Autoinhibition of Arf GTPase-activating protein activity by the BAR domain in ASAP1.
ASAP1 is an Arf GTPase-activating protein (GAP) that functions on membrane surfaces to catalyze the hydrolysis of GTP bound to Arf. ASAP1 contains a tandem of BAR, pleckstrin homology (PH), and Arf GAP domains and contributes to the formation of invadopodia and podosomes. The PH domain interacts with the catalytic domain influencing both the catalytic and Michaelis constants. Tandem BAR-PH domains have been found to fold into a functional unit. The results of sedimentation velocity studies were consistent with predictions from homology models in which the BAR and PH domains of ASAP1 fold together. We set out to test the hypothesis that the BAR domain of ASAP1 affects GAP activity by interacting with the PH and/or Arf GAP domains. Recombinant proteins composed of the BAR, PH, Arf GAP, and Ankyrin repeat domains (called BAR-PZA) and the PH, Arf GAP, and Ankyrin repeat domains (PZA) were compared. Catalytic power for the two proteins was determined using large unilamellar vesicles as a reaction surface. The catalytic power of PZA was greater than that of BAR-PZA. The effect of the BAR domain was dependent on the N-terminal loop of the BAR domain and was not the consequence of differential membrane association or changes in large unilamellar vesicle curvature. The Km for BAR-PZA was greater and the kcat was smaller than for PZA determined by saturation kinetics. Analysis of single turnover kinetics revealed a transition state intermediate that was affected by the BAR domain. We conclude that BAR domains can affect enzymatic activity through intraprotein interactions.
Jian
Xiaoying
X
Laboratory of Cellular and Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Brown
Patrick
P
Schuck
Peter
P
Gruschus
James M
JM
Balbo
Andrea
A
Hinshaw
Jenny E
JE
Randazzo
Paul A
PA
D020823:ADP-Ribosylation Factor 1 / Q000737:chemistry / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding / Q000502:physiology; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
C117891:ASAP1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D011994:Recombinant Proteins; D020823:ADP-Ribosylation Factor 1