Protein Interactions: the Multiple Solvent Crystal Structures of RNAse A and Analysis of the RalA and RalBP Complex

Show full item record

Title: Protein Interactions: the Multiple Solvent Crystal Structures of RNAse A and Analysis of the RalA and RalBP Complex
Author: Dechene, Michelle Christine
Advisors: Alexander Tropsha, Committee Member
Robert Rose, Committee Member
M. Celeste Sagui, Committee Member
Carla Mattos, Committee Chair
Abstract: In both structure and function, Ribonuclease A (RNAse A) and RalA are two very different proteins. RNAse A is an extracellular digestive enzyme that catalyzes the breakdown of 3’-5’ phosphodiester linkages in single stranded RNA. RalA is a small monomeric GTPase of the Ras family and is involved in a number of signaling pathways. While the basic fold of RalA is similar to the rest of the Ras family, Ral proteins have a distinct effector binding region and set of effector proteins. RalBP was the first RalA effector identified and it links RalA to receptor mediated-endocytosis and regulation of mitosis. RNAse A is a small kidney shaped protein with a well defined active site cleft running between the two lobes. The active site consists of several pockets, which are responsible for binding nucleotide bases and phosphate moieties of the RNA substrate. This enzyme is well studied and with over 40 years of structural information available, it is an excellent model protein for quantitatively defining the strengths of the Multiple Solvent Crystal Structures (MSCS) Method. MSCS is an experimental method using small organic solvent molecules to map the surface of proteins, and in addition to locating binding sites, provides information about patterns of protein hydration and plasticity. Twenty two solvent soaked structures were generated revealing 16 organic solvent molecules and 12 sulfate ions clustered in the active site, specifically in the two nucleotide-binding pockets, B1 and B2, and in the catalytic pocket, P1. A comparison of the solvent clusters and the available RNAseA-inhibitor structures revealed that the probe molecules interact with key hot spot residues necessary for ligand binding. Additionally, conserved water molecules were identified on the surface of RNAse A. Outside of the active site, many of these water molecules are involved in stabilizing interactions, or are associated with one of the three helices of RNAse A. In the active site, 9 well ordered water molecules, which stabilize the active site, bridge the interaction between the ligand and the active site residues, or are displaced upon ligand binding, were identified. These patterns of hydration are consistent with earlier analyses of RNAse A. Finally, RMSD and the hinge angle were used as tools to quantitate the plasticity observed at each residue and overall domain motions relative to one another, respectively. In addition to identifying rigid residues of the active site and those displaying more motion, it was found that the trends observed in the MSCS structures correlated well with those observed in other crystal and NMR structures of RNAse A. RalA interacts with effector proteins through its two flexible regions, termed switch I and II, which adopt different conformations in response to its nucleotide binding state. Effector proteins recognize RalA in the GTP-bound “on†state, and bind through these switch region. Where the Ras Binding Domains (RBD) of Ras effectors all adopt a similar fold and interact with active Ras through an intermolecular β-sheet involving switch I, the recent structures of RalA-effector complex structures of RalA-Sec5 and RalA-Exo84 reveal Ral effector Ral binding domains differ in structure and in the binding mode with RalA. In a third Ral effector, RalBP, the Ral-binding domain is predicted to be α-helical, which is different from the β-sandwich structures of Sec5 and Exo84, suggesting the RalA-RalBP interaction presents a previously unobserved binding mode. Furthermore, structural analysis using circular dichroism revealed that the Ral binding domain of RalBP is intrinsically disordered and folds upon binding to RalA. This is the first example of a Ras family effector with this behavior. Significant advances have been made towards the crystallizing of the RalA-RalBP complex, resulting in preliminary crystals.
Date: 2009-12-01
Degree: PhD
Discipline: Functional Genomics
URI: http://www.lib.ncsu.edu/resolver/1840.16/3190


Files in this item

Files Size Format View
etd.pdf 9.296Mb PDF View/Open

This item appears in the following Collection(s)

Show full item record