pH-dependent 11° F1FO ATP synthase sub-steps reveal insight into the FO torque generating mechanism

Most cellular ATP is made by rotary F1FO ATP synthases using proton translocation-generated clockwise torque on the FO c-ring rotor, while F1-ATP hydrolysis can force counterclockwise rotation and proton pumping. The FO torque-generating mechanism remains elusive even though the FO interface of stator subunit-a, which contains the transmembrane proton half-channels, and the c-ring is known from recent F1FO structures. Here, single-molecule F1FO rotation studies determined that the pKa values of the half-channels differ, show that mutations of residues in these channels change the pKa values of both half-channels, and reveal the ability of FO to undergo single c-subunit rotational stepping. These experiments provide evidence to support the hypothesis that proton translocation through FO operates via a Grotthuss mechanism involving a column of single water molecules in each half-channel linked by proton translocation-dependent c-ring rotation. We also observed pH-dependent 11° ATP synthase-direction sub-steps of the E. coli c10-ring of F1FO against the torque of F1-ATPase-dependent rotation that result from H+ transfer events from FO subunit-a groups with a low pKa to one c-subunit in the c-ring, and from an adjacent c-subunit to stator groups with a high pKa. These results support a mechanism in which alternating proton translocation-dependent 11° and 25° synthase-direction rotational sub-steps of the c10-ring occur to sustain F1FO ATP synthesis.

CRISPR/Cas9-Mediated Gene Editing of Vacuolar ATPase Subunit D Mediates Phytohormone Biosynthesis and Virus Resistance in Rice

Background: Vacuolar ATPases (v-ATPases) are proton pumps for proton translocation across membranes that utilize energy derived from ATP hydrolysis; Previous research revealed Osv-ATPases mediates phytohormes levels and resistance in rice. Osv-ATPase subunit d (Osv-ATPase d) is part of an integral, membrane-embedded V0 complex of V-ATPases complex, whether Osv-ATPase d involves in phytohormes biosynthesis and resistance in rice remains unknown.Finding: The knockout mutant line (line 5) of Osv-ATPase d was generated using the CRISPR/Cas9 system, mutation of Osv-ATPase d did not show any detrimental effect on plant growth or yield productivity. Transcriptomic results showed Osv-ATPase d probably involved in mediating the biosynthesis of plant hormones and resistance in rice. Mutation of Osv-ATPase d significantly increased JA and ABA biosynthesis than wild type. Compared to wild type, mutation of Osv-ATPase d increased the resistance against Southern rice black-streaked dwarf virus (SRBSDV), however, decreased the resistance against Rice stripe virus (RSV) in rice. Conclusion: Taken together, our data reveal the Osv-ATPase d mediates phytohormone biosynthesis and virus resistance in rice, which can be selected as a potential target for resistance breeding in rice.

Dynamic Coupling of Tyrosine 185 with the Bacteriorhodopsin Photocycle, as Revealed by Chemical Shifts, Assisted AF-QM/MM Calculations and Molecular Dynamic Simulations

Aromatic residues are highly conserved in microbial photoreceptors and play crucial roles in the dynamic regulation of receptor functions. However, little is known about the dynamic mechanism of the functional role of those highly conserved aromatic residues during the receptor photocycle. Tyrosine 185 (Y185) is a highly conserved aromatic residue within the retinal binding pocket of bacteriorhodopsin (bR). In this study, we explored the molecular mechanism of the dynamic coupling of Y185 with the bR photocycle by automated fragmentation quantum mechanics/molecular mechanics (AF-QM/MM) calculations and molecular dynamic (MD) simulations based on chemical shifts obtained by 2D solid-state NMR correlation experiments. We observed that Y185 plays a significant role in regulating the retinal cis–trans thermal equilibrium, stabilizing the pentagonal H-bond network, participating in the orientation switch of Schiff Base (SB) nitrogen, and opening the F42 gate by interacting with the retinal and several key residues along the proton translocation channel. Our findings provide a detailed molecular mechanism of the dynamic couplings of Y185 and the bR photocycle from a structural perspective. The method used in this paper may be applied to the study of other microbial photoreceptors.

A quantitative paradigm for water-assisted proton transport through proteins and other confined spaces

Water-assisted proton transport through confined spaces influences many phenomena in biomolecular and nanomaterial systems. In such cases, the water molecules that fluctuate in the confined pathways provide the environment and the medium for the hydrated excess proton migration via Grotthuss shuttling. However, a definitive collective variable (CV) that accurately couples the hydration and the connectivity of the proton wire with the proton translocation has remained elusive. To address this important challenge—and thus to define a quantitative paradigm for facile proton transport in confined spaces—a CV is derived in this work from graph theory, which is verified to accurately describe water wire formation and breakage coupled to the proton translocation in carbon nanotubes and the Cl−/H+ antiporter protein, ClC-ec1. Significant alterations in the conformations and thermodynamics of water wires are uncovered after introducing an excess proton into them. Large barriers in the proton translocation free-energy profiles are found when water wires are defined to be disconnected according to the new CV, even though the pertinent confined space is still reasonably well hydrated and—by the simple measure of the mere existence of a water structure—the proton transport would have been predicted to be facile via that oversimplified measure. In this paradigm, however, the simple presence of water is not sufficient for inferring proton translocation, since an excess proton itself is able to drive hydration, and additionally, the water molecules themselves must be adequately connected to facilitate any successful proton transport.

Cryo-EM of the yeast VO complex reveals distinct binding sites for macrolide V-ATPase inhibitors

Vacuolar-type adenosine triphosphatases (V-ATPases) are proton pumps found in almost all eukaryotic cells. These enzymes consist of a soluble catalytic V1 region that hydrolyzes ATP and a membrane-embedded VO region responsible for proton translocation. V-ATPase activity leads to acidification of endosomes, phagosomes, lysosomes, secretory vesicles, and the trans-Golgi network, with extracellular acidification occurring in some specialized cells. Small molecule inhibitors of V-ATPase have played a crucial role in elucidating numerous aspects of cell biology by blocking acidification of intracellular compartments, while therapeutic use of V-ATPase inhibitors has been proposed for treatment of cancer, osteoporosis, and some infections. Here, we determine structures of the isolated VO complex from Saccharomyces cerevisiae bound to two well-known macrolide inhibitors: bafilomycin A1 and archazolid A. The structures reveal different binding sites for the inhibitors on the surface of the proton-carrying c ring, with only a small amount of overlap between the two sites. Binding of both inhibitors is mediated primarily through van der Waals interactions in shallow pockets and suggests that the inhibitors block rotation of the ring. Together, these structures indicate the existence of a large chemical space available for V-ATPase inhibitors that block acidification by binding the c ring.

Structure and activation mechanism of the hexameric plasma membrane H+-ATPase

The S. cerevisiae plasma membrane H+-ATPase, Pma1, is a P3A-type ATPase and the primary protein component of the membrane compartment of Pma1 (MCP). Like other plasma membrane H+-ATPases, Pma1 assembles and functions as a hexamer, a property unique to this subfamily among the larger family of P-type ATPases. It has been unclear how Pma1 organizes the yeast membrane into MCP microdomains, or why it is that Pma1 needs to assemble into a hexamer to establish the membrane electrochemical proton gradient. Here we report a high-resolution cryo-EM study of native Pma1 hexamers embedded in endogenous lipids. Remarkably, we found that the Pma1 hexamer encircles a liquid-crystalline membrane domain composed of 57 ordered lipid molecules. The Pma1-encircled lipid patch structure likely serves as the building block of the MCP. At pH 7.4, the carboxyl-terminal regulatory α-helix binds to the phosphorylation domains of two neighboring Pma1 subunits, locking the hexamer in the autoinhibited state. The regulatory helix becomes disordered at lower pH, leading to activation of the Pma1 hexamer. The activation process is accompanied by a 6.7 Å downward shift and a 40° rotation of transmembrane helices 1 and 2 that line the proton translocation path. The conformational changes have enabled us to propose a detailed mechanism for ATP-hydrolysis-driven proton pumping across the plasma membrane. Our structures will facilitate the development of antifungal drugs that target this essential protein.

Leishmania type II dehydrogenase is essential for parasite viability irrespective of the presence of an active complex I

Type II NADH dehydrogenases (NDH2) are monotopic enzymes present in the external or internal face of the mitochondrial inner membrane that contribute to NADH/NAD+ balance by conveying electrons from NADH to ubiquinone without coupled proton translocation. Herein, we characterize the product of a gene present in all species of the human protozoan parasite Leishmania as a bona fide, matrix-oriented, type II NADH dehydrogenase. Within mitochondria, this respiratory activity concurs with that of type I NADH dehydrogenase (complex I) in some Leishmania species but not others. To query the significance of NDH2 in parasite physiology, we attempted its genetic disruption in two parasite species, exhibiting a silent (Leishmania infantum, Li) and a fully operational (Leishmania major, Lm) complex I. Strikingly, this analysis revealed that NDH2 abrogation is not tolerated by Leishmania, not even by complex I–expressing Lm species. Conversely, complex I is dispensable in both species, provided that NDH2 is sufficiently expressed. That a type II dehydrogenase is essential even in the presence of an active complex I places Leishmania NADH metabolism into an entirely unique perspective and suggests unexplored functions for NDH2 that span beyond its complex I–overlapping activities. Notably, by showing that the essential character of NDH2 extends to the disease-causing stage of Leishmania, we genetically validate NDH2—an enzyme without a counterpart in mammals—as a candidate target for leishmanicidal drugs.

MMPL-Family Proteins in Bacteria, Protozoa, Fungi, Plants and Animals: A Bioinformatics and Structural Investigation

Lipid transporters play an important role in most if not all organisms, ranging from bacteria to humans. For example, in Mycobacterium tuberculosis, the trehalose monomycolate transporter MmpL3 is involved in cell wall biosynthesis, while in humans, cholesterol transporters are involved in normal cell function as well as in disease. Here, using structural and bioinformatics information, we propose that there are proteins that also contain “MmpL3-like” (MMPL) transmembrane (TM) domains in many protozoa, including Trypanosoma cruzi, as well as in the bacterium Staphylococcus aureus, where the fatty acid transporter FarE has the same set of “active-site” residues as those found in the mycobacterial MmpL3s, and in T. cruzi. We also show that there are strong sequence and predicted structural similarities between the TM proton-translocation domain seen in the X-ray structures of mycobacterial MmpL3s and several human as well as fungal lipid transporters, leading to the proposal that there are similar proteins in apicomplexan parasites, and in plants. The animal, fungal, apicomplexan and plant proteins have larger extra-membrane domains than are found in the bacterial MmpL3, but they have a similar TM domain architecture, with the introduction of a (catalytically essential) Phe>His residue change, and a Ser/Thr H-bond network, involved in H -transport. Overall, the results are of interest since they show that MMPL-family proteins are present in essentially all life-forms: archaea, bacteria, protozoa, fungi, plants and animals and, where known, they are involved in “lipid” (glycolipid, phospholipid, sphingolipid, fatty acid, cholesterol, ergosterol) transport, powered by transmembrane molecular pumps having similar structures.

Structure of Escherichia coli respiratory complex I reconstituted into lipid nanodiscs reveals an uncoupled conformation

Respiratory complex I is a multi-subunit membrane protein complex that reversibly couples NADH oxidation and ubiquinone reduction with proton translocation against trans-membrane potential. Complex I from Escherichia coli is among the best functionally characterized complexes, but its structure remains unknown, hindering further mechanistic studies to understand the enzyme coupling mechanism. Here we describe the single particle cryo-electron microscopy (cryo-EM) structure of the entire catalytically active E. coli complex I reconstituted into lipid nanodiscs. The structure of this mesophilic bacterial complex I displays highly dynamic connection between the peripheral and membrane domains. The peripheral domain assembly is stabilized by unique terminal extensions and an insertion loop. The membrane domain structure reveals novel dynamic features. Unusual conformation of the conserved interface between the peripheral and membrane domains suggests an uncoupled conformation of the complex. Considering constraints imposed by the structural data we suggest a new simple hypothetical coupling mechanism for the molecular machine.

A Quantitative Paradigm for Water Assisted Proton Transport Through Proteins and Other Confined Spaces

Water assisted proton transport through confined spaces influences many phenomena in biomolecular and nanomaterial systems. In such cases, the water molecules that fluctuate in the confined pathways provide the environment and the medium for the hydrated excess proton migration via Grotthuss shuttling. However, a definitive collective variable (CV) that accurately couples the hydration and the connectivity of the proton wire with the proton translocation has remained elusive. To address this important challenge-and thus to define a new quantitative paradigm for facile proton transport in confined spaces-a CV is derived in this work from graph theory, which is verified to accurately describe water wire formation and breakage coupled to the proton translocation in carbon nanotubes and the Cl-/H+ antiporter protein, ClC-ec1. Significant alterations in the conformations and thermodynamics of water wires are uncovered after introducing an excess proton into them. Large barriers in the proton translocation free energy profiles are found when water wires are defined to be disconnected according to the new CV, even though the pertinent confined space is still reasonably well hydrated and-by the simple measure of the mere existence of a water structure-the proton transport would have been predicted to be facile via that oversimplified measure. In this new paradigm, however, the simple presence of water is not sufficient for inferring proton translocation since an excess proton itself is able to drive hydration and, additionally, the water molecules themselves must be adequately connected to facilitate any successful proton transport.