An active allosteric mechanism in ASAP1-mediated Arf1 GTP hydrolysis redefines PH domain function
GTPase-activating proteins are important regulators of small GTPases; among these, ASAP1 stimulates GTP hydrolysis on Arf1 and is implicated in cancer progression. ASAP1 contains a Pleckstrin Homology (PH) domain essential for maximum Arf·GTP hydrolysis. The prevailing view of PH domains is that they regulate proteins through passive mechanisms like membrane recruitment. In sharp contrast, we show that the PH domain of ASAP1 actively contributes to Arf1 GTP hydrolysis. By combining NMR, molecular dynamics simulations, kinetic assays, and mutational analysis, we find that the PH domain binds Arf·GTP at the membrane, to establish an active state primed for GTP hydrolysis. We identify key residues on the PH domain and Arf that drive this allosteric mechanism, which mathematical modeling shows contributes as much to GTPase activation as membrane recruitment. The finding that PH domains directly modulate small GTPases has broad implications for the Ras and Rho oncoprotein families.
Authors
- Alexander J. Sodt (ORCID: https://orcid.org/0000-0002-5570-8212)
- Bo Hu (ORCID: https://orcid.org/0000-0002-9491-4123)
- R. Andrew Byrd (ORCID: https://orcid.org/0000-0003-3625-4232)
- Margaret E. Johnson (ORCID: https://orcid.org/0000-0001-9881-291X)
- Olivier Soubias (ORCID: https://orcid.org/0000-0003-4819-2753)
- Frank Heinrich (ORCID: https://orcid.org/0000-0002-8579-553X)
- Rebekah Jackson (ORCID: https://orcid.org/0000-0002-9195-3186)
- Samuel L. Foley
- Eric Rosenberg (ORCID: https://orcid.org/0000-0001-9016-3839)
- Paul A. Randazzo (ORCID: https://orcid.org/0000-0001-5349-0881)
- Jess Li (ORCID: https://orcid.org/0000-0001-7708-8555)
- Xiaoying Jian (ORCID: https://orcid.org/0000-0003-3556-0553)
- Yue Zhang (ORCID: https://orcid.org/0000-0003-4367-4470)
Institutions
- National Institutes of Health (US)
- Johns Hopkins University (US)
- Johns Hopkins Medicine (US)
- Frederick National Laboratory for Cancer Research (US)
- National Cancer Institute (US)
- Eunice Kennedy Shriver National Institute of Child Health and Human Development (US)
- Center for Cancer Research (US)
- Ribon Therapeutics (United States) (US)
- NIST Center for Neutron Research (US)
- Carnegie Mellon University (US)
Publication Details
- Journal
- Nature Communications
- Published
- 2025-09-30
- DOI
- https://doi.org/10.1038/s41467-025-63764-w
- Citations
- 1
- Primary Topic
- Cellular transport and secretion
- Type
- article
- Field-Weighted Citation Impact
- 1.15
Funders
- U.S. Department of Commerce
- National Institutes of Health
- National Institute of Standards and Technology
- National Cancer Institute
- National Institute of Child Health and Human Development
- Eunice Kennedy Shriver National Institute of Child Health and Human Development