Structural Basis of the Sulphate Starvation Response in E. coli: Crystal Structure and Mutational Analysis of the Cofactor-binding Domain of the Cbl Transcriptional Regulator

VqsR is a quorum-sensing (QS) transcriptional regulator which controls QS systems (las,rhlandpqs) by directly downregulating the expression ofqscRinPseudomonas aeruginosa. As a member of the LuxR family of proteins, VqsR shares the common motif of a helix–turn–helix (HTH)-type DNA-binding domain at the C-terminus, while the function of its N-terminal domain remains obscure. Here, the crystal structure of the N-terminal domain of VqsR (VqsR-N; residues 1–193) was determined at a resolution of 2.1 Å. The structure is folded into a regular α–β–α sandwich topology, which is similar to the ligand-binding domain (LBD) of the LuxR-type QS receptors. Although their sequence similarity is very low, structural comparison reveals that VqsR-N has a conserved enclosed cavity which could recognize acyl-homoserine lactones (AHLs) as in other LuxR-type AHL receptors. The structure suggests that VqsR could be a potential AHL receptor.

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An enzyme captured in two conformational states: crystal structure ofS-adenosyl-L-homocysteine hydrolase fromBradyrhizobium elkanii

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004715018659 ◽

2015 ◽

Vol 71 (12) ◽

pp. 2422-2432 ◽

Cited By ~ 6

Author(s):

Tomasz Manszewski ◽

Kriti Singh ◽

Barbara Imiolczyk ◽

Mariusz Jaskolski

Keyword(s):

Crystal Structure ◽

Enzymatic Reaction ◽

Binding Domain ◽

Dimerization Domain ◽

Biologically Relevant ◽

Cofactor Binding ◽

Enzymatic Regulation ◽

Ligand Free ◽

Substrate Binding Domain ◽

Methyl Group Transfer

S-Adenosyl-L-homocysteine hydrolase (SAHase) is involved in the enzymatic regulation ofS-adenosyl-L-methionine (SAM)-dependent methylation reactions. After methyl-group transfer from SAM,S-adenosyl-L-homocysteine (SAH) is formed as a byproduct, which in turn is hydrolyzed to adenosine (Ado) and homocysteine (Hcy) by SAHase. The crystal structure of BeSAHase, an SAHase fromBradyrhizobium elkanii, which is a nitrogen-fixing bacterial symbiont of legume plants, was determined at 1.7 Å resolution, showing the domain organization (substrate-binding domain, NAD+cofactor-binding domain and dimerization domain) of the subunits. The protein crystallized in its biologically relevant tetrameric form, with three subunits in a closed conformation enforced by complex formation with the Ado product of the enzymatic reaction. The fourth subunit is ligand-free and has an open conformation. The BeSAHase structure therefore provides a unique snapshot of the domain movement of the enzyme induced by the binding of its natural ligands.

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Crystal Structure of the Sugar Binding Domain of the Archaeal Transcriptional Regulator TrmB

Journal of Biological Chemistry ◽

10.1074/jbc.m512809200 ◽

2006 ◽

Vol 281 (16) ◽

pp. 10976-10982 ◽

Cited By ~ 25

Author(s):

Michael Krug ◽

Sung-Jae Lee ◽

Kay Diederichs ◽

Winfried Boos ◽

Wolfram Welte

Keyword(s):

Crystal Structure ◽

Transcriptional Regulator ◽

Binding Domain ◽

Sugar Binding

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Mutations in the conserved C-terminal sequence in thyroid hormone receptor dissociate hormone-dependent activation from interference with AP-1 activity.

Molecular and Cellular Biology ◽

10.1128/mcb.17.8.4687 ◽

1997 ◽

Vol 17 (8) ◽

pp. 4687-4695 ◽

Cited By ~ 25

Author(s):

F Saatcioglu ◽

G Lopez ◽

B L West ◽

E Zandi ◽

W Feng ◽

...

Keyword(s):

Crystal Structure ◽

Thyroid Hormone ◽

Transcriptional Activation ◽

Hormone Receptor ◽

Thyroid Hormone Receptor ◽

Mutational Analysis ◽

Binding Domain ◽

Yeast Cells ◽

Terminal Sequence

A short C-terminal sequence that is deleted in the v-ErbA oncoprotein and conserved in members of the nuclear receptor superfamily is required for normal biological function of its normal cellular counterpart, the thyroid hormone receptor alpha (T3R alpha). We carried out an extensive mutational analysis of this region based on the crystal structure of the hormone-bound ligand binding domain of T3R alpha. Mutagenesis of Leu398 or Glu401, which are surface exposed according to the crystal structure, completely blocks or significantly impairs T3-dependent transcriptional activation but does not affect or only partially diminishes interference with AP-1 activity. These are the first mutations that clearly dissociate these activities for T3R alpha. Substitution of Leu400, which is also surface exposed, does not affect interference with AP-1 activity and only partially diminishes T3-dependent transactivation. None of the mutations affect ligand-independent transactivation, consistent with previous findings that this activity is mediated by the N-terminal domain of T3R alpha. The loss of ligand-dependent transactivation for some mutants can largely be reversed in the presence of GRIP1, which acts as a strong ligand-dependent coactivator for wild-type T3R alpha. There is excellent correlation between T3-dependent in vitro association of GRIP1 with T3R alpha mutants and their ability to support T3-dependent transcriptional activation. Therefore, GRIP1, previously found to interact with the glucocorticoid, estrogen, and androgen receptors, may also have a role in T3R alpha-mediated ligand-dependent transcriptional activation. When fused to a heterologous DNA binding domain, that of the yeast transactivator GAL4, the conserved C terminus of T3R alpha functions as a strong ligand-independent activator in both mammalian and yeast cells. However, point mutations within this region have drastically different effects on these activities compared to their effect on the full-length T3R alpha. We conclude that the C-terminal conserved region contains a recognition surface for GRIP1 or a similar coactivator that facilitates its interaction with the basal transcriptional apparatus. While important for ligand-dependent transactivation, this interaction surface is not directly involved in transrepression of AP-1 activity.

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Crystal structure of a chimaeric bacterial glutamate dehydrogenase

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x16007305 ◽

2016 ◽

Vol 72 (6) ◽

pp. 462-466 ◽

Cited By ~ 1

Author(s):

Tânia Oliveira ◽

Michael A. Sharkey ◽

Paul C. Engel ◽

Amir R. Khan

Keyword(s):

Crystal Structure ◽

Glutamate Dehydrogenase ◽

Rigid Bodies ◽

Nucleotide Binding Domain ◽

Oxidative Deamination ◽

E Coli ◽

Cofactor Binding ◽

Signature Sequences ◽

Clostridium Symbiosum ◽

Domain I

Glutamate dehydrogenases (EC 1.4.1.2–4) catalyse the oxidative deamination of L-glutamate to α-ketoglutarate using NAD(P)+as a cofactor. The bacterial enzymes are hexameric, arranged with 32 symmetry, and each polypeptide consists of an N-terminal substrate-binding segment (domain I) followed by a C-terminal cofactor-binding segment (domain II). The catalytic reaction takes place in the cleft formed at the junction of the two domains. Distinct signature sequences in the nucleotide-binding domain have been linked to the binding of NAD+versusNADP+, but they are not unambiguous predictors of cofactor preference. In the absence of substrate, the two domains move apart as rigid bodies, as shown by the apo structure of glutamate dehydrogenase fromClostridium symbiosum. Here, the crystal structure of a chimaeric clostridial/Escherichia colienzyme has been determined in the apo state. The enzyme is fully functional and reveals possible determinants of interdomain flexibility at a hinge region following the pivot helix. The enzyme retains the preference for NADP+cofactor from the parentE. colidomain II, although there are subtle differences in catalytic activity.

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Structural basis of hypoxic gene regulation by the Rv0081 transcription factor ofMycobacterium tuberculosis

10.1101/465575 ◽

2018 ◽

Author(s):

Ashwani Kumar ◽

Swastik Phulera ◽

Arshad Rizvi ◽

Parshuram Sonawane ◽

Hemendra Singh Panwar ◽

...

Keyword(s):

Gene Expression ◽

Crystal Structure ◽

Gene Regulation ◽

Transcription Factor ◽

Dna Binding ◽

Metal Binding ◽

Transcriptional Regulator ◽

Tuberculosis Infection ◽

Structural Basis ◽

Latent Phase

ABSTRACTThe transcription factor Rv0081 ofM. tuberculosiscontrols the hypoxic gene expression and acts as a regulatory hub in the latent phase of tuberculosis infection. We report here the crystal structure of Rv0081 at 3.3 Å resolution revealing that it belongs to the well-known ArsR/SmtB family proteins. ArsR/SmtB family transcriptional repressors exert gene regulation by reversible metal binding. Hypoxia in general is sensed by bacterial transcriptional regulators via metals or Cys-mediated thiol switches. Oxygen sensing typically leads to transcriptional repressor changing its conformational state with altered DNA-binding property under different oxygen levels. Surprisingly Rv0081 neither has a metal binding domain nor does it possess Cys residues suggesting an alternate mechanism of gene regulation. Our structural analysis identified Ser 48, Ser 49, Ser 52 and Gln 53 as potential residues of Rv0081 involved in DNA binding. We probed DNA-binding of Rv0081 with electrophoretic mobility shift assay (EMSA) as well as surface plasmon resonance (SPR), where the Alanine mutants of these residues showed diminished DNA binding. Similarly, Aspartate mutants of these Ser residues was shown to fail to bind to DNA. Since, phosphorylation of various regulatory proteins is one of the important controlling mechanisms, we expected the role of Ser-phosphorylation of Rv0081 in hypoxic condition. Probing Rv0081 with anti-phosphoserine antibodies inM. tuberculosiscell lysate showed marked enhancement in the phosphorylation of Rv0081 protein under hypoxia. Overall, our structural and biochemical analysis provides the molecular basis for the regulation of Rv0081 in the latent phase of tuberculosis infection.IMPORTANCETuberculosis is one of the deadliest infectious diseases caused by the bacteriumMycobacterium tuberculosis. In about 90% of the infected people,M. tuberculosisexists in a dormant or a latent stage which can be reactivated in favorable conditions. Hypoxia (low oxygen pressure) is one of causes of dormancy. Understanding hypoxic gene regulation inM. tuberculosisis therefore an important step to understand latency. Rv0081 is a transcriptional regulator of genes expressed during hypoxia. In order to understand the mechanism by which Rv00081 regulates gene expression during hypoxia, we have solved the crystal structure of Rv0081 and identified amino acid residues which are critical in its transcriptional regulator activity. The crystal structure is suggestive of mechanism of gene regulation under hypoxia.

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The complex of outer-arm dynein light chain-1 and the microtubule-binding domain of the γ heavy chain shows how axonemal dynein tunes ciliary beating

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011541 ◽

2020 ◽

Vol 295 (12) ◽

pp. 3982-3989 ◽

Cited By ~ 1

Author(s):

Akiyuki Toda ◽

Yosuke Nishikawa ◽

Hideaki Tanaka ◽

Toshiki Yagi ◽

Genji Kurisu

Keyword(s):

Binding Affinity ◽

Light Chain ◽

Mutational Analysis ◽

Molecular Model ◽

Binding Domain ◽

Structural Basis ◽

Dynein Light Chain ◽

Microtubule Binding ◽

Microtubule Binding Domain ◽

Axonemal Dynein

Axonemal dynein is a microtubule-based molecular motor that drives ciliary/flagellar beating in eukaryotes. In axonemal dynein, the outer-arm dynein (OAD) complex, which comprises three heavy chains (α, β, and γ), produces the main driving force for ciliary/flagellar motility. It has recently been shown that axonemal dynein light chain-1 (LC1) binds to the microtubule-binding domain (MTBD) of OADγ, leading to a decrease in its microtubule-binding affinity. However, it remains unclear how LC1 interacts with the MTBD and controls the microtubule-binding affinity of OADγ. Here, we have used X-ray crystallography and pulldown assays to examine the interaction between LC1 and the MTBD, identifying two important sites of interaction in the MTBD. Solving the LC1-MTBD complex from Chlamydomonas reinhardtii at 1.7 Å resolution, we observed that one site is located in the H5 helix and that the other is located in the flap region that is unique to some axonemal dynein MTBDs. Mutational analysis of key residues in these sites indicated that the H5 helix is the main LC1-binding site. We modeled the ternary structure of the LC1-MTBD complex bound to microtubules based on the known dynein-microtubule complex. This enabled us to propose a structural basis for both formations of the ternary LC1-MTBD-microtubule complex and LC1-mediated tuning of MTBD binding to the microtubule, suggesting a molecular model for how axonemal dynein senses the curvature of the axoneme and tunes ciliary/flagellar beating.

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