scholarly journals Structural insights into how GTP-dependent conformational changes in a metallochaperone UreG facilitate urease maturation

2017 ◽  
Vol 114 (51) ◽  
pp. E10890-E10898 ◽  
Author(s):  
Man Hon Yuen ◽  
Yu Hang Fong ◽  
Yap Shing Nim ◽  
Pak Ho Lau ◽  
Kam-Bo Wong
Keyword(s):  
Conformational Changes ◽  
Metal Binding ◽  
Sole Source ◽  
Planar Geometry ◽  
Binding Motif ◽  
Nickel Ion ◽  
Gtp Hydrolysis ◽  
Nickel Ions ◽  
Gtp Binding

The ability of metallochaperones to allosterically regulate the binding/release of metal ions and to switch protein-binding partners along the metal delivery pathway is essential to the metallation of the metalloenzymes. Urease, catalyzing the hydrolysis of urea into ammonia and carbon dioxide, contains two nickel ions bound by a carbamylated lysine in its active site. Delivery of nickel ions for urease maturation is dependent on GTP hydrolysis and is assisted by four urease accessory proteins UreE, UreF, UreG, and UreH(UreD). Here, we determined the crystal structure of the UreG dimer from Klebsiella pneumoniae in complex with nickel and GMPPNP, a nonhydrolyzable analog of GTP. Comparison with the structure of the GDP-bound Helicobacter pylori UreG (HpUreG) in the UreG2F2H2 complex reveals large conformational changes in the G2 region and residues near the 66CPH68 metal-binding motif. Upon GTP binding, the side chains of Cys66 and His68 from each of the UreG protomers rotate toward each other to coordinate a nickel ion in a square-planar geometry. Mutagenesis studies on HpUreG support the conformational changes induced by GTP binding as essential to dimerization of UreG, GTPase activity, in vitro urease activation, and the switching of UreG from the UreG2F2H2 complex to form the UreE2G2 complex with the UreE dimer. The nickel-charged UreE dimer, providing the sole source of nickel, and the UreG2F2H2 complex could activate urease in vitro in the presence of GTP. Based on our results, we propose a mechanism of how conformational changes of UreG during the GTP hydrolysis/binding cycle facilitate urease maturation.

scholarly journals Septin collar formation in budding yeast requires GTP binding and direct phosphorylation by the PAK, Cla4

2004 ◽  
Vol 164 (5) ◽  
pp. 701-715 ◽  
Author(s):  
Matthias Versele ◽  
Jeremy Thorner
Keyword(s):  
Wild Type ◽  
Gtp Hydrolysis ◽  
Filament Assembly ◽  
Gtp Binding ◽  
Type Mutant ◽  
Binding Residues ◽  
Septin Filament ◽  
Assembly Defect

Assembly at the mother–bud neck of a filamentous collar containing five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) is necessary for proper morphogenesis and cytokinesis. We show that Cdc10 and Cdc12 possess GTPase activity and appropriate mutations in conserved nucleotide-binding residues abrogate GTP binding and/or hydrolysis in vitro. In vivo, mutants unable to bind GTP prevent septin collar formation, whereas mutants that block GTP hydrolysis do not. GTP binding-defective Cdc10 and Cdc12 form soluble heteromeric complexes with other septins both in yeast and in bacteria; yet, unlike wild-type, mutant complexes do not bind GTP and do not assemble into filaments in vitro. Absence of a p21-activated protein kinase (Cla4) perturbs septin collar formation. This defect is greatly exacerbated when combined with GTP binding-defective septins; conversely, the septin collar assembly defect of such mutants is suppressed efficiently by CLA4 overexpression. Cla4 interacts directly with and phosphorylates certain septins in vitro and in vivo. Thus, septin collar formation may correspond to septin filament assembly, and requires both GTP binding and Cla4-mediated phosphorylation of septins.

scholarly journals Interaction of the Antiactivator FleN with the Transcriptional Activator FleQ Regulates Flagellar Number inPseudomonas aeruginosa

2001 ◽  
Vol 183 (22) ◽  
pp. 6636-6644 ◽  
Author(s):  
Nandini Dasgupta ◽  
Reuben Ramphal
Keyword(s):  
Binding Protein ◽  
Direct Interaction ◽  
Transcriptional Activator ◽  
Binding Motif ◽  
Yeast Two Hybrid System ◽  
Gtp Binding ◽  
C Terminus ◽  
Enhancer Binding Protein ◽  
Two Hybrid

ABSTRACT Flagellar number in Pseudomonas aeruginosa is controlled by FleN, a putative ATP/GTP binding protein. Disruption offleN results in multiflagellation of the otherwise monoflagellate strains PAK and PAO1 and is associated with a chemotactic defect. We propose that flagellar number is maintained by the antiactivator FleN, which downregulates flagellar genes by binding to their transcriptional activator, FleQ, an enhancer binding protein belonging to the NifA subfamily. In this report we demonstrate direct interaction of FleN and FleQ in the yeast two-hybrid system. Mutagenesis of the putative ATP/GTP binding motif in FleN24K→Q and truncation of FleN at either the N or C terminus abrogates this interaction. FleN does not inhibit the DNA binding ability of FleQ in vitro, thus indicating that it probably utilizes another mechanism(s) to serve as a FleQ antiactivator.

scholarly journals Conformation of the Solute-Binding Protein AdcAII Influences Zinc Uptake in Streptococcus pneumoniae

2021 ◽  
Vol 11 ◽  
Author(s):  
Marina L. Župan ◽  
Zhenyao Luo ◽  
Katherine Ganio ◽  
Victoria G. Pederick ◽  
Stephanie L. Neville ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Conformational Changes ◽  
Metal Binding ◽  
Binding Assays ◽  
Ligand Affinity ◽  
Atp Binding Cassette Transporter ◽  
Binding Residues ◽  
Biochemical Analyses ◽  
Putative Mechanism

Streptococcus pneumoniae scavenges essential zinc ions from the host during colonization and infection. This is achieved by the ATP-binding cassette transporter, AdcCB, and two solute-binding proteins (SBPs), AdcA and AdcAII. It has been established that AdcAII serves a greater role during initial infection, but the molecular details of how the protein selectively acquires Zn(II) remain poorly understood. This can be attributed to the refractory nature of metal-free AdcAII to high-resolution structural determination techniques. Here, we overcome this issue by separately mutating the Zn(II)-coordinating residues and performing a combination of structural and biochemical analyses on the variant proteins. Structural analyses of Zn(II)-bound AdcAII variants revealed that specific regions within the protein underwent conformational changes via direct coupling to each of the metal-binding residues. Quantitative in vitro metal-binding assays combined with affinity determination and phenotypic growth assays revealed that each of the four Zn(II)-coordinating residues contributes to metal binding by AdcAII. Intriguingly, the phenotypic growth impact of the mutant adcAII alleles was, in general, independent of affinity, suggesting that the Zn(II)-bound conformation of the SBP is crucial for efficacious metal uptake. Collectively, these data highlight the intimate coupling of ligand affinity with protein conformational change in ligand-receptor proteins and provide a putative mechanism for AdcAII. These findings provide further mechanistic insight into the structural and functional diversity of SBPs that is broadly applicable to other prokaryotes.

scholarly journals Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization

2004 ◽  
Vol 166 (7) ◽  
pp. 1003-1014 ◽  
Author(s):  
Gideon Lansbergen ◽  
Yulia Komarova ◽  
Mauro Modesti ◽  
Claire Wyman ◽  
Casper C. Hoogenraad ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Metal Binding ◽  
Intramolecular Interaction ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Scanning Force Microscopy ◽  
Binding Motif ◽  
Force Microscopy ◽  
Scanning Force ◽  
Metal Binding Motif

Cytoplasmic linker protein (CLIP)-170, CLIP-115, and the dynactin subunit p150Glued are structurally related proteins, which associate specifically with the ends of growing microtubules (MTs). Here, we show that down-regulation of CLIP-170 by RNA interference results in a strongly reduced accumulation of dynactin at the MT tips. The NH2 terminus of p150Glued binds directly to the COOH terminus of CLIP-170 through its second metal-binding motif. p150Glued and LIS1, a dynein-associating protein, compete for the interaction with the CLIP-170 COOH terminus, suggesting that LIS1 can act to release dynactin from the MT tips. We also show that the NH2-terminal part of CLIP-170 itself associates with the CLIP-170 COOH terminus through its first metal-binding motif. By using scanning force microscopy and fluorescence resonance energy transfer-based experiments we provide evidence for an intramolecular interaction between the NH2 and COOH termini of CLIP-170. This interaction interferes with the binding of the CLIP-170 to MTs. We propose that conformational changes in CLIP-170 are important for binding to dynactin, LIS1, and the MT tips.

LRRK2: from kinase to GTPase to microtubules and back

2017 ◽  
Vol 45 (1) ◽  
pp. 141-146 ◽  
Author(s):  
Marian Blanca Ramírez ◽  
Antonio Jesús Lara Ordóñez ◽  
Elena Fdez ◽  
Sabine Hilfiker
Keyword(s):  
Membrane Trafficking ◽  
Kinase Activity ◽  
Gtp Hydrolysis ◽  
Intact Cells ◽  
Gtp Binding ◽  
Lrrk2 Gene ◽  
Sporadic Parkinson’S Disease ◽  
Cellular Compartments ◽  
Kinase Substrates

Mutations in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene are intimately linked to both familial and sporadic Parkinson's disease. LRRK2 is a large protein kinase able to bind and hydrolyse GTP. A wealth of in vitro studies have established that the distinct pathogenic LRRK2 mutants differentially affect those enzymatic activities, either causing an increase in kinase activity without altering GTP binding/GTP hydrolysis, or displaying no change in kinase activity but increased GTP binding/decreased GTP hydrolysis. Importantly, recent studies have shown that all pathogenic LRRK2 mutants display increased kinase activity towards select kinase substrates when analysed in intact cells. To understand those apparently discrepant results, better insight into the cellular role(s) of normal and pathogenic LRRK2 is crucial. Various studies indicate that LRRK2 regulates numerous intracellular vesicular trafficking pathways, but the mechanism(s) by which the distinct pathogenic mutants may equally interfere with such pathways has largely remained elusive. Here, we summarize the known alterations in the catalytic activities of the distinct pathogenic LRRK2 mutants and propose a testable working hypothesis by which the various mutants may affect membrane trafficking events in identical ways by culminating in increased phosphorylation of select substrate proteins known to be crucial for membrane trafficking between specific cellular compartments.

The C-terminal domain is sufficient for endonuclease activity of Neisseria gonorrhoeae MutL

2009 ◽  
Vol 423 (2) ◽  
pp. 265-277 ◽  
Author(s):  
Viswanadham Duppatla ◽  
Chiranjeevi Bodda ◽  
Claus Urbanke ◽  
Peter Friedhoff ◽  
Desirazu N. Rao
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Metal Binding ◽  
Sedimentation Equilibrium ◽  
Velocity Sedimentation ◽  
Binding Motif ◽  
Endonuclease Activity ◽  
Dna Mismatch ◽  
Terminal Domain ◽  
Dependent Endonuclease

The mutL gene of Neisseria gonorrhoeae has been cloned and the gene product purified. We have found that the homodimeric N. gonorrhoeae MutL (NgoL) protein displays an endonuclease activity that incises covalently closed circular DNA in the presence of Mn2+, Mg2+ or Ca2+ ions, unlike human MutLα which shows endonuclease activity only in the presence of Mn2+. We report in the present paper that the C-terminal domain of N. gonorrhoeae MutL (NgoL-CTD) consisting of amino acids 460–658 exhibits Mn2+-dependent endonuclease activity. Sedimentation velocity, sedimentation equilibrium and dynamic light scattering experiments show NgoL-CTD to be a dimer. The probable endonucleolytic active site is localized to a metal-binding motif, DMHAX2EX4E, and the nicking endonuclease activity is dependent on the integrity of this motif. By in vitro comparison of wild-type and a mutant NgoL-CTD protein, we show that the latter protein exhibits highly reduced endonuclease activity. We therefore suggest that the mode of excision initiation in DNA mismatch repair may be different in organisms that lack MutH protein, but have MutL proteins that harbour the D[M/Q]HAX2EX4E motif.

scholarly journals A cysteine-rich metal-binding domain from rubella virus non-structural protein is essential for viral protease activity and virus replication

2008 ◽  
Vol 417 (2) ◽  
pp. 477-483 ◽  
Author(s):  
Yubin Zhou ◽  
Wen-Pin Tzeng ◽  
Yiming Ye ◽  
Yun Huang ◽  
Shunyi Li ◽  
...  
Keyword(s):  
Virus Replication ◽  
Conformational Changes ◽  
Rubella Virus ◽  
Metal Binding ◽  
Structural Integrity ◽  
Binding Motif ◽  
Structural Protein ◽  
Protease Domain ◽  
Apparent Dissociation Constant ◽  
Metal Binding Domain

The protease domain within the RUBV (rubella virus) NS (non-structural) replicase proteins functions in the self-cleavage of the polyprotein precursor into the two mature proteins which form the replication complex. This domain has previously been shown to require both zinc and calcium ions for optimal activity. In the present study we carried out metal-binding and conformational experiments on a purified cysteine-rich minidomain of the RUBV NS protease containing the putative Zn2+-binding ligands. This minidomain bound to Zn2+ with a stoichiometry of ≈0.7 and an apparent dissociation constant of <500 nM. Fluorescence quenching and 8-anilinonaphthalene-1-sulfonic acid fluorescence methods revealed that Zn2+ binding resulted in conformational changes characterized by shielding of hydrophobic regions from the solvent. Mutational analyses using the minidomain identified residues Cys1175, Cys1178, Cys1225 and Cys1227 were required for the binding of Zn2+. Corresponding mutational analyses using a RUBV replicon confirmed that these residues were necessary for both proteolytic activity of the NS protease and viability. The present study demonstrates that the CXXC(X)48CXC Zn2+-binding motif in the RUBV NS protease is critical for maintaining the structural integrity of the protease domain and essential for proteolysis and virus replication.

scholarly journals Molecular Modelling of the Ni(II)-Responsive Synechocystis PCC 6803 Transcriptional Regulator InrS in the Metal Bound Form

Inorganics ◽  
2019 ◽  
Vol 7 (6) ◽  
pp. 76 ◽  
Author(s):  
Elia Barchi ◽  
Francesco Musiani
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Metal Binding ◽  
Computational Study ◽  
Transcriptional Regulator ◽  
Planar Geometry ◽  
Synechocystis Pcc 6803 ◽  
Square Planar ◽  
Structural Details ◽  
Pcc 6803

InrS (internal nickel-responsive sensor) is a transcriptional regulator found in cyanobacteria that represses the transcription of the nickel exporter NrsD in the apo form and de-represses expression of the exporter upon Ni(II) binding. Although a crystal structure of apo-InrS from Synechocystis PCC 6803 has been reported, no structure of the protein with metal ions bound is available. Here we report the results of a computational study aimed to reconstruct the metal binding site by taking advantage of recent X-ray absorption spectroscopy (XAS) data and to envisage the structural rearrangements occurring upon Ni(II) binding. The modelled Ni(II) binding site shows a square planar geometry consistent with experimental data. The structural details of the conformational changes occurring upon metal binding are also discussed in the framework of trying to rationalize the different affinity of the apo- and holo-forms of the protein for DNA.

scholarly journals Dual Roles of Bradyrhizobium japonicumNickelin Protein in Nickel Storage and GTP-Dependent Ni Mobilization

2000 ◽  
Vol 182 (6) ◽  
pp. 1702-1705 ◽  
Author(s):  
Jonathan W. Olson ◽  
Robert J. Maier
Keyword(s):  
Gtpase Activity ◽  
Dual Roles ◽  
Nickel Ion ◽  
Nickel Ions ◽  
Hydrogenase Maturation ◽  
Nickel Binding ◽  
And Storage

ABSTRACT The hydrogenase accessory protein HypB, or nickelin, has two functions in the N2-fixing, H2-oxidizing bacterium Bradyrhizobium japonicum. One function of HypB involves the mobilization of nickel into hydrogenase. HypB also carries out a nickel storage/sequestering function in B. japonicum, binding nine nickel ions per monomer. Here we report that the two roles (nickel mobilization and storage) of HypB can be separated in vitro and in vivo using molecular and biochemical approaches. The role of HypB in hydrogenase maturation is completely dependent on its intrinsic GTPase activity; strains which produce a HypB protein that is severely deficient in GTPase activity but that fully retains nickel-sequestering ability cannot produce active hydrogenase even upon prolonged nickel supplementation. A HypB protein that lacks the nickel-binding polyhistidine region near the N terminus lacks only the nickel storage capacity function; it is still able to bind a single nickel ion and also retains complete GTPase activity.

scholarly journals Adenosine 5'-triphosphate--a new regulator of annexin function. A hypothesis.

1998 ◽  
Vol 45 (3) ◽  
pp. 735-744
Author(s):  
J Bandorowicz-Pikuła ◽  
S Pikuła
Keyword(s):  
Binding Motif ◽  
Dependent Manner ◽  
In Vitro Activity ◽  
Consensus Sequences ◽  
Calcium Dependent ◽  
Gtp Binding ◽  
Integral Proteins

The paradigm of annexins as phospholipid-binding proteins interacting with membranes in a calcium-dependent manner has been recently questioned in light of observations that some annexin isoforms may behave like membrane integral proteins or remain associated with their target membranes at low, resting, concentrations of Ca2+ in the cytoplasm. In addition, an evidence has been presented that some annexins (annexins I, VI and VII) bind in vitro ATP and GTP, and upon binding the nucleotide the in vitro activity of these proteins is modified. However, annexins do not contain Walker A and B consensus sequences for ATP/GTP binding. This review presents the hypothesis that a new ATP-binding motif exists within the annexin molecules and that ATP may play a role of functional ligand for annexins also in vivo.

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