Structure and Cu(I)-binding properties of the N-terminal soluble domains of Bacillus subtilis CopA

2008 ◽  
Vol 411 (3) ◽  
pp. 571-579 ◽  
Author(s):  
Chloe Singleton ◽  
Lucia Banci ◽  
Simone Ciofi-Baffoni ◽  
Leonardo Tenori ◽  
Margaret A. Kihlken ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Solution Structure ◽  
Gel Filtration ◽  
Protein Molecule ◽  
Binding Motif ◽  
Stable Form ◽  
Binding Motifs ◽  
Shift Analysis ◽  
P Type ◽  
Equilibrium Centrifugation

CopA, a P-type ATPase from Bacillus subtilis, plays a major role in the resistance of the cell to copper by effecting the export of the metal across the cytoplasmic membrane. The N-terminus of the protein features two soluble domains (a and b), that each contain a Cu(I)-binding motif, MTCAAC. We have generated a stable form of the wild-type two-domain protein, CopAab, and determined its solution structure. This was found to be similar to that reported previously for a higher stability S46V variant, with minor differences mostly confined to the Ser46-containing β3-strand of domain a. Chemical-shift analysis demonstrated that the two Cu(I)-binding motifs, located at different ends of the protein molecule, are both able to participate in Cu(I) binding and that Cu(I) is in rapid exchange between protein molecules. Surprisingly, UV–visible and fluorescence spectroscopy indicate very different modes of Cu(I) binding below and above a level of 1 Cu(I) per protein, consistent with a major structural change occurring above 1 Cu(I) per CopAab. Analytical equilibrium centrifugation and gel filtration results show that this is a result of Cu(I)-mediated dimerization of the protein. The resulting species is highly luminescent, indicating the presence of a solvent-shielded Cu(I) cluster.

Solution structure of the N-terminal domain of a potential copper-translocating P-type ATPase from Bacillus subtilis in the apo and Cu(I) loaded states

2002 ◽  
Vol 317 (3) ◽  
pp. 415-429 ◽  
Author(s):  
Lucia Banci ◽  
Ivano Bertini ◽  
Simone Ciofi-Baffoni ◽  
Mariapina D’Onofrio ◽  
Leonardo Gonnelli ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Solution Structure ◽  
Terminal Domain ◽  
P Type

scholarly journals CodY-Mediated Regulation of Guanosine Uptake in Bacillus subtilis

2011 ◽  
Vol 193 (22) ◽  
pp. 6276-6287 ◽  
Author(s):  
Boris R. Belitsky ◽  
Abraham L. Sonenshein
Keyword(s):  
Bacillus Subtilis ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Binding Motif ◽  
Downstream Site ◽  
Upstream Site ◽  
Binding Motifs ◽  
Content Type ◽  
A Minor

CodY is a global transcriptional regulator known to control expression of more than 100 genes and operons inBacillus subtilis. Some of the most strongly repressed targets of CodY, thenupNOPQ(formerly,yufNOPQ) genes, were found to encode a guanosine transporter. Using DNase I footprinting experiments, we identified two high-affinity CodY-binding sites in the regulatory region of thenupNgene. The two sites are located 50 bp upstream and 163 bp downstream of the transcription start site. The downstream site was responsible for 6- to 8-foldnupNrepression in the absence of the upstream site. When the upstream site was intact, however, only a minor contribution of the downstream site tonupNregulation could be detected under the conditions tested. Both sites contained 15-bp CodY-binding motifs with two mismatches each with respect to the consensus sequence, AATTTTCWGTTTTAA. However, the experimentally determined binding sites included additional sequences flanking the 15-bp CodY-binding motifs. An additional version of the 15-bp CodY-binding motif, with 5 mismatches with respect to the consensus but essential for efficient regulation by CodY, was found within the upstream site. The presence of multiple 15-bp motifs may be a common feature of CodY-binding sites.

A DENSITY FUNCTIONAL STUDY OF THE STRUCTURE OF SILANE COUPLING AGENT 3–AMINOPROPYLTRIETHOXYSILANE

2005 ◽  
Vol 04 (01) ◽  
pp. 117-126
Author(s):  
N. L. MA ◽  
P. WU
Keyword(s):  
Coupling Agent ◽  
Density Functional ◽  
Solution Structure ◽  
Silane Coupling Agent ◽  
Industrial Applications ◽  
Functional Study ◽  
Stable Form ◽  
Functional Theory ◽  
Silane Coupling ◽  
The Stability

Using density functional theory, we predicted the solution structure of the hydrolyzed 3–aminopropyltriethoxysilane (h–APS), which is a silane coupling agent commonly used in many industrial applications. We have located five stable minima on the potential energy surface of h–APS in which four of them are "neutral", and the remaining one is zwitterionic (dipolar) in nature. Our calculations suggested that the stability of the most stable form of h–APS in water (denoted as II_N) arose from strong intramolecular OH ⋯ N hydrogen bond. The least stable form is the zwitterionic form (I_ZW), which is estimated to be over 90 kJ mol -1 less stable than II_N. The factors governing the relative stabilities of different forms are discussed.

scholarly journals Structural and Functional Insights into Peptidoglycan Access for the Lytic Amidase LytA of Streptococcus pneumoniae

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Peter Mellroth ◽  
Tatyana Sandalova ◽  
Alexey Kikhney ◽  
Francisco Vilaplana ◽  
Dusan Hesek ◽  
...  
Keyword(s):  
Cell Wall ◽  
Streptococcus Pneumoniae ◽  
Solution Structure ◽  
Substrate Binding ◽  
Substrate Recognition ◽  
Lytic Activity ◽  
Site Directed Mutagenesis ◽  
Binding Motif ◽  
Content Type ◽  
Peptidoglycan Synthesis

ABSTRACT The cytosolic N-acetylmuramoyl-l-alanine amidase LytA protein of Streptococcus pneumoniae, which is released by bacterial lysis, associates with the cell wall via its choline-binding motif. During exponential growth, LytA accesses its peptidoglycan substrate to cause lysis only when nascent peptidoglycan synthesis is stalled by nutrient starvation or β-lactam antibiotics. Here we present three-dimensional structures of LytA and establish the requirements for substrate binding and catalytic activity. The solution structure of the full-length LytA dimer reveals a peculiar fold, with the choline-binding domains forming a rigid V-shaped scaffold and the relatively more flexible amidase domains attached in a trans position. The 1.05-Å crystal structure of the amidase domain reveals a prominent Y-shaped binding crevice composed of three contiguous subregions, with a zinc-containing active site localized at the bottom of the branch point. Site-directed mutagenesis was employed to identify catalytic residues and to investigate the relative impact of potential substrate-interacting residues lining the binding crevice for the lytic activity of LytA. In vitro activity assays using defined muropeptide substrates reveal that LytA utilizes a large substrate recognition interface and requires large muropeptide substrates with several connected saccharides that interact with all subregions of the binding crevice for catalysis. We hypothesize that the substrate requirements restrict LytA to the sites on the cell wall where nascent peptidoglycan synthesis occurs. IMPORTANCE Streptococcus pneumoniae is a human respiratory tract pathogen responsible for millions of deaths annually. Its major pneumococcal autolysin, LytA, is required for autolysis and fratricidal lysis and functions as a virulence factor that facilitates the spread of toxins and factors involved in immune evasion. LytA is also activated by penicillin and vancomycin and is responsible for the lysis induced by these antibiotics. The factors that regulate the lytic activity of LytA are unclear, but it was recently demonstrated that control is at the level of substrate recognition and that LytA required access to the nascent peptidoglycan. The present study was undertaken to structurally and functionally investigate LytA and its substrate-interacting interface and to determine the requirements for substrate recognition and catalysis. Our results reveal that the amidase domain comprises a complex substrate-binding crevice and needs to interact with a large-motif epitope of peptidoglycan for catalysis.

Tyrosine-protein kinase Src regulates Kv1.5 channel activity and membrane expression through interaction with the N-terminus of the channel

2020 ◽  
Author(s):  
Taylore Dodd ◽  
Tingzhong Wang ◽  
Shetuan Zhang
Keyword(s):  
Protein Kinase ◽  
Cell Voltage ◽  
Underlying Mechanism ◽  
Delayed Rectifier ◽  
Binding Motif ◽  
Membrane Expression ◽  
Binding Motifs ◽  
Delayed Rectifier Potassium Current ◽  
N Terminus ◽  
Tyrosine Protein Kinase

Kv1.5 is a voltage-gated potassium channel that generates the ultra-rapid delayed rectifier potassium current (IKur) important in the repolarization of the atrial action potential. Malfunction of the Kv1.5 channel often results in atrial fibrillation (AFib). A reduction in Kv1.5 current (IKv1.5) occurs upon activation of the endogenous tyrosine-protein kinase Src. The Src SH3 domain binds to proline-rich motifs located within the N-terminus of Kv1.5. Disruption of these binding motifs has been involved in the development of familial AFib. The mechanism underlying the reduction of IKv1.5 upon Src activation has not yet been established and the relationship between Kv1.5 and Src is poorly understood. Therefore, the present study aims to further elucidate the mechanism behind IKv1.5  reduction. The hypothesis that Src regulates Kv1.5 activity by altering the density of mature membrane-localized channels was tested using whole-cell voltage clamp and Western blot analysis. We demonstrate that Src tonically inhibits Kv1.5 activity and decreases the density of mature membrane-localized channels. Kv1.5 channels possessing mutations within the Src binding motifs were also investigated and it was determined that each binding motif contributes to the Kv1.5-Src relationship, however, the binding of Src to an individual motif is sufficiently effective. Our findings indicate that Src regulates Kv1.5 through an interaction with the N-terminal binding motifs and suggests that the inhibition of forward trafficking may be involved in the underlying mechanism. (Supported by the Heart and Stroke foundation of Canada and The Canadian Institutes of Health Research).

Crotalin, a vWF and GP Ib Cleaving Metalloproteinase from Venom of Crotalus atrox

2001 ◽  
Vol 86 (12) ◽  
pp. 1501-1511 ◽  
Author(s):  
Wen-Bin Wu ◽  
Hui-Chin Peng ◽  
Tur-Fu Huang
Keyword(s):  
Platelet Adhesion ◽  
Early Stage ◽  
Gel Filtration ◽  
Platelet Glycoprotein ◽  
Antithrombotic Effect ◽  
Binding Motifs ◽  
Sds Page ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryBinding of von Willebrand factor (vWF) to a variety of extracellular matrix (ECM) components and to platelet glycoprotein (GP) Ib-IX-V complex is important in mediating platelet adhesion and aggregation in the early stage of hemostasis. We previously purified a potent antithrombotic protein, named crotalin, functionally acting as a GP Ib antagonist (1). In this study, we further characterized crotalin as a P-I metalloproteinase with a molecular mass of 25 kDa as determined by gel filtration and two-dimensional SDS-PAGE. Crotalin is a vWF binding and cleaving metalloproteinase. In addition, crotalin cleaved platelet GP Ib as judged by flow cytometry and Western blotting. The multiple effects of crotalin on vWF and platelet GP Ib antagonized ristocetin-, but not collagen and thrombin-induced platelet aggregation, suggesting that its effect is specific. We also found that crotalin auto-proteolytically degraded to ~14 and ~10 kDa fragments in the presence of SDS. Interestingly, both degradation fragments, intact and reduced crotalin were able to bind vWF, suggesting the binding of crotalin to vWF is conformation-independent. In conclusion, the results presented further explain the potent antithrombotic effect of crotalin in vivo. In addition, the multiple effects of crotalin may be used as a tool to determine the binding motifs that are responsible for the vWF-ECMs or vWF-GP Ib interaction.

scholarly journals Tubulin-Dependent Transport of Connexin-36 Potentiates the Size and Strength of Electrical Synapses

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1146 ◽  
Author(s):  
Brown ◽  
del Corsso ◽  
Zoidl ◽  
Donaldson ◽  
Spray ◽  
...  
Keyword(s):  
Protein Transport ◽  
Mutational Analysis ◽  
Binding Motif ◽  
Dependent Manner ◽  
Electrical Synapses ◽  
Binding Motifs ◽  
Connexin 36 ◽  
Dependent Transport ◽  
Carboxy Terminal

Connexin-36 (Cx36) electrical synapses strengthen transmission in a calcium/calmodulin (CaM)/calmodulin-dependent kinase II (CaMKII)-dependent manner similar to a mechanism whereby the N-methyl-D-aspartate (NMDA) receptor subunit NR2B facilitates chemical transmission. Since NR2B–microtubule interactions recruit receptors to the cell membrane during plasticity, we hypothesized an analogous modality for Cx36. We determined that Cx36 binding to tubulin at the carboxy-terminal domain was distinct from Cx43 and NR2B by binding a motif overlapping with the CaM and CaMKII binding motifs. Dual patch-clamp recordings demonstrated that pharmacological interference of the cytoskeleton and deleting the binding motif at the Cx36 carboxyl-terminal (CT) reversibly abolished Cx36 plasticity. Mechanistic details of trafficking to the gap-junction plaque (GJP) were probed pharmacologically and through mutational analysis, all of which affected GJP size and formation between cell pairs. Lys279, Ile280, and Lys281 positions were particularly critical. This study demonstrates that tubulin-dependent transport of Cx36 potentiates synaptic strength by delivering channels to GJPs, reinforcing the role of protein transport at chemical and electrical synapses to fine-tune communication between neurons.

scholarly journals Chromatography of transforming deoxyribonucleic acid on methylated albumin and by gel filtration

1974 ◽  
Vol 137 (3) ◽  
pp. 543-546
Author(s):  
G. R. Barker ◽  
P. Hodges
Keyword(s):  
Molecular Weight ◽  
Bacillus Subtilis ◽  
High Molecular Weight ◽  
Deoxyribonucleic Acid ◽  
Degradation Products ◽  
Gel Filtration ◽  
Agarose Gel ◽  
Transforming Activity ◽  
Different Strains ◽  
Two Peaks

1. Native DNA from two strains of Bacillus subtilis was chromatographed by stepwise elution from MAK (methylated albumin on kieselguhr). 2. Transforming activity was confined to two out of the three main fractions, activity being distributed between the two peaks differently for DNA from the different strains. 3. Fractionation of DNA from both strains on 2% agarose gel gave two components. Approx. 75% of the material was eluted within the void volume of the column. Approx. 25% of the material consisted of degradation products of lower molecular weight. 4. Chromatography on MAK of the material of high molecular weight eluted from agarose gel gave a number of peaks differing in molecular weight, indicating that degradation of the DNA takes place during chromatography on MAK. 5. The distribution of transforming activity among the fractions from MAK suggests that degradation occurs preferentially in certain regions of the DNA.

scholarly journals Solution Structure of the Carbon Storage Regulator Protein CsrA from Escherichia coli

2005 ◽  
Vol 187 (10) ◽  
pp. 3496-3501 ◽  
Author(s):  
Pablo Gutiérrez ◽  
Yan Li ◽  
Michael J. Osborne ◽  
Ekaterina Pomerantseva ◽  
Qian Liu ◽  
...  
Keyword(s):  
Carbon Storage ◽  
Solution Structure ◽  
Rna Binding ◽  
Binding Motif ◽  
Binding Properties ◽  
Nmr Titration ◽  
C Terminus ◽  
Regulator Protein ◽  
Carbon Storage Regulator ◽  
Α Helix

ABSTRACT The carbon storage regulator A (CsrA) is a protein responsible for the repression of a variety of stationary-phase genes in bacteria. In this work, we describe the nuclear magnetic resonance (NMR)-based structure of the CsrA dimer and its RNA-binding properties. CsrA is a dimer of two identical subunits, each composed of five strands, a small α-helix and a flexible C terminus. NMR titration experiments suggest that the β1-β2 and β3-β4 loops and the C-terminal helix are important elements in RNA binding. Even though the β3-β4 loop contains a highly conserved RNA-binding motif, GxxG, typical of KH domains, our structure excludes CsrA from being a member of this protein family, as previously suggested. A mechanism for the recognition of mRNAs downregulated by CsrA is proposed.

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