P-selectin Targeting to Secretory Lysosomes of Rbl-2H3 Cells

Kaur, Jasber; Cutler, Daniel F.

doi:10.1074/jbc.m111293200

Cited by 16 publications

(21 citation statements)

References 46 publications

(20 reference statements)

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“…To examine this question, mutations in the P-loops of the two NBDs were combined with the M446W mutation individually and together. The G18R and G337R mutations have been shown to result in loss of resistance to arsenicals and loss of nucleotide binding in the A1 or A2 NBD, respectively (6,7,24). In M446W into which the G337R mutation was introduced there was no response of nucleotide under either unisite or multisite conditions (Fig.…”

Section: Under Unisite Conditions Trpmentioning

confidence: 99%

Unisite and Multisite Catalysis in the ArsA ATPase

Zhou

Shen

Liu

et al. 2002

Journal of Biological Chemistry

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The ars operon of plasmid R773 encodes an As(III)/ Sb(III) extrusion pump. The catalytic subunit, the ArsA ATPase, has two homologous halves, A1 and A2, each with a consensus nucleotide-binding sequence. ATP hydrolysis is slow in the absence of metalloid and is accelerated by metalloid binding. ArsA M446W has a single tryptophan adjacent to the A2 nucleotide-binding site. The ars operon of R-factor R773 encodes an arsenite extrusion system that confers resistance in Escherichia coli to the metalloids arsenite (As(III)) and antimonite (Sb(III)) (1). This efflux pump has a catalytic subunit, the ArsA ATPase, and a membrane subunit, the ArsB arsenite carrier (2, 3). ArsA has N-terminal (A1) and C-terminal (A2) halves that are homologous to each other, most likely as the result of ancestral gene duplication and fusion (4). The enzyme has two nucleotidebinding domains, NBD11 and NBD2, both of which are composed of residues from both the A1 and A2 halves (5). Both NBDs are required for metalloid resistance (6, 7).Other pumps with multiple NBDs that exhibit multisite catalysis such as F-type ATPases and ATP-binding cassette ATPases have been proposed to have mechanisms that involve catalytic alternation between the NBDs (where only one is active at a time) and a high degree of cooperativity between the sites (8, 9). Kaur (10) has suggested that ArsA exhibits both unisite and multisite catalysis in which only NBD1 participates in unisite catalysis and that a functional A1 NBD is required for NBD2 to participate in multisite catalysis. We have considered a similar alternating site model for the function of ArsA, where the two NBDs alternate between open and closed conformations in a concerted and interactive manner, coupling the energy of ATP hydrolysis to the transfer of As(III) or Sb(III) at the metal site of ArsA to the ArsB carrier (11, 12). In the absence of metalloid, ArsA catalyzes hydrolysis of ATP at a low basal rate (k ϭ 0.001 s Ϫ1 ) (13). Under presteady state conditions the addition of Sb(III) produces two bursts of phosphate liberation, one of which is ϳ250-fold faster than the other (k ϭ 49 and 0.2 s Ϫ1 ) (12). From these results, it appears that both NBDs hydrolyze ATP in the activated state. However, the two NBDs are not equivalent in either structure (5, 11) or catalytic rates (12), which raises the question of whether they are functionally equivalent or whether they play different roles in metalloid resistance.To examine the role of the individual NBDs, we previously constructed two single tryptophan ArsAs (F141W and W159) that report nucleotide occupancy and hydrolysis in NBD1 (14, 15). In each half there is a 12-residue sequence (DTAPTGH) that is found in all ArsA homologues from bacteria to humans (14). In the ArsA structure these are seen as extended regions that connect the single regulatory metalloid-binding domain with the two NBDs and probably function in signal transduction between the two substrate sites and the regulatory site (5 ATP (14,15). The response of spectroscopic signals to nucleoti...

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Section: Under Unisite Conditions Trpmentioning

confidence: 99%

Unisite and Multisite Catalysis in the ArsA ATPase

Zhou

Shen

Liu

et al. 2002

Journal of Biological Chemistry

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“…The 583-residue ArsA ATPase is composed of two homologous A1 and A2 halves that are connected by a short linker (3). Each of the two halves contains a single NBD (4), both of which are required for ATPase activity and resistance (5,6 (Fig. 10).…”

Section: Discussionmentioning

confidence: 99%

“…First, it could be vestigial and not involved in transport. These seems unlikely because mutagenesis of NBD2 resulted in loss of both As(III) resistance and ATPase activity (6). Second, one NBD could play a regulatory role with the energy for metalloid transport supplied by hydrolysis at the other NBD.…”

Section: Discussionmentioning

confidence: 99%

“…Each contains a nucleotide binding domain (NBD) 1 (4) (Fig. 1), and both NBDs are required for ATPase activity and metalloid transport (5,6). A novel metalloid binding domain located at the A1-A2 interface diametrically opposite to the NBDs modulates ATPase activity (4,7,8).…”

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confidence: 99%

“…Purification of [His] 6 -tagged ArsA ATPases-Wild type or ThArsA were purified from cultures of E. coli strain JM109 harboring either the wild type or tharsA plasmids. Cells were grown at 37°C in LuriaBertani medium to an A 600 of 0.6, at which point 0.1 mM isopropyl 1-thio-␤-D-galactopyranoside was added to induce ArsA expression.…”

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confidence: 99%

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Nonequivalence of the Nucleotide Binding Domains of the ArsA ATPase

Jiang

Bhattacharjee

Zhou

et al. 2005

Journal of Biological Chemistry

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The arsRDABC operon of Escherichia coli plasmid R773 encodes the ArsAB pump that catalyzes extrusion of the metalloids As(III) and Sb(III), conferring metalloid resistance. The catalytic subunit, ArsA, is an ATPase with two homologous halves, A1 and A2, connected by a short linker. Each half contains a nucleotide binding domain. The overall rate of ATP hydrolysis is slow in the absence of metalloid and is accelerated by metalloid binding. The results of photolabeling of ArsA with the ATP analogue 8-azidoadenosine 5-[␣-32 P]-triphosphate at 4°C indicate that metalloid stimulation correlates with a >10-fold increase in affinity for nucleotide. To investigate the relative contributions of the two nucleotide binding domains to catalysis, a thrombin site was introduced in the linker. This allowed discrimination between incorporation of labeled nucleotides into the two halves of ArsA. The results indicate that both the A1 and A2 nucleotide binding domains bind and hydrolyze trinucleotide, even in the absence of metalloid. Sb(III) increases the affinity of the A1 nucleotide binding domain to a greater extent than the A2 nucleotide binding domain. The ATP analogue labeled with 32 P at the ␥ position was used to measure hydrolysis of trinucleotide at 37°C. Under these catalytic conditions, both nucleotide binding domains hydrolyze ATP, but hydrolysis in A1 is stimulated to a greater degree by Sb(III) than A2. These results suggest that the two homologous halves of the ArsA may be functionally nonequivalent.In Escherichia coli the ArsAB pump encoded by the ars operon of plasmid R773 confers resistance to arsenicals and antimonials (1). ArsA is the catalytic subunit of the pump that hydrolyzes ATP in the presence of the trivalent forms of the two metalloids. ATP hydrolysis is coupled to extrusion of As(III) or Sb(III) through ArsB, which serves both as a membrane anchor for ArsA and as the substrate-conducting pathway. Upon overexpression, ArsA exists primarily as a soluble protein in the cytosol. Soluble ArsA has been purified and characterized and has been shown to be an As(III)/Sb(III)-stimulated ATPase (2).ArsA is composed of homologous N-terminal (A1) and Cterminal (A2) halves that are connected by a short linker (3). Each contains a nucleotide binding domain (NBD) 1 (4) (Fig. 1), and both NBDs are required for ATPase activity and metalloid transport (5, 6). A novel metalloid binding domain located at the A1-A2 interface diametrically opposite to the NBDs modulates ATPase activity (4,7,8). Two stretches of residues, D 142 TAPTGH 148 in A1 and D 447 TAPTGH 453 in A2, physically connect the metalloid binding domain to the A1 and A2 NBDs, respectively. The metalloid binding domain is composed of three metalloid atoms, each of which is coordinated by two protein ligands, one of which is donated by A1 and the other by A2. Thus the metalloid atoms serve as the "molecular glue" that brings the two halves of the protein together, activating catalysis. The basal rate of catalysis has been thought to reflect the activity of onl...

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