Browsing by Author "Carla Mattos, Committee Chair"

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    1.9A Crystal Structure of the Rap1a GTPase Bound to its Natural Ligand, GTP
    (2007-01-21) Miller, Christopher Michael; Carla Mattos, Committee Chair; Clay Clark, Committee Member; Robert Rose, Committee Member
    Rap1a is a small GTPase in the Ras superfamily whose most well known function is to antagonize the Ras. Rap1a and Ras share common effectors which allow Rap1a to either unproductively bind Ras' effectors forming an inactive complex or sequester Ras' effectors away from the plasma membrane where Ras is inserted by C-terminal post-translational modifications. To date, a 2.2Å crystal structure of Rap1a bound to the non-hydrolyzable GTP analogue, GMPPNP, and one of its effectors, Raf-1, has been solved. This thesis presents the 1.9Å monomeric form of Rap1a bound to its natural ligand, GTP. Comparisons made between the previously published Rap—Raf structure, Rap2a, H-Ras, and RalA shed some light on the functions for conserved areas of Rap1a. The presence of a unique salt bridge at the Rap⁄Raf interface, a new conformation of threonine 61, a possible link for switch the II residue phenylalanine 64 with GAP-induced GTPase activity, and a suggested role for α helix 4 contribute to the Rap1a story.
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    Application of the Multiple Solvent Crystal Structures Method to Analyze the Protein Binding Surface of H-Ras Protein
    (2006-05-02) Buhrman, Gregory Kale; Carla Mattos, Committee Chair
    H-Ras is a member of the small, monomeric GTPase protein superfamily. H-Ras functions as a 'molecular switch', using nucleotide dependent conformational changes to relay signals in a number of signal transduction pathways. Mutations in codons 12, 13 and 61 creates an oncogenic version of the protein which does not hydrolyze GTP, resulting in the constitutive activation of downstream effector proteins. Ras proteins participate in multiple protein : protein interactions in the cell, making Ras a good candidate protein to extend the Multiple Solvent Crystal Structures method (MSCS) to the analysis and prediction of protein binding surfaces. MSCS involves solving the crystal structure of the protein after soaking the protein crystal in a variety of organic solvent molecules. Replacing an aqueous solvent with an organic solvent affects the Ras protein structure in several ways. The disordered Switch II region of Ras is ordered in the presence of 2,2,2-trifluoroethanol or 1,6-hexanediol. Polar interactions that stabilize the ordered switch are enhanced in the presence of hydrophobic co-solvents. This suggests that hydrophobic solvents can be used in general to order short biologically relevant segments of disordered regions in protein crystals. We have used MSCS to study two crystal forms of active H-Ras bound to a nonhydrolyzable GTP analog (GMPPNP). We have also solved the structure of an oncogenic mutant of H-Ras (Q61L) in a non-canonical crystal form. This crystal form of H-Ras shows a new conformation for the flexible Switch II region that is not affected by crystal packing forces. This provides a structural explanation for the oncogenic properties of the Q61L mutation, showing that the Q61L mutation stabilizes a non-catalytic conformation of Switch II. MSCS analysis of Ras identifies the known Ras-effector binding domain as a site of protein: protein interaction and predicts a new protein binding site that is located in a large, solvent exposed pocket between Switch II and helix 3. In applying MSCS to the Ras protein, we show that by using polar organic solvent molecules as probes, we can identify binding sites that are highly charged and dynamic.
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    H-Ras and Its Oncogenic Mutants Ras G12V and Ras Q61L
    (2007-04-17) Holsenbeck, Stephanie Leah; Carla Mattos, Committee Chair; Linda Hanley-Bowdoin, Committee Member; Robert Rose, Committee Member
    The H-Ras protein is a GTPase important to cell cycle and differentiation. Mutations in this protein have been associated with 30% of cancers. A better understanding of this protein could lead to innovative treatments of cancers caused by the mutations. Solvent mapping of the crystallized Q61L mutant of the H-Ras protein with glycerol shows with potential areas of protein⁄protein interactions, areas that are of particular interest in the design of anti-cancer drugs. This study investigated whether glycerol can distinguish changes in the surface due to the mutation. A comparison was made between the wild-type and Q61L mutant H-Ras crystal structures in glycerol and aqueous solution. The structural analysis lead to the conclusion that the main changes observed were due to the solvent environment and not to the mutation.
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    Protein Interactions: the Multiple Solvent Crystal Structures of RNAse A and Analysis of the RalA and RalBP Complex
    (2009-12-01) Dechene, Michelle Christine; Alexander Tropsha, Committee Member; Robert Rose, Committee Member; M. Celeste Sagui, Committee Member; Carla Mattos, Committee Chair
    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.
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    Study of Protein Binding Sites on the GTPase RalA and the Sugar-binding Protein Hen Egg White Lysozyme
    (2006-08-10) Nicely, Nathan; Robert Kelly, Committee Member; Carla Mattos, Committee Chair; Robert Rose, Committee Member; Dennis Brown, Committee Member; William L. Miller, Committee Member
    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.