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Walker A Motif (walker a + motif)

Distribution by Scientific Domains
Life Sciences75%

Selected Abstracts

Uncoupling of the ATPase activity from the branch migration activity of RuvAB protein complexes containing both wild-type and ATPase-defective RuvB proteins

GENES TO CELLS, Issue 9 2003
Takashi Hishida
Background:,Escherichia coli RuvAB promotes branch migration of Holliday junctions during recombination repair and homologous recombination. RuvB forms a hexameric ring through which duplex DNA passes and is translocated in an ATP-dependent manner. ATPase-deficient RuvB mutant K68A has a mutation in the Walker A motif and exerts a dominant-negative effect on in vivo repair of UV-induced DNA damage. In this study, we examined RuvAB-dependent branch migration in the presence of a mutant RuvB, K68A. Results:, Mixing K68A with wild-type RuvB resulted in the formation of heterohexamers that showed unique properties of DNA binding, ATPase, and branch migration activities different from those of either wild-type or mutant homohexamers. RuvB heterohexamers inhibited branch migration and caused Holliday junctions to accumulate during RecA-mediated strand exchange. In the presence of RuvA, RuvB heterohexamers had Holliday junction-dependent ATPase activity, but did not promote branch migration. Conclusions:, These results suggest that functional cooperation among the subunits in the hexamers is required for branch migration, but inclusion of inactive subunits is tolerated for ATP hydrolysis. Therefore, we propose that an essential ATP hydrolysis-dependent functional cooperation is induced in RuvB hexamer subunits during RuvAB-mediated branch migration. [source]

Roles of the two ClpC ATP binding sites in the regulation of competence and the stress response

MOLECULAR MICROBIOLOGY, Issue 3 2001
Kürsad 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]

Structures of the nucleotide-binding domain of the human ABCB6 transporter and its complexes with nucleotides

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2010
Matthias 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]

Roles of the two ClpC ATP binding sites in the regulation of competence and the stress response

MOLECULAR MICROBIOLOGY, Issue 3 2001
Kürsad 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]