Structural Biology of GTPase Activating Proteins

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

In collaboration with Dr. Paul Randazzo, we are studying the structure and interactions between Arf proteins and a member of the ASAP family of Arf-GAP proteins. We have produced a variety of stable-isotope variants of these proteins and established conditions for NMR spectroscopic studies. Studies are underway to provide resonance assignments, structure confirmation based on existing known structures and to determine structures which are unknown, binding studies of cofactors, and to examine the assembly of larger multi-domain and multi-component systems. New findings emerged on the role of the PH domain within ASAP1, and an integrated multi-disciplinary study was published in Science Advances this year. We have developed methods to examine these proteins in a membrane environment. We will also combine the use of Neutron Reflectometry (NR) data with novel NMR and EPR methods to develop three-dimensional structural models of these complexes. We have utilized unique methodology in the NMR spectroscopy, structure calculation and isotopic labeling to obtain new structural information about ASAP1. These data are combined with binding information to activating lipids and membrane mimetics to pursue functional understanding of these important proteins. Previous developments include structural elucidation by X-ray and NMR of the PH domain from ASAP1 interacting with PIP2 lipid head groups. Our current work demonstrates binding to nano disk membrane mimetics, the equivalence of the mimetic and large unilamellar membrane standards, and we have described a unique cooperative binding mechanism of the ASAP1 PH domain with membrane surfaces. We have demonstrated that we can assemble functional and isotopic labeled (for NMR) myristoylated-Arf1 into membrane nano disks. This systems shows functional complexation with ASAP1 functional domains at the membrane surface. Current work in submission for publication describes the distribution of and conformational selection process involved in Arf1 recognition of ASAP1. Understanding the mechanism of interaction between Arf1 and ASAP1-PZA is progressing extremely well, with publications and molecular insight.