Interconnecting Flexibility, Structural Communication, and Function in RhoGEF Oncoproteins

Dbl family Rho guanine nucleotide exchange factors (RhoGEFs) play a central role in cell biology by catalyzing the exchange of guanosine 5'-triphosphate for guanosine 5'-diphosphate (GDP) on RhoA. Insights into the oncogenic constitutive activity of the Lbc RhoGEF were gained by analyzing the structure and dynamics of the protein in different functional states and in comparison with a close homologue, leukemia-associated RhoGEF. Higher intrinsic flexibility, less dense and extended structure network, and less stable allosteric communication pathways in Lbc, compared to the nonconstitutively active homologue, emerged as major determinants of the constitutive activity. Independent of the state, the essential dynamics of the two RhoGEFs is contributed by the last 10 amino acids of Dbl homology (DH) and the whole pleckstrin homology (PH) domains and tends to be equalized by the presence of RhoA. The catalytic activity of the RhoGEF relies on the scaffolding action of the DH domain that primarily turns the switch I (SWI) of RhoA on itself through highly conserved amino acids participating in the stability core and essential for function. Changes in the conformation of SWI and disorganization of the RhoA regions deputed to nucleotide binding are among the major RhoGEF effects leading to GDP release. Binding of RhoA reorganizes the allosteric communication on RhoGEF, strengthening the communication among the canonical RhoA binding site on DH, a secondary RhoA binding site on PH, and the binding site for heterotrimeric G proteins, suggesting dual roles for RhoA as a catalysis substrate and as a regulatory protein. The structure network-based analysis tool employed in this study proved to be useful for predicting potentially druggable regulatory sites in protein structures.