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 CAK-independent Activation of CDK6 by a Viral Cyclin

2001 ◽  
Vol 12 (12) ◽  
pp. 3987-3999 ◽  
Author(s):  
Philipp Kaldis ◽  
Päivi M. Ojala ◽  
Lily Tong ◽  
Tomi P. Mäkelä ◽  
Mark J. Solomon
Keyword(s):  
Conformational Changes ◽  
Kaposi's Sarcoma ◽  
Cell Cycle Progression ◽  
Kaposi’S Sarcoma ◽  
Binding Assays ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Induced Apoptosis ◽  
Kaposi’S Sarcoma Associated Herpesvirus

In normal cells, activation of cyclin-dependent kinases (cdks) requires binding to a cyclin and phosphorylation by the cdk-activating kinase (CAK). The Kaposi's sarcoma-associated herpesvirus encodes a protein with similarity to D-type cyclins. This KSHV-cyclin activates CDK6, alters its substrate specificity, and renders CDK6 insensitive to inhibition by the cdk inhibitor p16INK4a. Here we investigate the regulation of the CDK6/KSHV-cyclin kinase with the use of purified proteins and a cell-based assay. We find that KSHV-cyclin can activate CDK6 independent of phosphorylation by CAK in vitro. In addition, CAK phosphorylation decreased the p16INK4asensitivity of CDK6/KSHV-cyclin complexes. In cells, expression of CDK6 or to a lesser degree of a nonphosphorylatable CDK6T177Atogether with KSHV-cyclin induced apoptosis, indicating that CDK6 activation by KSHV-cyclin can proceed in the absence of phosphorylation by CAK in vivo. Coexpression of p16 partially protected cells from cell death. p16 and KSHV-cyclin can form a ternary complex with CDK6 that can be detected by binding assays as well as by conformational changes in CDK6. The Kaposi's sarcoma-associated herpesvirus has adopted a clever strategy to render cell cycle progression independent of mitogenic signals, cdk inhibition, or phosphorylation by CAK.

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 Defining binding motifs and dynamics of the multi-pocket FERM domain from ezrin, radixin, moesin and merlin

2020 ◽  
Author(s):  
Muhammad Ali ◽  
Alisa Khramushin ◽  
Vikash K Yadav ◽  
Ora Schueler-Furman ◽  
Ylva Ivarsson
Keyword(s):  
Conformational Changes ◽  
In Silico ◽  
Cell Cortex ◽  
List Type ◽  
Binding Assays ◽  
Ferm Domain ◽  
Binding Motifs ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions

AbstractThe ERMs (ezrin, radixin and moesin) and the closely related merlin (NF2) participate in signaling events at the cell cortex through interactions mediated by their conserved FERM domain. We systematically investigated the FERM domain mediated interactions with short linear motifs (SLiMs) by screening the FERM domains againsts a phage peptidome representing intrinsically disordered regions of the human proteome. We uncovered a diverse set of interacting partners with similar but distinct binding motifs (FYDF, xYxV, FY(D/E)L and LQE(I/L) that bind to distinct binding pockets. We validated interactions between moesin and merlin FERM domains and full-length FAM83G, HIF1A, LATS1, NOP53, PAK6, RRBP1 and ZNF622 through pull-down experiments. Using biophysical binding assays, we determined affinities of, and uncovered allosteric interdependencies between, different binding partners, suggesting that the FERM domain acts as a switchable interaction hub. Using Rosetta FlexPepDock computational peptide docking protocols, we investigated the energy landscapes of identified interactions, which provide a detailed molecular understanding of the binding of the distinct binding motifs, as well as possible allosteric interconnections. This study demonstrates how experimental and computational approaches together can unravel a complex system of protein-peptide interactions that includes a family of proteins with multiple binding sites that interact with similar but distinct binding motifs.HighlightsWe screened the human disorderome for motif-containing partners of the FERM domainsWe expand the ERM and merlin interactomes of the ERMs and merlinWe identify four distinct motif classes that bind the ERM and merlin FERM domains: FYDF, xYxV, FY(D/E)L and LQE(I/L)In-vitro and in-silico data suggest that the FYDF motif binds to the F3a site and that xYxV motif binds to the F3b siteIn-silico modelling sheds light on the underlying conformational changes responsible for ligand interdependenciesAbstract Figure

scholarly journals Activities of Ceftobiprole and Other Cephalosporins against Extracellular and Intracellular (THP-1 Macrophages and Keratinocytes) Forms of Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus

2009 ◽  
Vol 53 (6) ◽  
pp. 2289-2297 ◽  
Author(s):  
Sandrine Lemaire ◽  
Youri Glupczynski ◽  
Valérie Duval ◽  
Bernard Joris ◽  
Paul M. Tulkens ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Conformational Changes ◽  
Acidic Ph ◽  
Binding Assays ◽  
Methicillin Resistant ◽  
Partial Restoration ◽  
Drug Concentrations ◽  
Mrsa Strains ◽  
Hospital Acquired

ABSTRACT Staphylococcus aureus is an opportunistic intracellular organism. Although they poorly accumulate in eukaryotic cells, β-lactams show activity against intracellular methicillin (meticillin)-susceptible S. aureus (MSSA) if the exposure times and the drug concentrations are sufficient. Intraphagocytic methicillin-resistant S. aureus (MRSA) strains are susceptible to penicillins and carbapenems because the acidic pH favors the acylation of PBP 2a by these β-lactams through pH-induced conformational changes. The intracellular activity (THP-1 macrophages and keratinocytes) of ceftobiprole, which shows almost similar in vitro activities against MRSA and MSSA in broth, was examined against a panel of hospital-acquired and community-acquired MRSA strains (MICs, 0.5 to 2.0 mg/liter at pH 7.4 and 0.25 to 1.0 mg/liter at pH 5.5) and was compared with its activity against MSSA isolates. The key pharmacological descriptors {relative maximal efficacy (E max), relative potency (the concentration causing a reduction of the inoculum halfway between E 0 and E max [EC50]), and static concentration (Cs )} were measured. All strains showed sigmoidal dose-responses, with E max being about a 1 log10 CFU decrease from the postphagocytosis inoculum, and EC50 and Cs being 0.2 to 0.3× and 0.6 to 0.9× the MIC, respectively. Ceftobiprole effectively competed with Bocillin FL (a fluorescent derivative of penicillin V) for binding to PBP 2a at both pH 5.5 and pH 7.4. In contrast, cephalexin, cefuroxime, cefoxitin, or ceftriaxone (i) were less potent in PBP 2a competitive binding assays, (ii) showed only partial restoration of the activity against MRSA in broth at acidic pH, and (iii) were collectively less effective against MRSA in THP-1 macrophages and were ineffective in keratinocytes. The improved activity of ceftobiprole toward intracellular MRSA compared with the activities of conventional cephalosporins can be explained, at least in part, by its greater ability to bind to PBP 2a not only at neutral but also at acidic pH.

scholarly journals Streptococcus pneumoniae Choline-Binding Protein E Interaction with Plasminogen/Plasmin Stimulates Migration across the Extracellular Matrix

2007 ◽  
Vol 76 (2) ◽  
pp. 466-476 ◽  
Author(s):  
Cécile Attali ◽  
Cécile Frolet ◽  
Claire Durmort ◽  
Julien Offant ◽  
Thierry Vernet ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Streptococcus Pneumoniae ◽  
Solid Phase ◽  
Binding Protein ◽  
Physiological Role ◽  
Site Directed Mutagenesis ◽  
Binding Assays ◽  
Solid Phase Binding ◽  
Vitro Model

ABSTRACT The virulence mechanisms leading Streptococcus pneumoniae to convert from nasopharyngeal colonization to a tissue-invasive phenotype are still largely unknown. Proliferation of infection requires penetration of the extracellular matrix, which occurs by recruitment of host proteases to the bacterial cell surface. We present evidence supporting the role of choline-binding protein E (CBPE) (a member of the surface-exposed choline-binding protein family) as an important receptor for human plasminogen, the precursor of plasmin. The results of ligand overlay blot analyses, solid-phase binding assays, and surface plasmon resonance experiments support the idea of an interaction between CBPE and plasminogen. We have shown that the phosphorylcholine esterase (Pce) domain of CBPE interacts with the plasminogen kringle domains. Analysis of the crystal structure of the Pce domain, followed by site-directed mutagenesis, allowed the identification of the plasminogen-binding region composed in part by lysine residues, some of which map in a linear fashion on the surface of the Pce domain. The biological relevance of the CBPE-plasminogen interaction is supported by the fact that, compared to the wild-type strain, a mutant of pneumococcus with the cbpE gene deleted (i) displays a reduced level of plasminogen binding and plasmin activation and (ii) shows reduced ability to cross the extracellular matrix in an in vitro model. These results support the idea of a physiological role for the CBPE-plasminogen interaction in pneumococcal dissemination into human tissue.

scholarly journals Substrate-linked Conformational Change in the Periplasmic Component of a Cu(I)/Ag(I) Efflux System

2007 ◽  
Vol 282 (49) ◽  
pp. 35695-35702 ◽  
Author(s):  
Ireena Bagai ◽  
Wenbo Liu ◽  
Christopher Rensing ◽  
Ninian J. Blackburn ◽  
Megan M. McEvoy
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Metal Binding ◽  
Critical Role ◽  
Titration Calorimetry ◽  
Efflux System ◽  
Compact Structure ◽  
Resistance Nodulation Division ◽  
Methionine Residues

Gram-negative bacteria utilize dual membrane resistance nodulation division-type efflux systems to export a variety of substrates. These systems contain an essential periplasmic component that is important for assembly of the protein complex. We show here that the periplasmic protein CusB from the Cus copper/silver efflux system has a critical role in Cu(I) and Ag(I) binding. Isothermal titration calorimetry experiments demonstrate that one Ag(I) ion is bound per CusB molecule with high affinity. X-ray absorption spectroscopy data indicate that the metal environment is an all-sulfur 3-coordinate environment. Candidates for the metal-coordinating residues were identified from sequence analysis, which showed four conserved methionine residues. Mutations of three of these methionine residues to isoleucine resulted in significant effects on CusB metal binding in vitro. Cells containing these CusB variants also show a decrease in their ability to grow on copper-containing plates, indicating an important functional role for metal binding by CusB. Gel filtration chromatography demonstrates that upon binding metal, CusB undergoes a conformational change to a more compact structure. Based on these structural and functional effects of metal binding, we propose that the periplasmic component of resistance nodulation division-type efflux systems plays an active role in export through substrate-linked conformational changes.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

2019 ◽  
Vol 476 (21) ◽  
pp. 3141-3159 ◽  
Author(s):  
Meiru Si ◽  
Can Chen ◽  
Zengfan Wei ◽  
Zhijin Gong ◽  
GuiZhi Li ◽  
...  
Keyword(s):  
Stress Response ◽  
Ligand Binding ◽  
Corynebacterium Glutamicum ◽  
Conformational Changes ◽  
Cysteine Oxidation ◽  
Transcriptional Regulatory ◽  
Ligand Free ◽  
Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

scholarly journals Novel Transcriptional Regulons for Autotrophic Cycle Genes in Crenarchaeota

2015 ◽  
Vol 197 (14) ◽  
pp. 2383-2391 ◽  
Author(s):  
Semen A. Leyn ◽  
Irina A. Rodionova ◽  
Xiaoqing Li ◽  
Dmitry A. Rodionov
Keyword(s):  
Carbon Dioxide ◽  
Transcriptional Regulation ◽  
Comparative Genomics ◽  
Dna Binding ◽  
Transcriptional Control ◽  
Binding Assays ◽  
Promoter Regions ◽  
Content Type ◽  
Genome Context

ABSTRACTAutotrophic microorganisms are able to utilize carbon dioxide as their only carbon source, or, alternatively, many of them can grow heterotrophically on organics. Different variants of autotrophic pathways have been identified in various lineages of the phylumCrenarchaeota. Aerobic members of the orderSulfolobalesutilize the hydroxypropionate-hydroxybutyrate cycle (HHC) to fix inorganic carbon, whereas anaerobicThermoprotealesuse the dicarboxylate-hydroxybutyrate cycle (DHC). Knowledge of transcriptional regulation of autotrophic pathways inArchaeais limited. We applied a comparative genomics approach to predict novel autotrophic regulons in theCrenarchaeota. We report identification of two novel DNA motifs associated with the autotrophic pathway genes in theSulfolobales(HHC box) andThermoproteales(DHC box). Based on genome context evidence, the HHC box regulon was attributed to a novel transcription factor from the TrmB family named HhcR. Orthologs of HhcR are present in allSulfolobalesgenomes but were not found in other lineages. A predicted HHC box regulatory motif was confirmed byin vitrobinding assays with the recombinant HhcR protein fromMetallosphaera yellowstonensis. For the DHC box regulon, we assigned a different potential regulator, named DhcR, which is restricted to the orderThermoproteales. DhcR inThermoproteus neutrophilus(Tneu_0751) was previously identified as a DNA-binding protein with high affinity for the promoter regions of two autotrophic operons. The global HhcR and DhcR regulons reconstructed by comparative genomics were reconciled with available omics data inMetallosphaeraandThermoproteusspp. The identified regulons constitute two novel mechanisms for transcriptional control of autotrophic pathways in theCrenarchaeota.IMPORTANCELittle is known about transcriptional regulation of carbon dioxide fixation pathways inArchaea. We previously applied the comparative genomics approach for reconstruction of DtxR family regulons in diverse lineages ofArchaea. Here, we utilize similar computational approaches to identify novel regulatory motifs for genes that are autotrophically induced in microorganisms from two lineages ofCrenarchaeotaand to reconstruct the respective regulons. The predicted novel regulons in archaeal genomes control the majority of autotrophic pathway genes and also other carbon and energy metabolism genes. The HhcR regulon was experimentally validated by DNA-binding assays inMetallosphaeraspp. Novel regulons described for the first time in this work provide a basis for understanding the mechanisms of transcriptional regulation of autotrophic pathways inArchaea.

Ability of PknA, a mycobacterial eukaryotic-type serine/threonine kinase, to transphosphorylate MurD, a ligase involved in the process of peptidoglycan biosynthesis

2008 ◽  
Vol 415 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Meghna Thakur ◽  
Pradip K. Chakraborti
Keyword(s):  
Protein Kinases ◽  
Solid Phase ◽  
Morphological Changes ◽  
Binding Assays ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Peptidoglycan Biosynthesis ◽  
Vitro Binding ◽  
Solid Phase Binding

Eukaryotic-type serine/threonine protein kinases in bacteria have been implicated in controlling a host of cellular activities. PknA is one of eleven such protein kinases from Mycobacterium tuberculosis which regulates morphological changes associated with cell division. In the present study we provide the evidence for the ability of PknA to transphosphorylate mMurD (mycobacterial UDP-N-acetylmuramoyl-L-alanine:D-glutamate-ligase), the enzyme involved in peptidoglycan biosynthesis. Its co-expression in Escherichia coli along with PknA resulted in phosphorylation of mMurD. Consistent with these observations, results of the solid-phase binding assays revealed a high-affinity in vitro binding between the two proteins. Furthermore, overexpression of m-murD in Mycobacterium smegmatis yielded a phosphorylated protein. The results of the present study therefore point towards the possibility of mMurD being a substrate of PknA.

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