Association of the eukaryotic V1VO ATPase subunits a with d and d with A

AbstractOwing to the complex nature of V1VO ATPases, identification of neighboring subunits is essential for mechanistic understanding of this enzyme. Here, we describe the links between the V1 headpiece and the VO-domain of the yeast V1VO ATPase via subunit A and d as well as the VO subunits a and d using surface plasmon resonance and fluorescence correlation spectroscopy. Binding constants of about 60 and 200nM have been determined for the a–d and d–A assembly, respectively. The data are discussed in light of subunit a and d forming a peripheral stalk, connecting the catalytic A3B3 hexamer with VO.Structured summaryMINT-7012054: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by fluorescence correlation spectroscopy (MI:0052)MINT-7012041: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by surface plasmon resonance (MI:0107)MINT-7012028: d (uniprotkb:P32366) binds (MI:0407) to a (uniprotkb:P32563) by surface plasmon resonance (MI:0107

Structural Determination of Functional Units of the Nucleotide Binding Domain (NBD94) of the Reticulocyte Binding Protein Py235 of Plasmodium yoelii

Invasion of the red blood cells (RBC) by the merozoite of malaria parasites involves a large number of receptor ligand interactions. The reticulocyte binding protein homologue family (RH) plays an important role in erythrocyte recognition as well as virulence. Recently, it has been shown that members of RH in addition to receptor binding may also have a role as ATP/ADP sensor. A 94 kDa region named Nucleotide-Binding Domain 94 (NBD94) of Plasmodium yoelii YM, representative of the putative nucleotide binding region of RH, has been demonstrated to bind ATP and ADP selectively. Binding of ATP or ADP induced nucleotide-dependent structural changes in the C-terminal hinge-region of NBD94, and directly impacted on the RBC binding ability of RH.In order to find the smallest structural unit, able to bind nucleotides, and its coupling module, the hinge region, three truncated domains of NBD94 have been generated, termed NBD94(444-547), NBD94(566-663) and NBD94(674-793), respectively. Using fluorescence correlation spectroscopy NBD94(444-547) has been identified to form the smallest nucleotide binding segment, sensitive for ATP and ADP, which became inhibited by 4-Chloro-7-nitrobenzofurazan. The shape of NBD94(444-547) in solution was calculated from small-angle X-ray scattering data, revealing an elongated molecule, comprised of two globular domains, connected by a spiral segment of about 73.1 A in length. The high quality of the constructs, forming the hinge-region, NBD94(566-663) and NBD94(674-793) enabled to determine the first crystallographic and solution structure, respectively. The crystal structure of NBD94(566-663) consists of two helices with 97.8 A and 48.6 A in length, linked by a loop. By comparison, the low resolution structure of NBD94(674-793) in solution represents a chair-like shape with three architectural segments.These structures give the first insight into how nucleotide binding impacts on the overall structure of RH and demonstrates the potential use of this region as a novel drug target

Synthetic mimetics of the binding site of Mena EVH1 domain for proline rich ligands: Design, synthesis and investigation of binding behavior

Die Mena-EVH1-Domäne (mammalian enabled VASP homology 1) spielt eine wichtige Rolle bei der Organisation des Aktin-basierten Cytoskeletts und damit bei der Regulation von Bewegungsabläufen in Zellen. Das Protein ist 112 Aminosäuren lang und erkennt das prolinreiche Sequenzmotiv -FPPPP(. Dieses Motiv enthaltende Peptide wurden durch Kartierung mittels überlappender Peptide des Bakterien-Oberflächenproteins ActA von Listeria monocytogenes, einem natürlichen Liganden der Mena-EVH1-Domäne, identifiziert. Die Mena-EVH1-Domäne wurde mit dem Peptid-Liganden -FPPPP( co-kristallisiert, um die Kontaktstellen für diese Interaktion zu ermitteln. Die Tetraprolin-Sequenz des Liganden bindet hauptsächlich an die aromatischen Reste Tyr16, Trp23 und Phe77 von Mena-EVH1, die als aromatische Triade bezeichnet werden und eine sequenziell diskontinuierliche Bindungsstelle darstellen. Ziel dieser Arbeit war es, die diskontinuierliche Bindungsstelle der Mena-EVH1-Domäne durch zusammengesetzte synthetische Peptide nachzuahmen, in denen zwei Peptidfragmente, die die Reste der aromatischen Triade enthalten (Fragment A: 13VMVYDDANKKWVPA26, enthält Tyr16 und Trp23 und Fragment B: 70YNQATQTFHQWR81, enthält Phe77), in einem Molekül präsentiert werden. Beide Fragmente wurden sowohl als cyclische, als auch als lineare Peptide mit Hilfe der Festphasenpeptidsynthese synthetisiert. Die Fragmente wurden entweder mit einem Cystein-Rest (Fragment A) oder einem Bromacetyl-Rest (Fragment B) versehen, was eine regio-selektive Verknüpfung der Fragmente über einen Thioether ermöglicht.The Mena EVH1 domain (mammalian enabled VASP homology 1) plays an important role in the organization of the Actin based Cytoskeleton and therefor in the regulation of cell motility. The protein constits of 112 amino acids and recognizes the proline rich motive -FPPPP-. Peptides containing this motive were identified by mapping of bacteria-surface protein ActA of Listeria monocytogenes, a natural ligand of the Mena EVH1 domain, by overlapping peptides. The Mena EVH1 domain was co-crystallized with the Peptid ligand -FPPPP- to determine the contact site for this interaction. The tetraproline sequence of the ligand binds primarily to the aromatic residues Tyr16, Trp23 and Phe77 of Mena-EVH1 which are called ´aromatic triad´ and show a sequentiell discontinuous binding site. The purpose of this work was to mimic the discontinuous binding site of the Mena EVH1 domain by assembled synthetic peptide, in which two peptide fragments contain the residues of the aromatic triad (fragment A: 13VMVYDDANKKWVPA26, Tyr16 and Trp23 and fragment B: 70YNQATQTFHQWR81, Phe77) in a single molecule are presented. Both fragments were synthesized as cyclic and as linear peptides using solid phase peptide synthesis. The fragments were synthesized with a cysteine residue (fragment A) or a Bromacetyl residue (fragment B) which allows regioselective linkage of the fragments forming a thioether

Structure-based synthetic mimicry of discontinuous protein binding sites: inhibitors of the interaction of Mena EVH1 domain with proline-rich ligands.

The Mena EVH1 domain, a protein-interaction module involved in actin-based cell motility, recognizes proline-rich ligand motifs, which are also present in the sequence of the surface protein ActA of Listeria monocytogenes. The interaction of ActA with host Mena EVH1 enables the bacterium to actively recruit host actin in order to spread into neighboring cells. Based on the crystal structure of Mena EVH1 in complex with a polyproline peptide ligand, we have generated a range of assembled peptides presenting the Mena EVH1 fragments that make up its discontinuous binding site for proline-rich ligands. Some of these peptides were found to inhibit the interaction of Mena EVH1 with the ligand pGolemi. One of them was further characterized at the level of individual amino acid residues; this yielded information on the contribution of individual positions of the peptides to the interaction with the ligand and identified sites for future structure optimization

The structure of subunit E of the pyrococcus horikoshii OT3 A-ATP synthase gives insight into the elasticity of the peripheral stalk

A1AO ATP synthases are the major energy converters of archaea. They are composed of an A1 region that synthesizes ATP and an integral part AO that conducts ions. Subunit E is a component of the peripheral stalk that links the A1 with the AO part of the A-ATP synthase. We have determined the crystal structure of the entire subunit E (PhE) of the Pyrococcus horikoshii OT3 A-ATP synthase at 3.6 Å resolution. The structure reveals an extended S-shaped N-terminal α-helix with 112.29 Å in length, followed by a globular head group. The S-shaped feature, common in elastic connectors and spacers, would facilitate the storage of transient elastic energy during rotary motion in the enzyme. The structure has been superimposed into the asymmetric peripheral stalks of the three-dimensional reconstruction of the Pyrococcus furiosus enzyme, revealing that the S-shaped subunit PhE fits well into the bent peripheral stalk, whereas the previously solved E subunit structure (3.1 Å resolution) of Thermus thermophilus A-ATP synthase is well accommodated in the density of the straight stator domain. The different features of the two stalk subunits are discussed in light of a novel coupling mechanism in A-ATP synthases proposed to differ from the Wankel engine of F-ATP synthases

The solution structure of pGolemi, a high affinity Mena EVH1 binding miniature protein, suggests explanations for paralog-specific binding to Ena/VASP homology (EVH) 1 domains

Ena/VASP homology 1 (EVH1) domains are polyproline binding domains that are present in a wide range of adaptor proteins, among them Ena/VASP proteins involved in actin remodeling and axonal guidance. The interaction of ActA, a transmembrane protein from the food-borne pathogen Listeria monocytogenes, with EVH1 domains has been shown to be crucial for recruitment of the host's actin skeleton and, as a consequence, for the infectivity of this bacterium. We present the structure of a synthetic high-affinity Mena EVH1 ligand, pGolemi, capable of paralog-specific binding, solved by NMR spectroscopy. This peptide shares the common pancreatic peptide fold with its scaffold, avian pancreatic peptide, but shows pivotal differences in the amino-terminus. The interplay of spatial fixation and flexibility appears to be the reason for its high affinity towards Mena EVH1. Combined with earlier investigations, our structural data shed light on the specificity determinants of pGolemi and the importance of additional binding epitopes around the residues Thr74 and Phe32 on EVH1 domains regulating paralog specificity. Our results are expected to facilitate the design of other high-affinity, paralog-specific EVH1 domain ligands, and serve as a fundament for the investigation of the molecular mode of action of EVH1 domains

Relevance of the conserved histidine and asparagine residues in the phosphate-binding loop of the nucleotide binding subunit B of A1AO ATP synthases

The nucleotide binding sites in A-ATP synthases are located at the interfaces of subunit A and B, which is proposed to play a regulatory role. Differential binding of MgATP and -ADP to subunit B has been described, which does not exist in the related α and B subunits of F-ATP synthases and V-ATPases, respectively. The conserved phosphate loop residues, histidine and asparagine, of the A-ATP synthase subunit B have been proposed to be essential for γ-phosphate interaction. To investigate the role of these conserved P-loop residues in nucleotide-binding, subunit B residues H156 and N157 of the Methanosarcina mazei Gö1 A-ATP synthase were separately substituted with alanine. In addition, N157 was mutated to threonine, because it is the corresponding amino acid in the P-loop of F-ATP synthase subunit α. The structures of the subunit B mutants H156A, N157A/T were solved up to a resolution of 1.75 and 1.7 Å. The binding constants for MgATP and -ADP were determined, demonstrating that the H156A and N157A mutants have a preference to the nucleotide over the wild type and N157T proteins. Importantly, the ability to distinguish MgATP or -ADP was lost, demonstrating that the histidine and asparagine residues are crucial for nucleotide differentiation in subunit B. The structures reveal that the enhanced binding of the alanine mutants is attributed to the increased accessibility of the nucleotide binding cavity, explaining that the structural arrangement of the conserved H156 and N157 define the nucleotide-binding characteristics of the regulatory subunit B of A-ATP synthases

Association of the eukaryotic V1VO ATPase subunits a with d and d with A

AbstractOwing to the complex nature of V1VO ATPases, identification of neighboring subunits is essential for mechanistic understanding of this enzyme. Here, we describe the links between the V1 headpiece and the VO-domain of the yeast V1VO ATPase via subunit A and d as well as the VO subunits a and d using surface plasmon resonance and fluorescence correlation spectroscopy. Binding constants of about 60 and 200nM have been determined for the a–d and d–A assembly, respectively. The data are discussed in light of subunit a and d forming a peripheral stalk, connecting the catalytic A3B3 hexamer with VO.Structured summaryMINT-7012054: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by fluorescence correlation spectroscopy (MI:0052)MINT-7012041: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by surface plasmon resonance (MI:0107)MINT-7012028: d (uniprotkb:P32366) binds (MI:0407) to a (uniprotkb:P32563) by surface plasmon resonance (MI:0107