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 K_m for BAR-PZA was greater and the k_cat 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.The Bin, amphiphysin, RSV161/167 (BAR)² domain is a recently identified structural element in proteins that regulate membrane trafficking (1–7). The BAR superfamily comprises three subfamilies: F-BAR, I-BAR, and BAR. The BAR group can be further subdivided into BAR, N-BAR, PX-BAR, and BAR-pleckstrin homology (PH). The BAR group domains consist of three bundled α-helices that homodimerize to form a banana-shaped structure. The inner curved face can bind preferentially to surfaces with similar curvatures. As a consequence, BAR domains can function as membrane curvature sensors or as inducers of membrane curvature. BAR domains also bind to proteins (8, 9). Several proteins contain a BAR domain immediately N-terminal to a PH domain, which also mediates regulated membrane association (10–13). In the protein APPL1 (9), the BAR-PH domains fold together forming a binding site for the small GTP-binding protein Rab5. Arf GTPase-activating proteins (GAPs) are regulators of Arf family GTP-binding proteins (14–18). Two subtypes of Arf GAPs have N-terminal BAR and PH domains similar to that found in APPL1.Thirty-one genes encode Arf GAPs in humans (16–18). Each member of the family has an Arf GAP domain that catalyzes the hydrolysis of GTP bound to Arf family GTP-binding proteins. The Arf GAPs are otherwise structurally diverse. ASAP1 is an Arf GAP that affects membrane traffic and actin remodeling involved in cell movement and has been implicated in oncogenesis (19–22). ASAP1 contains, from the N terminus, BAR, PH, Arf GAP, Ankyrin repeat, proline-rich, and SH3 domains.ASAP1 contains a BAR domain immediately N-terminal to a PH domain. The PH domain of ASAP1 is functionally integrated with the Arf GAP domain and may form part of the substrate binding pocket (23, 24). The PH domain binds specifically to phosphatidylinositol 4,5-bisphosphate (PIP₂), a constituent of the membrane, leading to stimulation of GAP activity by a mechanism that is, in part, independent of recruitment to membranes (23, 25). The BAR domain of ASAP1 is critical for in vivo function of ASAP1, but the molecular functions of the BAR domain of ASAP1 have not been extensively characterized. Hypotheses related to membrane curvature have been examined. Recombinant ASAP1 can induce the formation of tubules from large unilamellar vesicles, which may be related to a function of ASAP1 in membrane traffic. The BAR domain might also regulate GAP activity of ASAP1. We have considered two mechanisms based on the known properties of BAR domains. First the BAR domain could regulate association of ASAP1 with membrane surfaces containing the substrate Arf1·GTP. The BAR domain could also affect GAP activity through an intramolecular association. In one BAR-PH protein that has been crystallized (APPL1), the two domains fold together to form a protein binding site (9). In ASAP1, the PH domain is functionally integrated with the GAP domain, raising the possibility that the BAR domain affects GAP activity by folding with the PH domain.Here we compared the kinetics of recombinant proteins composed of the PH, Arf GAP, and Ankyrin repeat (PZA)³ or BAR, PH, Arf GAP, and Ankyrin repeat (BAR-PZA) domains of ASAP1 to test the hypothesis that the BAR domain affects enzymatic activity. We found kinetic differences between the proteins that could not be explained by membrane association properties. The results were consistent with a model in which the BAR domain affects transition of ASAP1 through its catalytic cycle.