scholarly journals Genetic and Biochemical Analysis of Phosphatase Activity of Escherichia coli NRII (NtrB) and Its Regulation by the PII Signal Transduction Protein

2003 ◽  
Vol 185 (4) ◽  
pp. 1299-1315 ◽  
Author(s):  
Augen A. Pioszak ◽  
Alexander J. Ninfa
Keyword(s):  
Escherichia Coli ◽  
Signal Transduction ◽  
Binding Site ◽  
Phosphatase Activity ◽  
Atp Binding ◽  
Wild Type ◽  
Small Surface ◽  
Central Domain ◽  
Atp Binding Site ◽  
Terminal Domain

ABSTRACT Mutant forms of Escherichia coli NRII (NtrB) were isolated that retained wild-type NRII kinase activity but were defective in the PII-activated phosphatase activity of NRII. Mutant strains were selected as mimicking the phenotype of a strain (strain BK) that lacks both of the related PII and GlnK signal transduction proteins and thus has no mechanism for activation of the NRII phosphatase activity. The selection and screening procedure resulted in the isolation of numerous mutants that phenotypically resembled strain BK to various extents. Mutations mapped to the glnL (ntrB) gene encoding NRII and were obtained in all three domains of NRII. Two distinct regions of the C-terminal, ATP-binding domain were identified by clusters of mutations. One cluster, including the Y302N mutation, altered a lid that sits over the ATP-binding site of NRII. The other cluster, including the S227R mutation, defined a small surface on the “back” or opposite side of this domain. The S227R and Y302N proteins were purified, along with the A129T (NRII2302) protein, which has reduced phosphatase activity due to a mutation in the central domain of NRII, and the L16R protein, which has a mutation in the N-terminal domain of NRII. The S227R, Y302N, and L16R proteins were specifically defective in the PII-activated phosphatase activity of NRII. Wild-type NRII, Y302N, A129T, and L16R proteins bound to PII, while the S227R protein was defective in binding PII. This suggests that the PII-binding site maps to the “back” of the C-terminal domain and that mutation of the ATP-lid, central domain, and N-terminal domain altered functions necessary for the phosphatase activity after PII binding.

scholarly journals Mutational analysis of the ATP-binding site in HslU, the ATPase component of HslVU protease in Escherichia coli

FEBS Letters ◽  
1996 ◽  
Vol 398 (2-3) ◽  
pp. 151-154 ◽  
Author(s):  
Dong Hun Shin ◽  
Soon Ji Yoo ◽  
Yoon Kyung Shim ◽  
Jae Hong Seol ◽  
Man-Sik Kang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Mutational Analysis ◽  
Atp Binding ◽  
Atp Binding Site

scholarly journals The nucleotide-binding domain of the Zn2+-transporting P-type ATPase from Escherichia coli carries a glycine motif that may be involved in binding of ATP

2004 ◽  
Vol 377 (1) ◽  
pp. 95-105 ◽  
Author(s):  
Juha OKKERI ◽  
Liisa LAAKKONEN ◽  
Tuomas HALTIA
Keyword(s):  
Escherichia Coli ◽  
Sarcoplasmic Reticulum ◽  
Binding Site ◽  
Molecular Model ◽  
Nucleotide Binding ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Disease Mutations ◽  
Disease Protein ◽  
P Type

In P-type ATPases, the nucleotide-binding (N) domain is located in the middle of the sequence which folds into the phosphorylation (P) domain. The N domain of ZntA, a Zn2+-translocating P-type ATPase from Escherichia coli, is approx. 13% identical with the N domain of sarcoplasmic reticulum Ca2+-ATPase. None of the Ca2+-ATPase residues involved in binding of ATP are found in ZntA. However, the sequence G503SGIEAQV in the N domain of ZntA resembles the motif GxGxxG, which forms part of the ATP-binding site in protein kinases. This motif is also found in Wilson disease protein where several disease mutations cluster in it. In the present work, we have made a set of disease mutation analogues, including the mutants G503S (Gly503→Ser), G505R and A508F of ZntA. At low [ATP], these mutant ATPases are poorly phosphorylated. The phosphorylation defect of the mutants G503S and G505R can, however, be partially (G503S) or fully (G505R) compensated for by using a higher [ATP], suggesting that these mutations lower the affinity for ATP. In all three mutant ATPases, phosphorylation by Pi has become less sensitive to the presence of ATP, also consistent with the proposal that the Gly503 motif plays a role in ATP binding. In order to test this hypothesis, we have modelled the N domain of ZntA using the sarcoplasmic reticulum Ca2+-ATPase structure as a template. In the model, the Gly503 motif, as well as the residues Glu470 and His475, are located in the proximity of the ATP-binding site. In conclusion, the mutagenesis data and the molecular model are consistent with the idea that the two loops carrying the residues Glu470, His475, Gly503 and Gly505 play a role in ATP binding and activation.

A mutation at the ATP-binding site of pp60v-src abolishes kinase activity, transformation, and tumorigenicity

1985 ◽  
Vol 5 (7) ◽  
pp. 1772-1779
Author(s):  
M A Snyder ◽  
J M Bishop ◽  
J P McGrath ◽  
A D Levinson
Keyword(s):  
Binding Site ◽  
Kinase Activity ◽  
Atp Binding ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Atp Binding Site ◽  
Specific Immunoglobulin ◽  
Dependent Protein ◽  
Infected Cells

We constructed a mutant, called RSV-SF2, at the ATP-binding site of pp60v-src. In this mutant, lysine-295 is replaced with methionine. SF2 pp60v-src was found to have a half-life similar to that of wild-type pp60v-src and was localized in the membranous fraction of the cell. Rat cells expressing SF2 pp60v-src were morphologically untransformed and do not form tumors. The SF2 pp60v-src isolated from these cells lacked kinase activity with either specific immunoglobulin or other substrates, and expression of SF2 pp60v-src failed to cause an increase of total phosphotyrosine in the proteins of infected cells. Wild-type pp60v-src was phosphorylated on serine and tyrosine in infected cells, and the analogous phosphorylations could also be carried out in vitro. Phosphorylation of serine was catalyzed by a cyclic AMP-dependent protein kinase, and phosphorylation of tyrosine was perhaps catalyzed by pp60v-src itself. By contrast, SF2 pp60v-src could not be phosphorylated on serine or tyrosine either in infected cells or in vitro. These findings strengthen the belief that the phosphotransferase activity of pp60v-src is required for neoplastic transformation by the protein and suggest that the binding of ATP to pp60v-src elicits an allosteric change required for phosphorylation of serine in the protein.

scholarly journals The phosphatase activity of the isolated H4-H5 loop of Na+/K+ ATPase resides outside its ATP binding site

2004 ◽  
Vol 271 (19) ◽  
pp. 3923-3936 ◽  
Author(s):  
Rita Krumscheid ◽  
Rüdiger Ettrich ◽  
Zofie Sovová ◽  
Klára Sušánková ◽  
Zdeněk Lánský ◽  
...  
Keyword(s):  
Binding Site ◽  
Phosphatase Activity ◽  
Atp Binding ◽  
Atp Binding Site

Study of the ATP-binding site of helicase IV from Escherichia coli

2006 ◽  
Vol 341 (3) ◽  
pp. 828-836 ◽  
Author(s):  
Sandy Dubaele ◽  
Claude Lourdel ◽  
Patrick Chène
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Helicase Iv

scholarly journals Using NMR to identify binding regions for N and C-terminal Hsp90 inhibitors using Hsp90 domains

2021 ◽  
Vol 12 (3) ◽  
pp. 410-415
Author(s):  
Jeanette R. McConnell ◽  
H. Jane Dyson ◽  
Shelli R. McAlpine
Keyword(s):  
Side Effects ◽  
Binding Site ◽  
Atp Binding ◽  
Hsp90 Inhibitors ◽  
Allosteric Inhibitors ◽  
Atp Binding Site ◽  
Allosteric Inhibitor ◽  
Terminal Domain ◽  
Anti Cancer ◽  
Inhibitor Compounds

Allosteric inhibitors of Hsp90 have potential as anti-cancer agents without the side-effects that arise from targeting ATP-binding site in the N-terminal domain. This study gives NMR information on binding of allosteric inhibitor compounds to Hsp90.

The N-Terminal Domain of 2′,3′-Cyclic Nucleotide 3′-Phosphodiesterase Harbors a GTP/ATP Binding Site

2007 ◽  
Vol 70 (6) ◽  
pp. 502-510 ◽  
Author(s):  
Stefania Stingo ◽  
Mariorosario Masullo ◽  
Eugenia Polverini ◽  
Chiara Laezza ◽  
Immacolata Ruggiero ◽  
...  
Keyword(s):  
Binding Site ◽  
Cyclic Nucleotide ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Terminal Domain

scholarly journals N-ethylmaleimide-sensitive fusion protein: a trimeric ATPase whose hydrolysis of ATP is required for membrane fusion.

1994 ◽  
Vol 126 (4) ◽  
pp. 945-954 ◽  
Author(s):  
S W Whiteheart ◽  
K Rossnagel ◽  
S A Buhrow ◽  
M Brunner ◽  
R Jaenicke ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Binding Site ◽  
Reversible Inhibitor ◽  
Atp Binding ◽  
Wild Type ◽  
Atp Binding Site ◽  
Amino Terminal ◽  
Golgi Transport ◽  
D2 Domain

The NEM-sensitive fusion protein, NSF, together with SNAPs (soluble NSF attachment proteins) and the SNAREs (SNAP receptors), is thought to be generally used for the fusion of transport vesicles to their target membranes. NSF is a homotrimer whose polypeptide subunits are made up of three distinct domains: an amino-terminal domain (N) and two homologous ATP-binding domains (D1 and D2). Mutants of NSF were produced in which either the order or composition of the three domains were altered. These mutants could not support intra-Golgi transport, but they indicated that the D2 domain was required for trimerization of the NSF subunits. Mutations of the first ATP-binding site that affected either the binding (K266A) or hydrolysis (E329Q) of ATP completely eliminated NSF activity. The hydrolysis mutant was an effective, reversible inhibitor of Golgi transport with an IC50 of 125 ng/50 microliters assay. Mutants in the second ATP-binding site (binding, K549A; hydrolysis, D604Q) had either 14 or 42% the specific activity of the wild-type protein, respectively. Using coexpression of an inactive mutant with wild-type subunits, it was possible to produce a recombinant form of trimeric NSF that contained a mixture of subunits. The mixed NSF trimers were inactive, even when only one mutant subunit was present, suggesting that NSF action requires each of the three subunits in a concerted mechanism. These studies demonstrate that the ability of the D1 domain to hydrolyze ATP is required for NSF activity and, therefore is required for membrane fusion. The D2 domain is required for trimerization, but its ability to hydrolyze ATP is not absolutely required for NSF function.

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