • The transmembrane autophagy protein ATG9A is a domain-swapped homotrimer • Each ATG9A protomer comprises four transmembrane domains • The ATG9A homotrimer exhibits an internal network of branched cavities • Molecular dynamics simulations show that ATG9A trimers deform membranes Guardia et al. report a high-resolution cryo-EM structure of human ATG9A, the only transmembrane protein of the core autophagy machinery. The structure shows that ATG9A is a domain-swapped homotrimer with a complex network of internal cavities. Structure-based computational simulations predict that ATG9A has membrane-bending properties. [ABSTRACT FROM AUTHOR]

Ceramides play a key modulatory role in many cellular processes, which results from their effect on the structure and dynamics of biological membranes. In this study, we investigate the influence of C16-ceramide (C16) on the biophysical properties of DMPC lipid bilayers using solid-state NMR and atomistic molecular dynamics (MD) simulations. MD simulations and NMR measurements were carried out for a pure DMPC bilayer and for a 20% DMPC-C16 mixture. Calculated key structural properties, namely area per lipid, chain order parameters, and mass density profiles, indicate that C16 has an ordering effect on the DMPC bilayer. Furthermore, the simulations predict that specific hydrogen-bonds form between DMPC and C16 molecules. Multi-nuclear solid-state NMR was used to verify these theoretical predictions. Chain order parameters extracted from 13C¹H dipole couplings were measured for both lipid and ceramide and follow the trend suggested by the MD simulations. Furthermore, ¹H-MAS NMR experiments showed a direct contact between ceramide and lipids. [ABSTRACT FROM AUTHOR]

Abstract: CD1 proteins mediate the trafficking and presentation of a diverse range of lipid antigens to T-cell receptors, and thus play a key role in our adaptive immune system. Crystal structures of several CD1 isoforms reveal a highly conserved tertiary structure, but also great variability in the anatomy of their binding pockets, reflecting their distinct ligand specificity. The structure of one important member of the family, CD1c, remains unknown. CD1c is of great interest as it can present an unusual and potent lipid antigen, mannosyl-β1-phosphomycoketide (MPM) from Mycobacterium tuberculosis, the causative agent of tuberculosis. CD1c has also been reported to present acetylated 12-amino-acid-long peptides (lipo-12), an observation with broad immunological implications but difficult to rationalize on structural grounds. To gain insights into the structural basis for the ligand specificity of CD1c, we have generated an atomic model of its binding domain using a detailed position-specific multiple-template homology modeling approach. This model reveals structural features unique to this isoform, particularly with regard to the so-called pocket F′, which provide a compelling rationale for the ability of CD1c to bind not only branched alkyl chains such as in MPM, but also long lipopeptides comparable to those presented by MHC proteins. A model of CD1c with bound MPM was constructed and analyzed through molecular dynamics simulations, showing marked structural stability in the time-scale of 100ns. A model of CD1c in complex with lipo-12 is also presented. [Copyright &y& Elsevier]

In both prokaryotes and eukaryotes, multidrug and toxiccompound extrusion (MATE) transporters catalyze the efflux of a broad range of cytotoxic compounds, including humanmade antibiotics and anticancer drugs. MATEs are secondary-active antiporters, i.e., their drug-efflux activity is coupled to, and powered by, the uptake of ions down a preexisting transmembrane electrochemical gradient. Key aspects of this mechanism, however, remain to be delineated, such as its ion specificity and stoichiometry. We previously revealed the existence of a Na+-binding site in a MATE transporter from Pyroccocus furiosus (PfMATE) and hypothesized that this site might be broadly conserved among prokaryotic MATEs. Here, we evaluate this hypothesis by analyzing VcmN and ClbM, which along with PfMATE are the only three prokaryotic MATEs whose molecular structures have been determined at atomic resolution, i.e. better than 3 Å. Reinterpretation of existing crystallographic data and molecular dynamics simulations indeed reveal an occupied Na+-binding site in the Nterminal lobe of both structures, analogous to that identified in PfMATE. We likewise find this site to be strongly selective against K+, suggesting it is mechanistically significant. Consistent with these computational results, DEER spectroscopy measurements for multiple doubly-spin-labeled VcmN constructs demonstrate Na+-dependent changes in protein conformation. The existence of this binding site in three MATE orthologs implicates Na+ in the ion-coupled drug-efflux mechanisms of this class of transporters. These results also imply that observations of H+-dependent activity likely stem either from a site elsewhere in the structure, or from H+ displacing Na+ under certain laboratory conditions, as has been noted for other Na+-driven transport systems. [ABSTRACT FROM AUTHOR]

CD1 proteins mediate the presentation of endogenous and foreign lipids on the cell surface for recognition by T cell receptors. To sample a diverse antigen pool, CD1 proteins are repeatedly internalized and recycled, assisted, in some cases, by lipid transfer proteins such as saposins. The specificity of each CD1 isoform is, therefore, conferred in part by its intracellular pathway but also by distinct structural features of the antigen-binding domain. Crystal structures of CD1-lipid complexes reveal hydrophobic grooves and pockets within these binding domains that appear to be specialized for different lipids. However, the mechanism of lipid loading and release remains to be characterized. Here we gain insights into this mechanism through a metaanalysis of the five human CD1 isoforms, in the lipid-bound and lipid-free states, using all-atom molecular dynamics simulations. Strikingly, for isoforms CD1b through CD1e, our simulations show the near-complete collapse of the hydrophobic cavities in the absence of the antigen. This event results from the spontaneous closure of the binding domain entrance, flanked by two α-helices. Accordingly, we show that the anatomy of the binding cavities is restored if these α-helices are repositioned extrinsically, suggesting that helper proteins encountered during recycling facilitate lipid exchange allosterically. By contrast, we show that the binding cavity of CD1a is largely preserved in the unliganded state because of persistent electrostatic interactions that keep the portal α-helices at a constant separation. The robustness of this binding groove is consistent with the observation that lipid exchange in CD1a is not dependent on cellular internalization. [ABSTRACT FROM AUTHOR]

The ion-driven membrane rotors of ATP synthases consist of multiple copies of subunit c, forming a closed ring. Subunit c typically comprises two transmembrane helices, and the c ring features an ion-binding site in between each pair of adjacent subunits. Here, we use experimental and computational methods to study the structure and specificity of an archaeal c subunit more akin to those of V-type ATPases, namely that from Pyrococcus furiosus. The c subunit was purified by chloroform/methanol extraction and determined to be 15.8 kDa with four predicted transmembrane helices. However, labeling withDCCDas well as Na+-DCCD competition experiments revealed only one binding site for DCCD and Na+, indicating that the mature c subunit of this A1AO ATP synthase is indeed of the V-type. A structural model generated computationally revealed one Na+-binding site within each of the c subunits, mediated by a conserved glutamate side chain alongside other coordinating groups. An intriguing second glutamate located in-between adjacent c subunits was ruled out as a functional Na+-binding site. Molecular dynamics simulations indicate that the c ring of P. furiosus is highly Na+-specific under in vivo conditions, comparable with the Na+-dependent V1VO ATPase from Enterococcus hirae. Interestingly, the same holds true for the c ring from the methanogenic archaeon Methanobrevibacter ruminantium, whose c subunits also feature a V-type architecture but carry two Na+-binding sites instead. These findings are discussed in light of their physiological relevance and with respect to the mode of ion coupling in A1AO ATP synthases. [ABSTRACT FROM AUTHOR]

Abstract: The functional mechanism of the F1Fo ATP synthase, like many membrane transporters and pumps, entails a conformational cycle that is coupled to the movement of H+ or Na+ ions across its transmembrane domain, down an electrochemical gradient. This coupling is an efficient means of energy transduction and regulation, provided that ion binding to the membrane domain, known as Fo, is appropriately selective. In this study we set out to establish the structural and energetic basis for the ion-binding selectivity of the membrane-embedded Fo rotors of two representative ATP synthases. First, we use a biochemical approach to demonstrate the inherent binding selectivity of these rotors, that is, independently from the rest of the enzyme. We then use atomically detailed computer simulations of wild-type and mutagenized rotors to calculate and rationalize their selectivity, on the basis of the structure, dynamics and coordination chemistry of the binding sites. We conclude that H+ selectivity is most likely a robust property of all Fo rotors, arising from the prominent presence of a conserved carboxylic acid and its intrinsic chemical propensity for protonation, as well as from the structural plasticity of the binding sites. In H+-coupled rotors, the incorporation of hydrophobic side chains to the binding sites enhances this inherent H+ selectivity. Size restriction may also favor H+ over Na+, but increasing size alone does not confer Na+ selectivity. Rather, the degree to which Fo rotors may exhibit Na+ coupling relies on the presence of a sufficient number of suitable coordinating side chains and/or structural water molecules. These ligands accomplish a shift in the relative binding energetics, which under some physiological conditions may be sufficient to provide Na+ dependence. [Copyright &y& Elsevier]