Biochemistry and Molecular Pharmacology
Program in Molecular Medicine
First Thesis Advisor
David Lambright, PhD
Catalytic Domain, Cell Membrane, GTP Phosphohydrolases, Guanine Nucleotide Exchange Factors, Phosphatidylinositols
Dissertations, UMMS; Catalytic Domain; Cell Membrane; GTP Phosphohydrolases; Guanine Nucleotide Exchange Factors; Phosphatidylinositols
Guanine nucleotide exchange factors (GEFs) regulate and organize diverse cellular processes through their role in converting GTPases from the inactive GDP bound state to the active GTP bound state. An increasing number of GEFs undergo autoregulatory mechanisms through complex intramolecular interactions. Relief of autoinhibition involves specific phosphorylation or binding to lipid and/or effector proteins at sites distal from the catalytic domain, and is often coupled to membrane recruitment. In Cytohesin Arf GEFs, the catalytic Sec7 domain is autoinhibited by a linker region and C-terminal helix flanking a Pleckstrin Homology (PH) domain. Upon binding of the PH domain to low abundance phosphoinositides, the GTPase Arf6-GTP can both relieve autoinhibition and recruit Cytohesins to the plasma membrane. This thesis focuses on determining the molecular mechanism underlying both these functions.
The structural mechanisms by which Arf6-GTP binding relieves autoinhibition were studied using biochemical and crystallographic studies. The crystal structure of the Grp1 PH domain in complex with Arf6 revealed that Arf6-GTP binding relieves autoinhibition through competitive sequestration of the inhibitory elements into grooves formed at the periphery of the interface. Importantly, the interaction orients all known membrane targeting components to a common surface. Detailed biochemical studies showed a common mode of binding among Cytohesin family members in which phosphoinositide head group binding primes the interaction with Arf6, and membrane recruitment of both stimulatory and substrate Arf enhances the effect.
To assess changes in the Sec7 domain conformation upon activation, Size Exclusion Chromatography in line with Small Angle X-Ray Scattering (SEC-SAXS) was performed. The unique nature of this data led to the development of a novel data analysis and processing strategy. A graphically based, python-extensible software package was created for data normalization, buffer correction, Guinier Analysis, and constant background subtraction. As an unbiased substitute for traditional buffer subtraction, a method to reconstruct the protein scattering through singular value decomposition (SVD) and linear combination of the basis vectors was developed. These methods produced exceptional data quality and allowed versatility for application to other data collection techniques or systems, especially those lacking confident buffer matching or low signal.
SEC-SAXS confirmed the overall structure of autoinhibited Grp1 in solution and showed only slight overall changes upon activation by deletion of the autoinhibitory Cterminal helix. Fusion of Arf6 with Grp1 produced a consistently elongated shape in the active state that was incompatible with the autoinhibited or theoretical active positions of the Sec7 domain. Monte Carlo and rigid body modeling using known structural domains revealed a requirement for Sec7-PH linker flexibility in addition to Sec7 domain mobility. These data support an integrated structural model whereby phosphoinositides and Arf-GTP support nucleotide exchange at membranes through allosteric activation, membrane recruitment, and large-scale rearrangement of the Sec7 domain. Overall, these findings offer insight into Cytohesin function that can be applied to assess relief of autoinhibition in the context of other GEFs and GTPases.
Malaby, AW. An Integrated Structural Mechanism for Relief of Autoinhibition and Membrane Targeting in Cytohesin Family Guanine Nucleotide Exchange Factors: A Dissertation. (2014). University of Massachusetts Medical School. GSBS Dissertations and Theses. Paper 703. DOI: 10.13028/M2D60X. http://escholarship.umassmed.edu/gsbs_diss/703
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