scholarly journals Small-molecule sensitization of RecBCD helicase–nuclease to a Chi hotspot-activated state

2020 ◽  
Vol 48 (14) ◽  
pp. 7973-7980
Author(s):  
Ahmet C Karabulut ◽  
Ryan T Cirz ◽  
Andrew F Taylor ◽  
Gerald R Smith
Keyword(s):  
Binding Site ◽  
Small Molecule ◽  
Molecular Mechanisms ◽  
Strand Exchange ◽  
Atp Binding ◽  
Dna Unwinding ◽  
Wild Type ◽  
Atp Binding Site ◽  
Activated State ◽  
Complex Enzymes

Abstract Coordinating multiple activities of complex enzymes is critical for life, including transcribing, replicating and repairing DNA. Bacterial RecBCD helicase–nuclease must coordinate DNA unwinding and cutting to repair broken DNA. Starting at a DNA end, RecBCD unwinds DNA with its fast RecD helicase on the 5′-ended strand and its slower RecB helicase on the 3′-ended strand. At Chi hotspots (5′ GCTGGTGG 3′), RecB’s nuclease cuts the 3′-ended strand and loads RecA strand-exchange protein onto it. We report that a small molecule NSAC1003, a sulfanyltriazolobenzimidazole, mimics Chi sites by sensitizing RecBCD to cut DNA at a Chi-independent position a certain percent of the DNA substrate's length. This percent decreases with increasing NSAC1003 concentration. Our data indicate that NSAC1003 slows RecB relative to RecD and sensitizes it to cut DNA when the leading helicase RecD stops at the DNA end. Two previously described RecBCD mutants altered in the RecB ATP-binding site also have this property, but uninhibited wild-type RecBCD lacks it. ATP and NSAC1003 are competitive; computation docks NSAC1003 into RecB’s ATP-binding site, suggesting NSAC1003 acts directly on RecB. NSAC1003 will help elucidate molecular mechanisms of RecBCD-Chi regulation and DNA repair. Similar studies could help elucidate other DNA enzymes with activities coordinated at chromosomal sites.

scholarly journals Small-molecule sensitization of RecBCD helicase-nuclease to a Chi hotspot-activated state

2020 ◽  
Author(s):  
Ahmet C. Karabulut ◽  
Ryan T. Cirz ◽  
Gerald R. Smith
Keyword(s):  
Binding Site ◽  
Small Molecule ◽  
Molecular Mechanisms ◽  
Strand Exchange ◽  
Atp Binding ◽  
Dna Unwinding ◽  
Wild Type ◽  
Atp Binding Site ◽  
Activated State ◽  
Complex Enzymes

ABSTRACTCoordination of multiple activities of complex enzymes is critical for life, including transcribing, replicating, and repairing DNA. Bacterial RecBCD helicase-nuclease must coordinate DNA unwinding and cutting to repair broken DNA. Starting at a DNA end, RecBCD unwinds DNA with its fast RecD helicase on the 5’-ended strand and its slower RecB helicase on the 3’-ended strand. At Chi hotspots (5’GCTGGTGG3’), RecB’s nuclease cuts the 3’-ended strand and loads RecA strand-exchange protein onto it. We report here that a small molecule NSAC1003, a sulfanyltriazolobenzimidazole, mimics Chi sites by sensitizing RecBCD to cut DNA at a Chi-independent position a certain percent of the DNA substrate’s length. This percent decreases with increasing NSAC1003 concentration. Our data indicate that NSAC1003 slows RecB and sensitizes it to cut DNA when the leading helicase RecD stops at the DNA distal end. Two previously described RecBCD mutants altered in the RecB ATP-binding site also have this property, but uninhibited wild-type RecBCD lacks it. Computation docks NSAC1003 into the ATP-binding site, suggesting that NSAC1003 acts directly on RecB. NSAC1003 will help elucidate the molecular mechanisms of RecBCD-Chi regulation and DNA repair. Similar studies could help elucidate other DNA enzymes whose activities are coordinated at chromosomal sites.

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.

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 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.

scholarly journals Contribution of the ATP Binding Site of ParE to Susceptibility to Novobiocin and Quinolones in Streptococcus pneumoniae

2005 ◽  
Vol 187 (4) ◽  
pp. 1536-1540 ◽  
Author(s):  
Philippe Dupont ◽  
Alexandra Aubry ◽  
Emmanuelle Cambau ◽  
Laurent Gutmann
Keyword(s):  
Streptococcus Pneumoniae ◽  
Binding Site ◽  
Quinolone Resistance ◽  
Atp Binding ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Atp Binding Site ◽  
Open Conformation ◽  
A Subunit

ABSTRACT In Streptococcus pneumoniae, an H103Y substitution in the ATP binding site of the ParE subunit of topoisomerase IV was shown to confer quinolone resistance and hypersensitivity to novobiocin when associated with an S84F change in the A subunit of DNA gyrase. We reconstituted in vitro the wild-type topoisomerase IV and its ParE mutant. The ParE mutant enzyme showed a decreased activity for decatenation at subsaturating ATP levels and was more sensitive to inhibition by novobiocin but was as sensitive to quinolones. These results show that the ParE alteration H103Y alone is not responsible for quinolone resistance and agree with the assumption that it facilitates the open conformation of the ATP binding site that would lead to novobiocin hypersensitivity and to a higher requirement of ATP.

scholarly journals 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.

New Mechanistic Insight on the PIM-1 Kinase Inhibitor AZD1208 Using Multidrug Resistant Human Erythroleukemia Cell Lines and Molecular Docking Simulations

2019 ◽  
Vol 19 (11) ◽  
pp. 914-926 ◽  
Author(s):  
Maiara Bernardes Marques ◽  
Michael González-Durruthy ◽  
Bruna Félix da Silva Nornberg ◽  
Bruno Rodrigues Oliveira ◽  
Daniela Volcan Almeida ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Binding Site ◽  
Cell Lines ◽  
Chemotherapeutic Agents ◽  
Atp Binding ◽  
Human Leukemia ◽  
Atp Binding Site ◽  
Mechanistic Insight ◽  
Multiple Drugs

Background:PIM-1 is a kinase which has been related to the oncogenic processes like cell survival, proliferation, and multidrug resistance (MDR). This kinase is known for its ability to phosphorylate the main extrusion pump (ABCB1) related to the MDR phenotype.Objective:In the present work, we tested a new mechanistic insight on the AZD1208 (PIM-1 specific inhibitor) under interaction with chemotherapy agents such as Daunorubicin (DNR) and Vincristine (VCR).Materials and Methods:In order to verify a potential cytotoxic effect based on pharmacological synergism, two MDR cell lines were used: Lucena (resistant to VCR) and FEPS (resistant to DNR), both derived from the K562 non-MDR cell line, by MTT analyses. The activity of Pgp was ascertained by measuring accumulation and the directional flux of Rh123. Furthermore, we performed a molecular docking simulation to delve into the molecular mechanism of PIM-1 alone, and combined with chemotherapeutic agents (VCR and DNR).Results:Our in vitro results have shown that AZD1208 alone decreases cell viability of MDR cells. However, co-exposure of AZD1208 and DNR or VCR reverses this effect. When we analyzed the ABCB1 activity AZD1208 alone was not able to affect the pump extrusion. Differently, co-exposure of AZD1208 and DNR or VCR impaired ABCB1 activity, which could be explained by compensatory expression of abcb1 or other extrusion pumps not analyzed here. Docking analysis showed that AZD1208 is capable of performing hydrophobic interactions with PIM-1 ATP- binding-site residues with stronger interaction-based negative free energy (FEB, kcal/mol) than the ATP itself, mimicking an ATP-competitive inhibitory pattern of interaction. On the same way, VCR and DNR may theoretically interact at the same biophysical environment of AZD1208 and also compete with ATP by the PIM-1 active site. These evidences suggest that AZD1208 may induce pharmacodynamic interaction with VCR and DNR, weakening its cytotoxic potential in the ATP-binding site from PIM-1 observed in the in vitro experiments.Conclusion:Finally, the current results could have a pre-clinical relevance potential in the rational polypharmacology strategies to prevent multiple-drugs resistance in human leukemia cancer therapy.

scholarly journals Identification of the ATP binding site in tyrocidine synthetase 1 by selective modification with fluorescein 5'-isothiocyanate.

1994 ◽  
Vol 269 (21) ◽  
pp. 14962-14966
Author(s):  
M. Pavela-Vrancic ◽  
E. Pfeifer ◽  
W. Schröder ◽  
H. von Döhren ◽  
H. Kleinkauf
Keyword(s):  
Binding Site ◽  
Atp Binding ◽  
Atp Binding Site

scholarly journals Crizotinib acts as ABL1 inhibitor combining ATP-binding with allosteric inhibition and is active against native BCR-ABL1 and its resistance and compound mutants BCR-ABL1T315I and BCR-ABL1T315I-E255K

2021 ◽  
Author(s):  
Afsar Ali Mian ◽  
Isabella Haberbosch ◽  
Hazem Khamaisie ◽  
Abed Agbarya ◽  
Larissa Pietsch ◽  
...  
Keyword(s):  
Binding Site ◽  
Kinase Inhibitors ◽  
Therapeutic Potential ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Binding Pocket ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Lung Cancer Patients

AbstractResistance remains the major clinical challenge for the therapy of Philadelphia chromosome–positive (Ph+) leukemia. With the exception of ponatinib, all approved tyrosine kinase inhibitors (TKIs) are unable to inhibit the common “gatekeeper” mutation T315I. Here we investigated the therapeutic potential of crizotinib, a TKI approved for targeting ALK and ROS1 in non-small cell lung cancer patients, which inhibited also the ABL1 kinase in cell-free systems, for the treatment of advanced and therapy-resistant Ph+ leukemia. By inhibiting the BCR-ABL1 kinase, crizotinib efficiently suppressed growth of Ph+ cells without affecting growth of Ph− cells. It was also active in Ph+ patient-derived long-term cultures (PD-LTCs) independently of the responsiveness/resistance to other TKIs. The efficacy of crizotinib was confirmed in vivo in syngeneic mouse models of BCR-ABL1- or BCR-ABL1T315I-driven chronic myeloid leukemia–like disease and in BCR-ABL1-driven acute lymphoblastic leukemia (ALL). Although crizotinib binds to the ATP-binding site, it also allosterically affected the myristol binding pocket, the binding site of GNF2 and asciminib (former ABL001). Therefore, crizotinib has a seemingly unique double mechanism of action, on the ATP-binding site and on the myristoylation binding pocket. These findings strongly suggest the clinical evaluation of crizotinib for the treatment of advanced and therapy-resistant Ph+ leukemia.

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