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|Title: ||Study of Protein Binding Sites on the GTPase RalA and the Sugar-binding Protein Hen Egg White Lysozyme|
|Authors: ||Nicely, Nathan|
|Advisors: ||Robert Kelly, Committee Member|
Carla Mattos, Committee Chair
Robert Rose, Committee Member
Dennis Brown, Committee Member
William L. Miller, Committee Member
|Keywords: ||protein interfaces|
protein binding sites
|Issue Date: ||10-Aug-2006|
|Abstract: ||Hen egg white lysozyme and simian RalA are two very different proteins by function and category. Lysozyme is an extracellular enzyme that catalyzes the hydrolysis of the β-linkage between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) subunits in the peptidoglycan polymers that compose some Gram-positive bacterial cell walls. RalA is a Ras-related GTPase involved in multiple distinct signaling pathways. The structure and sequence of its core domain is similar to Ras and Rap (another Ras-related GTPase), but they have mutually exclusive sets of upstream activators and downstream effectors. Furthermore, RalA is activated by calcium-loaded Calmodulin through its carboxy-terminal domain, and it binds phospholipase D constitutively through its amino-terminal domain; both traits are unique within the Ras subfamily.
Lysozyme has a deep active site cleft between two subdomains which is responsible for binding the saccharide substrate. This binding site is small in its surface area compared to the total accessible surface area of lysozyme. It is relatively well-ordered and pre-formed, with good shape complementarity to the substrate. We employ the Multiple Solvent Crystal Structures method which uses small organic solvent molecules as probes to map the functional surface of the protein. Of ten solvent-soaked crystal structures, 11 solvent molecules were identified as bound to lysozyme in a total of six sites. Nine of these 11 solvent molecules bound in the active site cleft in well defined clusters corresponding to the established subsites in which the NAM/NAG subunits of the natural substrates bind. Five of these nine bind in subsite C, which has the most favorable binding energy of the six subsites. Two bind in subsite D and one each in subsites E and F. The positions and orientations of the bound solvent molecules mimic the acetamido functional groups on the NAM/NAG subunits, especially in subsite C. Of the two organic solvent molecules which bound outside the active site cleft, one bound at a two-fold crystal contact and the other on the edge of the epitope for an anti-lysozyme antibody.
RalA has two large segments, termed the switch regions (I & II), that experience disorder-to-order transitions upon complexation with binding partners. These regions are responsible for significant structural changes across a large patch of the protein's accessible surface. We have solved the crystal structures of RalA in both its GDP- ("off;" inactivated) and GTP analog-bound ("on;" activated) forms. Disorder-to-order transitions occur in both switch regions upon protein-protein interaction in the form of crystallographic and noncrystallographic symmetry contacts; however, in the absence of such protein-protein contacts, both switches are disordered. This indicates a departure from the behavior of Ras in which the presence of GTP analog alone is sufficient to order switch I. Also, we identify two possible sites for protein-protein interaction on the surface of RalA by comparing structural features of the protein with the available data regarding amino acid residues important for its biochemical functions and including the experimental functionality map for Ras generated by the Multiple Solvent Crystal Structures method.
A thorough analysis of the binding sites on RalA and lysozyme reveal some trends which agree with recent hypotheses on the nature of protein-ligand interfaces. First, all the binding sites on both proteins tend to have centers which are relatively invariant in terms of structural plasticity. These cores are surrounded by residues which exhibit conformational flexibility. Second, the binding sites are sparsely hydrated; any bound water molecules at our binding sites can be displaced by solvent molecules. Conversely, the switch regions of RalA are well hydrated at protein-protein contacts, reflecting the ability of water molecules to contribute to the close packing of atoms and charge complementarity in protein-ligand interfaces.|
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