Modulation of the Rcs-mediated signal transfer by conformational flexibility

2008 ◽  
Vol 36 (6) ◽  
pp. 1427-1432 ◽  
Author(s):  
Vladimir V. Rogov ◽  
Kerstin Schmöe ◽  
Fank Löhr ◽  
Natalia Yu. Rogova ◽  
Frank Bernhard ◽  
...  
Keyword(s):  
Active Site ◽  
Regulatory Networks ◽  
Helical Structure ◽  
Conformational Flexibility ◽  
Conformational Exchange ◽  
Phosphoryl Transfer ◽  
Phosphate Binding ◽  
Signal Transfer ◽  
Sensor Kinases ◽  
The Individual

The Rcs (regulator of capsule synthesis) signalling complex comprises the membrane-associated hybrid sensor kinases RcsC and RcsD, the transcriptional regulator RcsB and the two co-inducers RcsA and RcsF. Acting as a global regulatory network, the Rcs phosphorelay controls multiple cellular pathways including capsule synthesis, cell division, motility, biofilm formation and virulence mechanisms. Signal-dependent communication of the individual Rcs domains showing histidine kinase, phosphoreceiver, phosphoryl transfer and DNA-binding activities is characteristic and essential for the modulation of signal transfer. We have analysed the structures of core elements of the Rcs network including the RcsC-PR (phosphoreceiver domain of RcsC) and the RcsD-HPt (histidine phosphotransfer domain of RcsD), and we have started to characterize the dynamics and recognition mechanisms of the proteins. RcsC-PR represents a typical CheY-like α/β/α sandwich fold and it shows a large conformational flexibility near the active-site residue Asp875. NMR analysis revealed that RcsC-PR is able to adopt preferred conformations upon Mg2+ co-ordination, BeF3− activation, phosphate binding and RcsD-HPt recognition. In contrast, the α-helical structure of RcsD-HPt is conformationally stable and contains a recognition area in close vicinity to the active-site His842 residue. Our studies indicate the importance of protein dynamics and conformational exchange for the differential response to the variety of signals perceived by complex regulatory networks.

scholarly journals It takes two (Las1 HEPN endoribonuclease domains) to cut RNA correctly

2020 ◽  
Vol 295 (18) ◽  
pp. 5857-5870 ◽  
Author(s):  
Monica C. Pillon ◽  
Kevin H. Goslen ◽  
Jacob Gordon ◽  
Melissa L. Wells ◽  
Jason G. Williams ◽  
...  
Keyword(s):  
Active Site ◽  
Ribosome Biogenesis ◽  
Nuclease Activity ◽  
Conformational Flexibility ◽  
Neurological Dysfunction ◽  
Rna Cleavage ◽  
Higher Eukaryotes ◽  
The Individual

The ribosome biogenesis factor Las1 is an essential endoribonuclease that is well-conserved across eukaryotes and a newly established member of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain-containing nuclease family. HEPN nucleases participate in diverse RNA cleavage pathways and share a short HEPN nuclease motif (RφXXXH) important for RNA cleavage. Most HEPN nucleases participate in stress-activated RNA cleavage pathways; Las1 plays a fundamental role in processing pre-rRNA. Underscoring the significance of Las1 function in the cell, mutations in the human LAS1L (LAS1-like) gene have been associated with neurological dysfunction. Two juxtaposed HEPN nuclease motifs create Las1's composite nuclease active site, but the roles of the individual HEPN motif residues are poorly defined. Here using a combination of in vivo experiments in Saccharomyces cerevisiae and in vitro assays, we show that both HEPN nuclease motifs are required for Las1 nuclease activity and fidelity. Through in-depth sequence analysis and systematic mutagenesis, we determined the consensus HEPN motif in the Las1 subfamily and uncovered its canonical and specialized elements. Using reconstituted Las1 HEPN-HEPN′ chimeras, we defined the molecular requirements for RNA cleavage. Intriguingly, both copies of the Las1 HEPN motif were important for nuclease function, revealing that both HEPN motifs participate in coordinating the RNA within the Las1 active site. We also established that conformational flexibility of the two HEPN domains is important for proper nuclease function. The results of our work reveal critical information about how dual HEPN domains come together to drive Las1-mediated RNA cleavage.

Structure and activity of phosphoglycerate mutase

1981 ◽  
Vol 293 (1063) ◽  
pp. 121-130 ◽  
Keyword(s):  
Active Site ◽  
Transfer Reaction ◽  
Chemical Kinetic ◽  
Phosphoglycerate Mutase ◽  
Phosphoryl Transfer ◽  
X Ray ◽  
Histidine Residues ◽  
Ping Pong ◽  
Available Information ◽  
Structure And Activity

The structure of yeast phosphoglycerate mutase determined by X-ray crystallographic and amino acid sequence studies has been interpreted in terms of the chemical, kinetic and mechanistic observations made on this enzyme. There are two histidine residues at the active site, with imidazole groups almost parallel to each other and approximately 0.4 nm apart, positioned close to the 2 and 3 positions of the substrate. The simplest interpretation of the available information suggests that a ping-pong type mechanism operates in which at least one of these histidine residues participates in the phosphoryl transfer reaction. The flexible C-terminal region also plays an important role in the enzymic reaction.

Folding of the polypeptide chain(s), conformational flexibility and reactivity of the metal active site of hemocyanin and tyrosinase

1990 ◽  
Vol 3 (2) ◽  
pp. 93-97 ◽  
Author(s):  
Mariano Beltramini ◽  
Meri Santamaria ◽  
Benedetto Salvato ◽  
Konrad Lerche
Keyword(s):  
Active Site ◽  
Polypeptide Chain ◽  
Conformational Flexibility

scholarly journals Residual Helicity at the Active Site of the Histidine Phosphocarrier, HPr, Modulates Binding Affinity to Its Natural Partners

2021 ◽  
Vol 22 (19) ◽  
pp. 10805
Author(s):  
José L. Neira ◽  
David Ortega-Alarcón ◽  
Bruno Rizzuti ◽  
Martina Palomino-Schätzlein ◽  
Adrián Velázquez-Campoy ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Active Site ◽  
Antibacterial Properties ◽  
Helical Structure ◽  
Titration Calorimetry ◽  
Its Sequence ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Residual Structure ◽  
Α Helix

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. The first proteins in the cascade are common to all organisms (EI and HPr). The active site of HPr involves a histidine (His15) located immediately before the beginning of the first α-helix. The regulator of sigma D (Rsd) protein also binds to HPr. The region of HPr comprising residues Gly9-Ala30 (HPr9–30), involving the first α-helix (Ala16-Thr27) and the preceding active site loop, binds to both the N-terminal region of EI and intact Rsd. HPr9–30 is mainly disordered. We attempted to improve the affinity of HPr9–30 to both proteins by mutating its sequence to increase its helicity. We designed peptides that led to a marginally larger population in solution of the helical structure of HPr9–30. Molecular simulations also suggested a modest increment in the helical population of mutants, when compared to the wild-type. The mutants, however, were bound with a less favorable affinity than the wild-type to both the N-terminal of EI (EIN) or Rsd, as tested by isothermal titration calorimetry and fluorescence. Furthermore, mutants showed lower antibacterial properties against Staphylococcus aureus than the wild-type peptide. Therefore, we concluded that in HPr, a compromise between binding to its partners and residual structure at the active site must exist to carry out its function.

scholarly journals Kinetic probes for inter-domain co-operation in human somatic angiotensin-converting enzyme

2005 ◽  
Vol 391 (3) ◽  
pp. 641-647 ◽  
Author(s):  
Olga E. Skirgello ◽  
Peter V. Binevski ◽  
Vladimir F. Pozdnev ◽  
Olga A. Kost
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Enzymatic Activity ◽  
Active Site ◽  
The Other ◽  
Converting Enzyme ◽  
Human Enzyme ◽  
Independent Functions ◽  
The Common ◽  
The Individual ◽  
Hydrolysis Of

s-ACE (the somatic form of angiotensin-converting enzyme) consists of two homologous domains (N- and C-domains), each bearing a catalytic site. Negative co-operativity between the two domains has been demonstrated for cow and pig ACEs. However, for the human enzyme there are conflicting reports in the literature: some suggest possible negative co-operativity between the domains, whereas others indicate independent functions of the domains within s-ACE. We demonstrate here that a 1:1 stoichiometry for the binding of the common ACE inhibitors, captopril and lisinopril, to human s-ACE is enough to abolish enzymatic activity towards FA {N-[3-(2-furyl)acryloyl]}-Phe-GlyGly, Cbz (benzyloxycarbonyl)-Phe-His-Leu or Hip (N-benzoylglycyl)-His-Leu. The kinetic parameters for the hydrolysis of seven tripeptide substrates by human s-ACE appeared to represent average values for parameters obtained for the individual N- and C-domains. Kinetic analysis of the simultaneous hydrolysis of two substrates, Hip-His-Leu (S1) and Cbz-Phe-His-Leu (S2), with a common product (His-Leu) by s-ACE at different values for the ratio of the initial concentrations of these substrates (i.e. σ=[S2]0/[S1]0) demonstrated competition of these substrates for binding to the s-ACE molecule, i.e. binding of a substrate at one active site makes the other site unavailable for either the same or a different substrate. Thus the two domains within human s-ACE exhibit strong negative co-operativity upon binding of common inhibitors and in the hydrolysis reactions of tripeptide substrates.

scholarly journals Insights into the phosphoryl transfer catalyzed by cAMP-dependent protein kinase

2014 ◽  
Vol 70 (a1) ◽  
pp. C449-C449
Author(s):  
Oksana Gerlits ◽  
Amit Das ◽  
Jianhui Tian ◽  
Malik Keshwani ◽  
Susan Taylor ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Active Site ◽  
Phosphate Group ◽  
Nucleoside Triphosphate ◽  
Phosphoryl Transfer ◽  
Hydrogen Atoms ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

Protein kinases are involved in a number of cell signaling pathways. They catalyze phosphorylation of proteins and regulate the majority of cellular processes (such as growth, differentiation, lipid metabolism, regulation of sugar, nucleic acid synthesis, etc.). Chemically, protein kinases covalently transfer the gamma-phosphate group of a nucleoside triphosphate (e.g. ATP) to a hydroxyl group of a Ser, Thr or Tyr residue of substrate protein or peptide. The reaction involves moving hydrogen atoms between the enzyme, substrate and nucleoside. The unanswered question is whether the proton transfer from the Ser residue happens before the phosphoryl transfer using the general acid-base catalyst, Asp166, or after the reaction went through the transition state by directly protonating the phosphate group. To address this key question about the phosphoryl transfer, we determined a number of X-ray structures of ternary complexes of catalytic subunit of cAMP-dependent protein kinase (PKAc) with various substrates, nucleotides and cofactors. Importantly, we were able to trap and mimic the initial (Michaelis complex) and final (product complex) stages of the reaction. The results demonstrate that Mg2+, Ca2+, Sr2+, and Ba2+ metal ions bind to the active site and facilitate the reaction to produce ADP and a phosphorylated peptide. The study also revealed that metal-free PKAc can facilitate the phosphoryl transfer reaction; a result that was confirmed with single turnover enzyme kinetics measurements. Comparison of the product and the pseudo-Michaelis complex structures, in conjunction with molecular dynamics simulations, reveals conformational, coordination, and hydrogen bonding changes that help further our understanding of the mechanism, roles of metals, and active site residues involved in PKAc activity.

scholarly journals Transmembrane signaling and cytoplasmic signal conversion by dimeric transmembrane helix 2 and a linker domain of the DcuS sensor kinase

2020 ◽  
pp. jbc.RA120.015999
Author(s):  
Marius Stopp ◽  
Philipp Aloysius Steinmetz ◽  
Christopher Schubert ◽  
Christian Griesinger ◽  
Dirk Schneider ◽  
...  
Keyword(s):  
Signal Transmission ◽  
Transmembrane Helix ◽  
Helical Structure ◽  
Kinase Domain ◽  
Sensor Kinase ◽  
Transmembrane Signaling ◽  
Activated State ◽  
Sensor Kinases ◽  
And Function ◽  
Cytoplasmic Side

Transmembrane signaling is a key process of membrane bound sensor kinases. The C4-dicarboxylate (fumarate) responsive sensor kinase DcuS of Escherichia coli is anchored by transmembrane helices TM1 and TM2 in the membrane. Signal transmission across the membrane relies on the piston-type movement of the periplasmic part of TM2. To define the role of TM2 in transmembrane signaling, we use oxidative Cys cross-linking to demonstrate that TM2 extends over the full distance of the membrane and forms a stable transmembrane homodimer in both the inactive and fumarate-activated state of DcuS. A S186xxxGxxxG194 motif is required for the stability and function of the TM2 homodimer. The TM2 helix further extends on the periplasmic side into the α6-helix of the sensory PASP domain, and on the cytoplasmic side into the α1-helix of PASC. PASC has to transmit the signal to the C-terminal kinase domain. A helical linker on the cytoplasmic side connecting TM2 with PASC contains a LxxxLxxxL sequence. The dimeric state of the linker was relieved during fumarate activation of DcuS, indicating structural rearrangements in the linker. Thus, DcuS contains a long α-helical structure reaching from the sensory PASP (α6) domain across the membrane to α1(PASC). Taken together, the results suggest piston-type transmembrane signaling by the TM2-homodimer from PASP across the full TM region, whereas the fumarate-destabilized linker dimer converts the signal on the cytoplasmic side for PASC and kinase regulation.

Non-homologous end-joining partners in a helical dance: structural studies of XLF–XRCC4 interactions

2011 ◽  
Vol 39 (5) ◽  
pp. 1387-1392 ◽  
Author(s):  
Qian Wu ◽  
Takashi Ochi ◽  
Dijana Matak-Vinkovic ◽  
Carol V. Robinson ◽  
Dimitri Y. Chirgadze ◽  
...  
Keyword(s):  
Gel Filtration ◽  
Coiled Coil ◽  
Helical Structure ◽  
Complementation Group ◽  
Helical Axis ◽  
Structural Studies ◽  
End Joining ◽  
Individual Crystal ◽  
Non Homologous End Joining ◽  
The Individual

XRCC4 (X-ray cross-complementation group 4) and XLF (XRCC4-like factor) are two essential interacting proteins in the human NHEJ (non-homologous end-joining) pathway that repairs DNA DSBs (double-strand breaks). The individual crystal structures show that the dimeric proteins are homologues with protomers containing head domains and helical coiled-coil tails related by approximate two-fold symmetry. Biochemical, mutagenesis, biophysical and structural studies have identified the regions of interaction between the two proteins and suggested models for the XLF–XRCC4 complex. An 8.5 Å (1 Å=0.1 nm) resolution crystal structure of XLF–XRCC4 solved by molecular replacement, together with gel filtration and nano-ESI (nano-electrospray ionization)–MS results, demonstrates that XLF and XRCC4 dimers interact through their head domains and form an alternating left-handed helical structure with polypeptide coiled coils and pseudo-dyads of individual XLF and XRCC4 dimers at right angles to the helical axis.

On the mechanism of phosphoenolpyruvate synthetase (PEPs) and its inhibition by sodium fluoride: potential magnesium and aluminum fluoride complexes of phosphoryl transfer

2015 ◽  
Vol 93 (3) ◽  
pp. 236-240 ◽  
Author(s):  
Nicole E. McCormick ◽  
David L. Jakeman
Keyword(s):  
Sodium Fluoride ◽  
Active Site ◽  
Carbon Sources ◽  
Indirect Evidence ◽  
Intermediate Formation ◽  
Phosphoryl Transfer ◽  
Metal Fluoride ◽  
Enzyme Active Site ◽  
Antibiotic Development ◽  
Fluoride Complexes

Phosphoenolpyruvate synthase (PEPs) catalyzes the conversion of pyruvate to phosphoenolpyruvate (PEP) using a two-step mechanism invoking a phosphorylated-His intermediate. Formation of PEP is an initial step in gluconeogenesis, and PEPs is essential for growth of Escherichia coli on 3-carbon sources such as pyruvate. The production of PEPs has also been linked to bacterial virulence and antibiotic resistance. As such, PEPs is of interest as a target for antibiotic development, and initial investigations of PEPs have indicated inhibition by sodium fluoride. Similar inhibition has been observed in a variety of phospho-transfer enzymes through the formation of metal fluoride complexes within the active site. Herein we quantify the inhibitory capacity of sodium fluoride through a coupled spectrophotometric assay. The observed inhibition provides indirect evidence for the formation of a MgF3−complex within the enzyme active site and insight into the phospho-transfer mechanism of PEPs. The effect of AlCl3on PEPs enzyme activity was also assessed and found to decrease substrate binding and turnover.

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