Structural and Mutational Analysis of the PhoQ Histidine Kinase Catalytic Domain

ABSTRACT The Bacillus anthracis BA2291 gene codes for a sensor histidine kinase involved in the induction of sporulation. Genes for orthologs of the sensor domain of the BA2291 kinase exist in virulence plasmids in this organism, and these proteins, when expressed, inhibit sporulation by converting BA2291 to an apparent phosphatase of the sporulation phosphorelay. Evidence suggests that the sensor domains inhibit BA2291 by titrating its activating signal ligand. Studies with purified BA2291 revealed that this kinase is uniquely specific for GTP in the forward reaction and GDP in the reverse reaction. The G1 motif of BA2291 is highly modified from ATP-specific histidine kinases, and modeling this motif in the structure of the kinase catalytic domain suggested how guanine binds to the region. A mutation in the putative coiled-coil linker between the sensor domain and the catalytic domains was found to decrease the rate of the forward autophosphorylation reaction and not affect the reverse reaction from phosphorylated Spo0F. The results suggest that the activating ligand for BA2291 is a critical signal for sporulation and in a limited concentration in the cell. Decreasing the response to it either by slowing the forward reaction through mutation or by titration of the ligand by expressing the plasmid-encoded sensor domains switches BA2291 from an inducer to an inhibitor of the phosphorelay and sporulation.

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Phosphatase activity of histidine kinase EnvZ without kinase catalytic domain

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.97.14.7808 ◽

2000 ◽

Vol 97 (14) ◽

pp. 7808-7813 ◽

Cited By ~ 89

Author(s):

Y. Zhu ◽

L. Qin ◽

T. Yoshida ◽

M. Inouye

Keyword(s):

Phosphatase Activity ◽

Histidine Kinase ◽

Catalytic Domain ◽

Kinase Catalytic Domain

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Interhelical H-Bonds Modulate the Activity of a Polytopic Transmembrane Kinase

Biomolecules ◽

10.3390/biom11070938 ◽

2021 ◽

Vol 11 (7) ◽

pp. 938

Author(s):

Juan Cruz Almada ◽

Ana Bortolotti ◽

Jean Marie Ruysschaert ◽

Diego de Mendoza ◽

María Eugenia Inda ◽

...

Keyword(s):

Histidine Kinase ◽

Adaptive Response ◽

Catalytic Domain ◽

Membrane Lipids ◽

Signal Transmission ◽

Expression System ◽

Lipid Homeostasis ◽

Transmembrane Helices ◽

Transmembrane Region

DesK is a Histidine Kinase that allows Bacillus subtilis to maintain lipid homeostasis in response to changes in the environment. It is located in the membrane, and has five transmembrane helices and a cytoplasmic catalytic domain. The transmembrane region triggers the phosphorylation of the catalytic domain as soon as the membrane lipids rigidify. In this research, we study how transmembrane inter-helical interactions contribute to signal transmission; we designed a co-expression system that allows studying in vivo interactions between transmembrane helices. By Alanine-replacements, we identified a group of polar uncharged residues, whose side chains contain hydrogen-bond donors or acceptors, which are required for the interaction with other DesK transmembrane helices; a particular array of H-bond- residues plays a key role in signaling, transmitting information detected at the membrane level into the cell to finally trigger an adaptive response.

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Mutational analysis of a ras catalytic domain

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2646-2654.1986 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2646-2654

Author(s):

B M Willumsen ◽

A G Papageorge ◽

H F Kung ◽

E Bekesi ◽

T Robins ◽

...

Keyword(s):

Catalytic Domain ◽

Mutational Analysis ◽

Nucleotide Binding ◽

Membrane Localization ◽

Wide Range ◽

Murine Sarcoma Virus ◽

Sarcoma Virus ◽

Transforming Activity ◽

Type V

We used linker insertion-deletion mutagenesis to study the catalytic domain of the Harvey murine sarcoma virus v-rasH transforming protein, which is closely related to the cellular rasH protein. The mutants displayed a wide range of in vitro biological activity, from those that induced focal transformation of NIH 3T3 cells with approximately the same efficiency as the wild-type v-rasH gene to those that failed to induce any detectable morphologic changes. Correlation of transforming activity with the location of the mutations enabled us to identify three nonoverlapping segments within the catalytic domain that were dispensable for transformation and six other segments that were required for transformation. Segments that were necessary for guanosine nucleotide (GDP) binding corresponded to three of the segments that were essential for transformation; two of the three segments share strong sequence homology with other purine nucleotide-binding proteins. Loss of GDP binding was associated with apparent instability of the protein. Lesions in two of the three other required regions significantly reduced GDP binding, while small lesions in the last required region did not impair GDP binding or membrane localization. We speculate that this latter region interacts with the putative cellular target of ras. The results suggest that transforming ras proteins require membrane localization, guanosine nucleotide binding, and an additional undefined function that may represent interaction with their target.

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Insights into the regulation of eukaryotic elongation factor 2 kinase and the interplay between its domains

Biochemical Journal ◽

10.1042/bj20111536 ◽

2012 ◽

Vol 442 (1) ◽

pp. 105-118 ◽

Cited By ~ 30

Author(s):

Craig R. Pigott ◽

Halina Mikolajek ◽

Claire E. Moore ◽

Stephen J. Finn ◽

Curtis W. Phippen ◽

...

Keyword(s):

Protein Kinase ◽

Catalytic Domain ◽

Functional Organization ◽

Elongation Factor ◽

Kinase Domain ◽

Conserved Residues ◽

Elongation Factor 2 ◽

Eukaryotic Elongation Factor 2 ◽

Kinase Catalytic Domain ◽

Eukaryotic Elongation Factor

eEF2K (eukaryotic elongation factor 2 kinase) is a Ca2+/CaM (calmodulin)-dependent protein kinase which regulates the translation elongation machinery. eEF2K belongs to the small group of so-called ‘α-kinases’ which are distinct from the main eukaryotic protein kinase superfamily. In addition to the α-kinase catalytic domain, other domains have been identified in eEF2K: a CaM-binding region, N-terminal to the kinase domain; a C-terminal region containing several predicted α-helices (resembling SEL1 domains); and a probably rather unstructured ‘linker’ region connecting them. In the present paper, we demonstrate: (i) that several highly conserved residues, implicated in binding ATP or metal ions, are critical for eEF2K activity; (ii) that Ca2+/CaM enhance the ability of eEF2K to bind to ATP, providing the first insight into the allosteric control of eEF2K; (iii) that the CaM-binding/α-kinase domain of eEF2K itself possesses autokinase activity, but is unable to phosphorylate substrates in trans; (iv) that phosphorylation of these substrates requires the SEL1-like domains of eEF2K; and (v) that highly conserved residues in the C-terminal tip of eEF2K are essential for the phosphorylation of eEF2, but not a peptide substrate. On the basis of these findings, we propose a model for the functional organization and control of eEF2K.

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Regulation of Escherichia coli RelA Requires Oligomerization of the C-Terminal Domain

Journal of Bacteriology ◽

10.1128/jb.183.2.570-579.2001 ◽

2001 ◽

Vol 183 (2) ◽

pp. 570-579 ◽

Cited By ~ 66

Author(s):

Michal Gropp ◽

Yael Strausz ◽

Miriam Gross ◽

Gad Glaser

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Catalytic Domain ◽

Mutational Analysis ◽

Amino Acid Starvation ◽

E Coli ◽

Terminal Domain ◽

Negative Effect ◽

And Control ◽

Two Hybrid

ABSTRACT The E. coli RelA protein is a ribosome-dependent (p)ppGpp synthetase that is activated in response to amino acid starvation. RelA can be dissected both functionally and physically into two domains: The N-terminal domain (NTD) (amino acids [aa] 1 to 455) contains the catalytic domain of RelA, and the C-terminal domain (CTD) (aa 455 to 744) is involved in regulating RelA activity. We used mutational analysis to localize sites important for RelA activity and control in these two domains. We inserted two separate mutations into the NTD, which resulted in mutated RelA proteins that were impaired in their ability to synthesize (p)ppGpp. When we caused the CTD inrelA + cells to be overexpressed, (p)ppGpp accumulation during amino acid starvation was negatively affected. Mutational analysis showed that Cys-612, Asp-637, and Cys-638, found in a conserved amino acid sequence (aa 612 to 638), are essential for this negative effect of the CTD. When mutations corresponding to these residues were inserted into the full-length relA gene, the mutated RelA proteins were impaired in their regulation. In attempting to clarify the mechanism through which the CTD regulates RelA activity, we found no evidence for competition for ribosomal binding between the normal RelA and the overexpressed CTD. Results from CyaA complementation experiments of the bacterial two-hybrid system fusion plasmids (G. Karimova, J. Pidoux, A. Ullmann, and D. Ladant, Proc. Natl. Acad. Sci. USA 95:5752–5756, 1998) indicated that the CTD (aa 564 to 744) is involved in RelA-RelA interactions. Our findings support a model in which RelA activation is regulated by its oligomerization state.

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Mutational Analysis of the Catalytic Domain of the Murine Dnmt3a DNA-(cytosine C5)-methyltransferase

Journal of Molecular Biology ◽

10.1016/j.jmb.2006.01.035 ◽

2006 ◽

Vol 357 (3) ◽

pp. 928-941 ◽

Cited By ~ 65

Author(s):

Humaira Gowher ◽

Panida Loutchanwoot ◽

Olga Vorobjeva ◽

Vikas Handa ◽

Renata Z. Jurkowska ◽

...

Keyword(s):

Catalytic Domain ◽

Mutational Analysis

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Abstract 3678: Protein Kinase C-zeta (PKCζ) Is A Novel Mediator Of Endothelial Dysfunction By Inhibiting The ERK5/ Kruppel-like Factor 2 (KLF2) Pathway

Circulation ◽

10.1161/circ.118.suppl_18.s_464-b ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Patrizia Nigro ◽

Chang Hoon Woo ◽

Maria Antonietta Belisario ◽

Carolyn McClain ◽

Jun-ichi Abe ◽

...

Keyword(s):

Shear Stress ◽

Endothelial Dysfunction ◽

Transcriptional Repression ◽

Transcriptional Activity ◽

Catalytic Domain ◽

Mutational Analysis ◽

Critical Role ◽

Enos Expression ◽

Interaction Domain ◽

Pb1 Domain

Background: Shear stress-induced activation of ERK5 and KLF2 leads to eNOS expression maintaining normal endothelial cell (EC) function. PKCζ has emerged as a pathologic mediator of EC dysfunction based on 1) a positive correlation between PKCζ activity and disturbed flow pattern; and 2) PKCζ activation is essential for TNFα stimulation of JNK, caspase-3 and EC apoptosis. Therefore we hypothesized that TNFα and its pathologic mediator ONOO − would inhibit ERK5 transcriptional activity and eNOS expression induced by flow. Results: TNFα and ONOO − significantly inhibited ERK5 transcriptional activity, KLF2 promoter activity, and eNOS expression induced by flow (shear stress = 12 dyn/cm2, 24 hrs). Both TNFαand ONOO − increased PKCζ activity. Transfection of wild type PKCζ (WT-PKCζ) and catalytic domain of PKCζ (CATζ) significantly inhibited ERK5 transcriptional activity (38±1.4% and 57±2.8% respectively; p<0.01, p<0.005). Also, transfection of PKCζ siRNA reversed TNFα-mediated inhibition of ERK5 transcriptional activity (control vs PKCz siRNA, 28±2.5% vs 9±0.3%, p<0.05), suggesting a critical role for PKCζ in ERK5 transcriptional repression. Surprisingly, TNFα and ONOO − did not significantly decrease ERK5 phosphorylation, suggesting that inhibition occurred downstream of ERK5 phosphorylation. Previously we reported that ERK5-SUMOylation inhibited flow-mediated eNOS expression, but we could not detect increased ERK5-SUMOylation in WT-PKCζ or CATζ transfected cells. Importantly, we found that ONOO − significantly increased PKCζ-ERK5 interaction. PKCζ is known to contain a PB1 domain, a well studied protein-protein interaction domain. However, mutational analysis demonstrated that the ERK5 binding site in PKCζ was within the catalytic domain of PKCζ, not the PB1 domain. These data suggest that the PKCζ-ERK5 interaction likely inhibits ERK5 transcriptional activity by direct phosphorylation of ERK5 by PKCζ kinase. Conclusion: PKCζ is a novel mediator of TNFα and ONOO − induced endothelial dysfunction by inhibiting ERK5 transcriptional activity independent of kinase activity and ERK5-SUMOylation.

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Insights into histidine kinase activation mechanisms from the monomeric blue light sensor EL346

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1813586116 ◽

2019 ◽

Vol 116 (11) ◽

pp. 4963-4972 ◽

Cited By ~ 5

Author(s):

Igor Dikiy ◽

Uthama R. Edupuganti ◽

Rinat R. Abzalimov ◽

Peter P. Borbat ◽

Madhur Srivastava ◽

...

Keyword(s):

Blue Light ◽

Conformational Changes ◽

Histidine Kinase ◽

Structural Changes ◽

Molecular Mechanisms ◽

Catalytic Domain ◽

Response Regulator ◽

Phosphoryl Group ◽

Dark State ◽

Light Sensing

Translation of environmental cues into cellular behavior is a necessary process in all forms of life. In bacteria, this process frequently involves two-component systems in which a sensor histidine kinase (HK) autophosphorylates in response to a stimulus before subsequently transferring the phosphoryl group to a response regulator that controls downstream effectors. Many details of the molecular mechanisms of HK activation are still unclear due to complications associated with the multiple signaling states of these large, multidomain proteins. To address these challenges, we combined complementary solution biophysical approaches to examine the conformational changes upon activation of a minimal, blue-light–sensing histidine kinase from Erythrobacter litoralis HTCC2594, EL346. Our data show that multiple conformations coexist in the dark state of EL346 in solution, which may explain the enzyme’s residual dark-state activity. We also observe that activation involves destabilization of the helices in the dimerization and histidine phosphotransfer-like domain, where the phosphoacceptor histidine resides, and their interactions with the catalytic domain. Similar light-induced changes occur to some extent even in constitutively active or inactive mutants, showing that light sensing can be decoupled from activation of kinase activity. These structural changes mirror those inferred by comparing X-ray crystal structures of inactive and active HK fragments, suggesting that they are at the core of conformational changes leading to HK activation. More broadly, our findings uncover surprising complexity in this simple system and allow us to outline a mechanism of the multiple steps of HK activation.

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