Conservation of structure and mechanism in primary and secondary transporters exemplified by SiaP, a sialic acid binding virulence factor from Haemophilus influenzae

Extracytoplasmic solute receptors (ESRs) are important components of solute uptake systems in bacteria, having been studied extensively as parts of ATP binding cassette transporters. Herein we report the first crystal structure of an ESR protein from a functionally characterized electrochemical ion gradient-dependent secondary transporter. This protein, SiaP, forms part of a tripartite ATP-independent periplasmic transporter specific for sialic acid in Haemophilus influenzae. Surprisingly, the structure reveals an overall topology similar to ATP binding cassette ESR proteins, which is not apparent from the sequence, demonstrating that primary and secondary transporters can share a common structural component. The structure of SiaP in the presence of the sialic acid analogue 2,3-didehydro-2-deoxyN-acetylneuraminic acid reveals the ligand bound in a deep cavity with its carboxylate group forming a salt bridge with a highly conserved Arg residue. Sialic acid binding, which obeys simple bimolecular association kinetics as determined by stopped-flow fluorescence spectroscopy, is accompanied by domain closure about a hinge region and the kinking of an alpha-helix hinge component. The structure provides insight into the evolution, mechanism, and substrate specificity of ESR-dependent secondary transporters that are widespread in prokaryotes

NOX Inhibitors - A Promising Avenue for Ischemic Stroke.

NADPH-oxidase (NOX) mediated superoxide originally found on leukocytes, but now recognized in several types of cells in the brain. It has been shown to play an important role in the progression of stroke and related cerebrovascular disease. NOX is a multisubunit complex consisting of 2 membrane-associated and 4 cytosolic subunits. NOX activation occurs when cytosolic subunits translocate to the membrane, leading to transport electrons to oxygen, thus producing superoxide. Superoxide produced by NOX is thought to function in long-term potentiation and intercellular signaling, but excessive production is damaging and has been implicated to play an important role in the progression of ischemic brain. Thus, inhibition of NOX activity may prove to be a promising treatment for ischemic brain as well as an adjunctive agent to prevent its secondary complications. There is mounting evidence that NOX inhibition in the ischemic brain is neuroprotective, and targeting NOX in circulating immune cells will also improve outcome. This review will focus on therapeutic effects of NOX assembly inhibitors in brain ischemia and stroke. However, the lack of specificity and toxicities of existing inhibitors are clear hurdles that will need to be overcome before this class of compounds could be translated clinically

From the Ca2+-activated F1FO-ATPase to the mitochondrial permeability transition pore: an overview

Based on recent advances on the Ca2+-activated F1FO-ATPase features, a novel multistep mechanism involving the mitochondrial F1FO complex in the formation and opening of the still enigmatic mitochondrial permeability transition pore (MPTP), is proposed. MPTP opening makes the inner mitochondrial membrane (IMM) permeable to ions and solutes and, through cascade events, addresses cell fate to death. Since MPTP forms when matrix Ca2+ concentration rises and ATP is hydrolyzed by the F1FO-ATPase, conformational changes, triggered by Ca2+ insertion in F1, may be transmitted to FO and locally modify the IMM curvature. These events would cause F1FO-ATPase dimer dissociation and MPTP opening

Exploring the directionality of <i>Escherichia coli </i>formate hydrogenlyase:a membrane-bound enzyme capable of fixing carbon dioxide to organic acid

During mixed‐acid fermentation Escherichia coli produces formate, which is initially excreted out the cell. Accumulation of formate, and dropping extracellular pH, leads to biosynthesis of the formate hydrogenlyase (FHL) complex. FHL consists of membrane and soluble domains anchored within the inner membrane. The soluble domain comprises a [NiFe] hydrogenase and a formate dehydrogenase that link formate oxidation directly to proton reduction with the release of CO (2) and H(2). Thus, the function of FHL is to oxidize excess formate at low pH. FHL subunits share identity with subunits of the respiratory Complex I. In particular, the FHL membrane domain contains subunits (HycC and HycD) that are homologs of NuoL/M/N and NuoH, respectively, which have been implicated in proton translocation. In this work, strain engineering and new assays demonstrate unequivocally the nonphysiological reverse activity of FHL in vivo and in vitro. Harnessing FHL to reduce CO (2) to formate is biotechnologically important. Moreover, assays for both possible FHL reactions provide opportunities to explore the bioenergetics using biochemical and genetic approaches. Comprehensive mutagenesis of hycC did not identify any single amino acid residues essential for FHL operation. However, the HycD E199, E201, and E203 residues were found to be critically important for FHL function

Structure and biogenesis of the chloroplast NAD(P)H dehydrogenase complex

AbstractEleven genes (ndhA-ndhK) encoding proteins homologous to the subunits of bacterial and mitochondrial NADH dehydrogenase (complex I) were found in the plastid genome of most land plants. These genes encode subunits of the chloroplast NAD(P)H dehydrogenase (NDH) complex involved in photosystem I (PSI) cyclic electron transport and chlororespiration. Although the chloroplast NDH is believed to be closely and functionally related to the cyanobacterial NDH-1L complex, extensive proteomic, genetic and bioinformatic studies have discovered many novel subunits that are specific to higher plants. On the basis of extensive mutant characterization, the chloroplast NDH complex is divided into four parts, the A, B, membrane and lumen subcomplexes, of which subunits in the B and lumen subcomplexes are specific to higher plants. These results suggest that the structure of NDH has been drastically altered during the evolution of land plants. Furthermore, chloroplast NDH interacts with multiple copies of PSI to form the unique NDH–PSI supercomplex. Two minor light-harvesting-complex I (LHCI) proteins, Lhca5 and Lhca6, are required for the specific interaction between NDH and PSI. The evolution of chloroplast NDH in land plants may be required for development of the function of NDH to alleviate oxidative stress in chloroplasts. In this review, we summarize recent progress on the subunit composition and structure of the chloroplast NDH complex, as well as the information on some factors involved in its assembly. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts

Assembly of the peripheral stalk of ATP synthase in human mitochondria.

The adenosine triphosphate (ATP) synthase in human mitochondria is a membrane bound assembly of 29 proteins of 18 kinds organized into F1-catalytic, peripheral stalk (PS), and c8-rotor ring modules. All but two membrane components are encoded in nuclear genes, synthesized on cytoplasmic ribosomes, imported into the mitochondrial matrix, and assembled into the complex with the mitochondrial gene products ATP6 and ATP8. Intermediate vestigial ATPase complexes formed by disruption of nuclear genes for individual subunits provide a description of how the various domains are introduced into the enzyme. From this approach, it is evident that three alternative pathways operate to introduce the PS module (including associated membrane subunits e, f, and g). In one pathway, the PS is built up by addition to the core subunit b of membrane subunits e and g together, followed by membrane subunit f. Then this b-e-g-f complex is bound to the preformed F1-c8 module by subunits OSCP and F6 The final component of the PS, subunit d, is added subsequently to form a key intermediate that accepts the two mitochondrially encoded subunits. In another route to this key intermediate, first e and g together and then f are added to a preformed F1-c8-OSCP-F6-b-d complex. A third route involves the addition of the c8-ring module to the complete F1-PS complex. The key intermediate then accepts the two mitochondrially encoded subunits, stabilized by the addition of subunit j, leading to an ATP synthase complex that is coupled to the proton motive force and capable of making ATP

In vitro reconstitution of dynamically interacting integral membrane subunits of energy-coupling factor transporters

Energy-coupling factor (ECF) transporters mediate import of micronutrients in prokaryotes. They consist of an integral membrane S-component (that binds substrate) and ECF module (that powers transport by ATP hydrolysis). It has been proposed that different S-components compete for docking onto the same ECF module, but a minimal liposome-reconstituted system, required to substantiate this idea, is lacking. Here, we co-reconstituted ECF transporters for folate (ECF-FolT2) and pantothenate (ECF-PanT) into proteoliposomes, and assayed for crosstalk during active transport. The kinetics of transport showed that exchange of S-components is part of the transport mechanism. Competition experiments suggest much slower substrate association with FolT2 than with PanT. Comparison of a crystal structure of ECF-PanT with previously determined structures of ECF-FolT2 revealed larger conformational changes upon binding of folate than pantothenate, which could explain the kinetic differences. Our work shows that a minimal in vitro system with two reconstituted transporters recapitulates intricate kinetics behaviour observed in vivo

From the Structural and (Dys)Function of ATP Synthase to Deficiency in Age-Related Diseases

open3noThis research was funded by CARISBO Foundation, grants numbers 2021.0058 to C.G and 2021.0173 to S.N.; Italian Minister of University and Research—Rita Levi Montalcini Program-Rientro cervelli RLM2017 to C.G.The ATP synthase is a mitochondrial inner membrane complex whose function is essential for cell bioenergy, being responsible for the conversion of ADP into ATP and playing a role in mitochondrial cristae morphology organization. The enzyme is composed of 18 protein subunits, 16 nuclear DNA (nDNA) encoded and two mitochondrial DNA (mtDNA) encoded, organized in two domains, FO and F1. Pathogenetic variants in genes encoding structural subunits or assembly factors are responsible for fatal human diseases. Emerging evidence also underlines the role of ATP-synthase in neurodegenerative diseases as Parkinson’s, Alzheimer’s, and motor neuron diseases such as Amyotrophic Lateral Sclerosis. Post-translational modification, epigenetic modulation of ATP gene expression and protein level, and the mechanism of mitochondrial transition pore have been deemed responsible for neuronal cell death in vivo and in vitro models for neurodegenerative diseases. In this review, we will explore ATP synthase assembly and function in physiological and pathological conditions by referring to the recent cryo-EM studies and by exploring human disease models.openGarone C.; Pietra A.; Nesci S.Garone C.; Pietra A.; Nesci S