AREA 1. MEMBRANE PROTEINS IN BIOENERGETICS The research focus in Area 1 has been the ATP synthase, a membrane-bound enzyme that produces most of the ATP used by cells. ATP synthases use a unique turbine-like rotary mechanism to facilitate the transmembrane flow protons down an electrochemical gradient, and harness the resulting energy gain to power the chemical reactions required for ATP production. Key aspects of this process of energy conversion, however, remain poorly characterized - particularly the mechanism of the membrane domain. The involvement of the ATP synthase in other physiological processes is also an area of active research. In FY16/17 we have continued our efforts to advance our understanding of the mechanism and physiological roles of this essential system. In one study (Leone et al. JGP 2016) we developed a model of the molecular structure of a subcomplex of the membrane domain - specifically of the two protein elements known to be key for proton permeation and energy transduction, known as the c-ring and subunit-a. To do so, we used quantitative molecular-modeling methods to integrate a wide range of experimental and bioinformatic information - namely cryo-EM data, extensive cross-linking and cysteine-accessibility measurements and an analysis of correlated mutations within each protein and at their interface. This systematic, integrative approach enabled us to unambiguously delineate the topology of subunit-a and its relationship with the c-ring. The resulting structure reveals important clues in regard to the pathway followed by protons as they traverse the membrane through the complex, and explains the directionality of the rotary mechanism and its strict coupling to the electrochemical proton gradient. Lastly, the structure also provides insights into the mode of action of known inhibitors - e.g. antibiotics that selectively target the bacterial or mitochondrial enzymes. This study is a stepping-stone towards establishing the mechanism of this enzyme at the atomic level. In a second study (Zhou et al. JPCB 2017), we also utilized computational methods to construct structural models of two prototypical c-rings at atomic resolution. Specifically, we modeled the c-rings of the ATP synthase from Saccharomyces cerevisiae and Escherichia coli, which are prototypical of the enzymes in mitochondria (e.g. human) and pathogenic bacteria, respectively. Our models capture the structures of these c-rings in a state in which they are loaded with protons, in the context of the membrane; that is, in the state that would mediate the recognition of a potential inhibitor. Thus, we anticipate that these structural models will be will be of value in future efforts to improve the potency and specificity of antibiotics targeting these enzymes. In our more recent study (Zhou et al, eLife 2017), we evaluated the possibility that the ATP synthase mediates the so-called mitochondrial permeability transition (MPT), a specific metabolic state characteristic of some diseased states and that ultimately leads to cell death. The MPT is believed to be triggered by the opening of an as-yet-unidentified, non-selective, large-conductance channel in the innermost mitochondrial membrane. Researchers in this field had proposed that the abovementioned c-ring is this channel. However, using advanced computer simulations, we demonstrated that the functional characteristics of the MPT pore are completely inconsistent with those of the c-ring, unequivocally disproving its direct involvement in this important process. AREA 2. TRANSPORT AND SIGNALING ACROSS BIOLOGICAL MEMBRANES Two proteins of considerable biomedical significance have been the focus in Area 2. First, the Na+/Ca2+ (NCX) exchanger, which is key for the control of calcium signals in many cell types, e.g. in cardiac cells controlling the heartbeat. Second, the so-called anion-exchanger AE1, which plays a central role in the process carbon-dioxide clearance via the blood stream. Both NCX and AE1 are members of a large class of membrane proteins known as secondary-active transporters. These proteins catalyze the translocation of substrates across the membrane against their concentration gradient. To harness the necessary energy, this process is directly or indirectly coupled to the translocation of another substrate, typically an ion, down a pre-existing concentration gradient. In antiporters like NCX and AE1, the two transported substrates traverse the membrane in opposite directions and in two different, yet interdependent steps of the transport cycle. In the NCX project, we continued our investigations of the structure and mechanism of NCX_Mj, a bacterial member of the family. NCX_Mj is thought to be a valid model system of the cardiac Na+/Ca2+ exchanger, e.g. in future efforts in pharmacological research. Our previous computational and structural studies, carried in collaboration with Prof. Y. Jiang (UT Southwestern/HHMI), had resulted in a clear-cut prediction in regard to the mode by which Na+ and Ca2+ are recognized by this protein, competing with each other. One aspect of that prediction is the exchange stoichiometry, namely 3Na+:1Ca2+. In FY16/17, we sought to evaluate this prediction through the experimental functional studies of NCX_Mj, which we designed and conducted in collaboration with Dr. J. Mindell (NINDS). Specifically, NCX_Mj was purified and reconstituted in liposomes and its functional characteristics were evaluated through 45Ca2+ flux assays under a broad range of well-defined experimental conditions. Among other important findings, this study demonstrated that, as predicted, the transport stoichiometry of NCX_Mj is 3Na+:1Ca2+ (Shlosman et al., in preparation). These findings provide the foundation for future investigations of the mechanism of ion translocation. In a second study (Ficici et al, submitted), we sought to outline a mechanistic framework with which to begin to explain the functional properties of AE1. This study was a collaboration with Dr. L Forrest (NINDS) and Prof. M. Jennings (U Arkansas). Our investigation was based on a recently-determined crystal structure, which captured the so-called outward-facing conformation, i.e. that exposing the substrate-binding sites to the extracellular space. A detailed analysis of this structure revealed that it features an internal quasi-symmetry; this feature is shared by many other secondary-active transporters, and has been shown to explain the ability of these proteins to adopt two major conformational state, each exposing the substrate-binding sites to only one side of the membrane. Following this reasoning, we developed and analyzed a model of the inward-facing conformation, by inverting the internal quasi-symmetry of the crystal structure. Comparison of both conformations reveals an elevator-like transport mechanism, whereby a subdomain of the structure that contains the substrate-binding site moves perpendicularly to the membrane plane. Though unexpected, this structural mechanism is in fact consistent with a broad range of biochemical and functional data, and forms the basis for future research. AREA 3. DEVELOPMENT OF SIMULATION METHODS The focus in Area 3 has been on improving a methodology we previously developed known an Ensemble-Biased Metadynamics. This maximum-entropy method biases a conventional molecular simulation to sample a conformational ensemble that is exactly consistent with one or more probability distributions known a priori, e.g., from EPR, FRET or analogous measurements, thereby providing a faithful structural interpretation of the input data. In this improved formulation, tested in collaboration with Profs. H. Mchaourab and E. Hustedt (Vanderbilt), the experimental error of the target probability distributions is considered explicitly (Hustedt, Marinelli et al., in preparation).
Showing the most recent 10 out of 17 publications
