The dynamics underlying enhancement of E2 ligase activity by E3 ligases in sumoylation

Abstract

Covalent attachment of SUMO (Small Ubiquitin Like Modifier) to proteins, sumoylation, is a posttranslational modification that can alter intracellular localization, interactions with other proteins or lead to modifications by other post-translational modifiers. Defects in sumoylation pathway are related to many neurological diseases, such as Huntington’s disease, Parkinson’s disease and more. Additionally, sumoylation is a part of cancer related pathways. Similar to other ubiquitin like modifier (Ubl) conjugation mechanisms, the conjunction of SUMO to targtes involves three groups of enzymes: E1 ligase, Aos1/Ub2 hetero-dimer; E2 ligase, Ubc9; E3 ligases, one of which is RanBP2. Differing from the other Ubl conjunction paths, the E2 ligase, Ubc9, can function without the E3 enzymes but with lower reaction efficiency. One of the target proteins that can be efficiently sumoylated by Ubc9 only, is RanGAP1. Although there are suggested models, it is not clear to date how the E3 enzyme, RanBP2, enhances sumoylation. This thesis mainly aims to identify the conformational/configurational restrictions and allosteric effects that RanBP2 may have on Ubc9-SUMO complex to increase sumoylation rate. Along, the structural motion that drives Ubc9-SUMO complex into association with RanBP2 is also addressed. For this, Ubc9-SUMO and Ubc9- SUMO-RanBP2 complexes from Ubc9-SUMO-RanGAP1-RanBP2 crystal structure are studied by Molecular Dynamics (MD) simulations. The conformational dynamics are elaborated by various means to reflect the equilibrium and dynamic behavior of these complex structures. The results in general suggest that RanBP2 restricts the conformational space of Ubc9-SUMO complex and as well as the orientational space of its monomers with respect to each other. The differences in the network of interactions between Ubc9 and SUMO residues in RanBP2 bound and unbound states suggest the determinants of the restriction in the motion observed. The correlations between the fluctuations of the residues associated with the catalytic activity and the residues that are responsible for the specific target recognition in Ubc9 are shown to be stabilized with RanBP2 binding. The comparative analysis of the dynamics with and without RanBP2 identifies a possible allosteric effect of RanBP2 binding on the mobility and flexibility of specific Ubc9 residues, Asp100 and Lys101, which are functional in target recognition. Additionally, it is seen that the dynamics of Ubc9-SUMO complex displays a pre-existing behavior for the binding to RanBP2. This may in general imply that the dynamics of structures set the sequence of events in the association with others to form complex structures.