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Nucleotide-binding Domain (nucleotide-binding + domain)
Selected AbstractsNucleotide-binding domain 1 of cystic fibrosis transmembrane conductance regulatorFEBS JOURNAL, Issue 17 2000Production of a suitable protein for structural studies Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). This protein belongs to the large ATP-binding cassette (ABC) family of transporters. Most patients with cystic fibrosis bear a mutation in the nucleotide-binding domain 1 (NBD1) of CFTR, which plays a key role in the activation of the channel function of CFTR. Determination of the three dimensional structure of NBD1 is essential to better understand its structure,function relationship, and relate it to the biological features of CFTR. In this paper, we report the first preparation of recombinant His-tagged NBD1, as a soluble, stable and isolated domain. The method avoids the use of renaturing processes or fusion constructs. ATPase activity assays show that the recombinant domain is functional. Using tryptophan intrinsic fluorescence, we point out that the local conformation, in the region of the most frequent mutation ,F508, could differ from that of the nucleotide-binding subunit of histidine permease, the only available ABC structure. We have undertaken three dimensional structure determination of NBD1, and the first two dimensional 15N- 1H NMR spectra demonstrate that the domain is folded. The method should be applicable to the structural studies of NBD2 or of other NBDs from different ABC proteins of major biological interest, such as multidrug resistance protein 1 or multidrug resistance associated protein 1. [source] Functional role of the linker region in purified human P-glycoproteinFEBS JOURNAL, Issue 13 2009Tomomi Sato Human P-glycoprotein (P-gp), which conveys multidrug resistance, is an ATP-dependent drug efflux pump that transports a wide variety of structurally unrelated compounds out of cells. P-gp possesses a ,linker region' of , 75 amino acids that connects two homologous halves, each of which contain a transmembrane domain followed by a nucleotide-binding domain. To investigate the role of the linker region, purified human P-gp was cleaved by proteases at the linker region and then compared with native P-gp. Based on a verapamil-stimulated ATP hydrolase assay, size-exclusion chromatography analysis and a thermo-stability assay, cleavage of the P-gp linker did not directly affect the preservation of the overall structure or the catalytic process in ATP hydrolysis. However, linker cleavage increased the kcat values both with substrate (ksub) and without substrate (kbasal), but decreased the ksub/kbasal values of all 10 tested substrates. The former result indicates that cleaving the linker activates P-gp, while the latter result suggests that the linker region maintains the tightness of coupling between the ATP hydrolase reaction and substrate recognition. Inspection of structures of the P-gp homolog, MsbA, suggests that linker-cleaved P-gp has increased ATP hydrolase activity because the linker interferes with a conformational change that accompanies the ATP hydrolase reaction. Moreover, linker cleavage affected the specificity constants [ksub/Km(D)] for some substrates (i.e. linker cleavage probably shifts the substrate specificity profile of P-gp). Thus, this result also suggests that the linker region regulates the inherent substrate specificity of P-gp. [source] Nucleotide-binding domain 1 of cystic fibrosis transmembrane conductance regulatorFEBS JOURNAL, Issue 17 2000Production of a suitable protein for structural studies Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). This protein belongs to the large ATP-binding cassette (ABC) family of transporters. Most patients with cystic fibrosis bear a mutation in the nucleotide-binding domain 1 (NBD1) of CFTR, which plays a key role in the activation of the channel function of CFTR. Determination of the three dimensional structure of NBD1 is essential to better understand its structure,function relationship, and relate it to the biological features of CFTR. In this paper, we report the first preparation of recombinant His-tagged NBD1, as a soluble, stable and isolated domain. The method avoids the use of renaturing processes or fusion constructs. ATPase activity assays show that the recombinant domain is functional. Using tryptophan intrinsic fluorescence, we point out that the local conformation, in the region of the most frequent mutation ,F508, could differ from that of the nucleotide-binding subunit of histidine permease, the only available ABC structure. We have undertaken three dimensional structure determination of NBD1, and the first two dimensional 15N- 1H NMR spectra demonstrate that the domain is folded. The method should be applicable to the structural studies of NBD2 or of other NBDs from different ABC proteins of major biological interest, such as multidrug resistance protein 1 or multidrug resistance associated protein 1. [source] Recent advances in the discovery of flavonoids and analogs with high-affinity binding to P-glycoprotein responsible for cancer cell multidrug resistanceMEDICINAL RESEARCH REVIEWS, Issue 5 2002Ahcène Boumendjel Abstract P-glycoprotein (P-gp) is a plasma membrane glycoprotein that confers multidrug resistance on cells by virtue of its ability to exclude cytotoxic drugs in an ATP-dependent manner. The most commonly considered hypothesis is that P-gp acts as an ATP-driven drug-export pump, the mechanism of which is not understood in detail. Therefore, a tremendous effort is being made to find out modulator molecules to inhibit P-gp. We have been developing flavonoid derivatives as a new class of promising modulators using a new in vitro rational-screening assay based on measurements of the binding-affinity toward the C-terminal nucleotide-binding domain (NBD2) of P-gp. This review is focused on our results obtained with a variety of flavonoids. Structure,activity relationships of flavonoids as potential MDR modulators are reported. © 2002 Wiley Periodicals, Inc. Med Res Rev, 22, No. 5, 512,529, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/med.10015 [source] Roles of the two ClpC ATP binding sites in the regulation of competence and the stress responseMOLECULAR MICROBIOLOGY, Issue 3 2001Kürsad Turgay MecA targets the competence transcription factor ComK to ClpC. As a consequence, this factor is degraded by the ClpC/ClpP protease. ClpC is a member of the Clp/HSP100 family of ATPases and possesses two ATP binding sites. We have individually modified the Walker A motifs of these two sites and have also deleted a putative substrate recognition domain of ClpC at the C-terminus. The effects of these mutations were studied in vitro and in vivo. Deletion of the C-terminal domain resulted in a decreased binding affinity for MecA, a decreased ATPase activity in response to MecA addition and decreased degradative activity in vitro. In vivo, this deletion resulted in a failure to degrade ComK and in a decrease in thermal resistance for growth. Mutation of the N-terminal Walker A box (K214Q) caused a drastically decreased ATPase activity in vitro, but did not interfere with MecA binding. In vivo, this mutation had no effect on thermal resistance, but had a clpC null phenotype with respect to competence. Mutation of the C-terminal Walker A motif (K551Q) caused essentially the reverse phenotype both in vivo and in vitro. Although binding to MecA was only moderately impaired with 2 mM ATP, this mutant protein displayed no response to 0.2 mM ATP, unlike the wild-type ClpC and the K214Q mutant protein. The ATPase activity of the K551Q mutant protein, induced by the addition of MecA plus ComS, was decreased about 10-fold but was not eliminated. In vivo, the K551Q mutation showed a partial defect with respect to competence and a profound loss of thermal resistance. Sporulation was reduced drastically by the K551Q and less so by the K214Q mutation, but remained unaffected by deletion of the C-terminal domain. Although the evidence suggests that the functions of the two ATP-binding domains overlap, it appears that the N-terminal nucleotide-binding domain of ClpC is particularly concerned with MecA-related functions, whereas the C-terminal domain plays a more general role in the activities of ClpC. [source] Integrated biophysical studies implicate partial unfolding of NBD1 of CFTR in the molecular pathogenesis of F508del cystic fibrosisPROTEIN SCIENCE, Issue 10 2010Chi Wang Abstract The lethal genetic disease cystic fibrosis is caused predominantly by in-frame deletion of phenylalanine 508 in the cystic fibrosis transmembrane conductance regulator (CFTR). F508 is located in the first nucleotide-binding domain (NBD1) of CFTR, which functions as an ATP-gated chloride channel on the cell surface. The F508del mutation blocks CFTR export to the surface due to aberrant retention in the endoplasmic reticulum. While it was assumed that F508del interferes with NBD1 folding, biophysical studies of purified NBD1 have given conflicting results concerning the mutation's influence on domain folding and stability. We have conducted isothermal (this paper) and thermal (accompanying paper) denaturation studies of human NBD1 using a variety of biophysical techniques, including simultaneous circular dichroism, intrinsic fluorescence, and static light-scattering measurements. These studies show that, in the absence of ATP, NBD1 unfolds via two sequential conformational transitions. The first, which is strongly influenced by F508del, involves partial unfolding and leads to aggregation accompanied by an increase in tryptophan fluorescence. The second, which is not significantly influenced by F508del, involves full unfolding of NBD1. Mg-ATP binding delays the first transition, thereby offsetting the effect of F508del on domain stability. Evidence suggests that the initial partial unfolding transition is partially responsible for the poor in vitro solubility of human NBD1. Second-site mutations that increase the solubility of isolated F508del-NBD1 in vitro and suppress the trafficking defect of intact F508del-CFTR in vivo also stabilize the protein against this transition, supporting the hypothesize that it is responsible for the pathological trafficking of F508del-CFTR. [source] Determinants of activation kinetics in mammalian hyperpolarization-activated cation channelsTHE JOURNAL OF PHYSIOLOGY, Issue 1 2001Takahiro M. Ishii 1The structural basis for the different activation kinetics of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels was investigated with the whole-cell patch clamp technique by using HCN1, HCN4, chimeric channels and mutants in a mammalian expression system (COS,7). 2The activation time constant of HCN4 was about 40-fold longer than that of HCN1 when compared at ,100 mV. 3In chimeras between HCN1 and HCN4, the region of the S1 transmembrane domain and the exoplasmic S1-S2 linker markedly affected the activation kinetics. The cytoplasmic region between S6 and the cyclic nucleotide-binding domain (CNBD) also significantly affected the activation kinetics. 4The S1 domain and S1-S2 linker of HCN1 differ from those of HCN4 at eight amino acid residues, and each single point mutation of them changed the activation kinetics less than 2-fold. However, the effects of those mutations were additive and the substitution of the whole S1 and S1-S2 region of HCN1 by that of HCN4 resulted in a 10, to 20-fold slowing. 5The results indicate that S1 and S1-S2, and S6-CNBD are the crucial components for the activation gating of HCN channels. [source] Structures of the nucleotide-binding domain of the human ABCB6 transporter and its complexes with nucleotidesACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2010Matthias Haffke The human ATP-binding cassette (ABC) transporter ABCB6 is involved in haem-precursor transport across the mitochondrial membrane. The crystal structure of its nucleotide-binding domain (NBD) has been determined in the apo form and in complexes with ADP, with ADP and Mg2+ and with ATP at high resolution. The overall structure is L-shaped and consists of two lobes, consistent with other reported NBD structures. Nucleotide binding is mediated by the highly conserved Tyr599 and the Walker A motif, and induces notable structural changes. Structural comparison with other structurally characterized NBDs and full-length ABC transporters gives the first insight into the possible catalytic mechanism of ABCB6 and the role of the N-terminal helix ,1 in full-length ABCB6. [source] Direct inter-subdomain interactions switch between the closed and open forms of the Hsp70 nucleotide-binding domain in the nucleotide-free stateACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2010Meiri Shida The 70,kDa heat-shock proteins (Hsp70s) are highly conserved chaperones that are involved in several cellular processes, such as protein folding, disaggregation and translocation. In this study, the crystal structures of the human Hsp70 nucleotide-binding domain (NBD) fragment were determined in the nucleotide-free state and in complex with adenosine 5,-(,,,-imido)triphosphate (AMPPNP). The structure of the nucleotide-free NBD fragment is similar to that of the AMPPNP-bound NBD fragment and is designated as the `closed form'. In the nucleotide-free NBD fragment the closed form is intrinsically supported through interactions between Tyr15, Lys56 and Glu268 which connect subdomains IA, IB and IIB at the centre of the protein. Interaction with the substrate-binding domain (SBD) of Hsp70 or the BAG domain of BAG1 impairs this subdomain connection and triggers the rotation of subdomain IIA around a hydrophobic helix from subdomain IA. The subdomain rotation is limited by Asp199 and Asp206 from subdomain IIA and clearly defines the open form of the NBD. The open form is further stabilized by a new interaction between Gly230 from subdomain IIB and Ser340 from subdomain IIA. The structure of the NBD in the nucleotide-free state is determined by switching of the inter-subdomain interactions. [source] The structure of Mg-ATPase nucleotide-binding domain at 1.6,Å resolution reveals a unique ATP-binding motifACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2009Kjell O. Håkansson The structure of the nucleotide-binding domain of the Mg-ATPase MgtA from Escherichia coli has been solved and refined to 1.6,Å resolution. The structure is made up of a six-stranded ,-sheet and a bundle of three ,-helices, with the nucleotide-binding site sandwiched in between. The MgtA nucleotide-binding domain is shorter and more compact compared with that of the related Ca-ATPase and lacks one of the ,-strands at the edge of the ,-sheet. The ATP-binding pocket is surrounded by three sequence and structural motifs known from other P-type ATPases and a fourth unique motif that is found only in Mg-ATPases. This motif consists of a short polypeptide stretch running very close to the ATP-binding site, while in Ca-ATPase the binding site is more open, with the corresponding polypeptide segment folded away from the active site. [source] Cloning, expression, purification and preliminary X-ray crystallographic studies of Escherichia coli Hsp100 nucleotide-binding domain 2 (NBD2)ACTA CRYSTALLOGRAPHICA SECTION D, Issue 6-2 2002Jingzhi Li Escherichia coli Hsp100 ClpB has been identified recently as playing critical roles in multi-chaperone systems. ClpB binds and disaggregates denatured polypeptides by employing ATP hydrolysis and allows other molecular chaperones such as Hsp70 DnaK and Hsp40 DnaJ to refold the non-native polypeptides. ClpB contains two nucleotide-binding domains (NBD1 and NBD2) in its primary sequence. Walker A and Walker B motifs exist in both nucleotide-binding domains. Therefore, ClpB belongs to the large ATPase family known as ATPase associated with various cellular activities (AAA). The mechanisms by which NBD1 and NBD2 function to support the ClpB molecular-chaperone activity are currently unknown. To investigate how NBD2 participates in ClpB function to disaggregate denatured proteins, ClpB NBD2 has been cloned and crystallized. The ClpB NBD2 crystals diffract X-rays to 2.5,Å using synchrotron X-ray sources. The crystals belong to space group P212121, with unit-cell parameters a = 99.57, b = 149.34, c = 164.69,Å. [source] Structure of the nucleotide-binding domain of Plasmodium falciparum Rab6 in the GDP-bound formACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2000Debasish Chattopadhyay Rab proteins are small Ras-like GTPases which play important roles in regulating intracellular vesicle trafficking. The nucleotide-binding domain of Rab6 from the malaria parasite Plasmodium falciparum was crystallized with GDP bound to the active site. The MAD phasing technique was used to determine the crystal structure to 2.3,Å resolution. Comparisons of the structure of GDP-bound PfRab6 with the recently determined structures of Rab3A in complex with either a GTP analog or with GTP and Rabphillin present structural evidence supporting the traditional model for the molecular GTP/GDP switch in Rab proteins. PfRab6 residues homologous to those distinguishing human Rab6 isoforms, which differ in binding to Rabkinesin-6 in human cells, are located next to the recognized complementarity-determining region (CDR) and constitute a conceptual broadening of that domain. Despite significant observable differences in Golgi ultrastructure, the Rab6 core structure and switch mechanism appear highly conserved when compared with murine Rab3a structures. A significant difference between the PfRab6 and higher eukaryotic Rabs may be the lack of CDR features that allow binding interactions with Rabkinesin-type effectors. [source] Crystallization and data collection of the nucleotide-binding domain of Mg-ATPaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Kjell O. Håkansson Understanding of how P-type ATPases work would greatly benefit from the elucidation of more high-resolution structures. The nucleotide-binding domain of Mg-ATPase was selected for structural studies because Mg-ATPase is closely related to eukaryotic Ca-ATPase and Na,K-ATPase while the nucleotide-binding domain itself has diverged substantially. Two fragments of Mg-ATPase were cloned in Escherichia coli and purified. The entire cytoplasmic loop (residues 367,673), consisting of the phosphorylation and nucleotide-binding domains, expressed well and was purified in large quantities. The smaller 19.5,kDa nucleotide-binding domain (residues 383,545) expressed less well but formed crystals that diffracted to a resolution of 1.53,Å which will be used for molecular replacement. [source] Cloning, expression, purification and preliminary X-ray crystallographic studies of Escherichia coli Hsp100 nucleotide-binding domain 2 (NBD2)ACTA CRYSTALLOGRAPHICA SECTION D, Issue 6-2 2002Jingzhi Li Escherichia coli Hsp100 ClpB has been identified recently as playing critical roles in multi-chaperone systems. ClpB binds and disaggregates denatured polypeptides by employing ATP hydrolysis and allows other molecular chaperones such as Hsp70 DnaK and Hsp40 DnaJ to refold the non-native polypeptides. ClpB contains two nucleotide-binding domains (NBD1 and NBD2) in its primary sequence. Walker A and Walker B motifs exist in both nucleotide-binding domains. Therefore, ClpB belongs to the large ATPase family known as ATPase associated with various cellular activities (AAA). The mechanisms by which NBD1 and NBD2 function to support the ClpB molecular-chaperone activity are currently unknown. To investigate how NBD2 participates in ClpB function to disaggregate denatured proteins, ClpB NBD2 has been cloned and crystallized. The ClpB NBD2 crystals diffract X-rays to 2.5,Å using synchrotron X-ray sources. The crystals belong to space group P212121, with unit-cell parameters a = 99.57, b = 149.34, c = 164.69,Å. [source] Crystallization and data collection of the nucleotide-binding domain of Mg-ATPaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Kjell O. Håkansson Understanding of how P-type ATPases work would greatly benefit from the elucidation of more high-resolution structures. The nucleotide-binding domain of Mg-ATPase was selected for structural studies because Mg-ATPase is closely related to eukaryotic Ca-ATPase and Na,K-ATPase while the nucleotide-binding domain itself has diverged substantially. Two fragments of Mg-ATPase were cloned in Escherichia coli and purified. The entire cytoplasmic loop (residues 367,673), consisting of the phosphorylation and nucleotide-binding domains, expressed well and was purified in large quantities. The smaller 19.5,kDa nucleotide-binding domain (residues 383,545) expressed less well but formed crystals that diffracted to a resolution of 1.53,Å which will be used for molecular replacement. [source] | |||||||||||||