Open Access Dissertation
Date of Award
Biochemistry and Molecular Biology
Protein structural elucidation by means of X-ray crystallography is a powerful approach for both insight into a proteins biophysical properties and function. To date X-ray crystallography remains the gold standard in high resolution structural determination and serves as the basis for rational drug design for the purpose of combating a number of human diseases. Such an approach also allows for the exploitation of how various proteins interact with their substrates providing a molecular basis for their physiological function. In this dissertation, using X-ray crystallographic analysis along with other biophysical characterization methods, we seek to understand the mechanistic foundation for which scaffolding proteins such as NHERF1 and NHERF2 interact with their substrates in which leads to a wide variety of critical cellular events. Additionally, we attempt elucidate the structure and function of a recently identified membrane protein, NgBR, with hopes to understand the molecular basis in which it influences the activation of the infamous oncoprotein Ras.
Protein scaffolding is a term that denotes the coming together of two or more proteins in which results in the formation of macromolecular complexes. Such events are known to regulate several cellular processes including membrane protein recycling, protein stabilization, cell to cell adhesion, and regulation of signaling pathways. There are several families of scaffolding proteins with one of the most known to be the PDZ domain containing family. Two proteins within this family, NHERF1 and NHERF2, contain two PDZ domains in which are responsible for a variety of signaling events. The second PDZ domain of NHERF1 has been shown to complex with chemokine receptor CXCR2 and mediate the formation of the CXCR2-NHERF1-PLCβ2 complex. This complex formation has been shown to be influential in chemokine induced neutrophil migration and infiltration in regions of inflammation leading to the exacerbation of a variety of inflammatory diseases. Additionally, the first PDZ domain of NHERF2 has been shown to mediate the scaffolding of the CFTR-NHERF2-LAP2 complex which has been shown to inhibit CFTR activity by the LPA2 mediated inhibition of adenylate cyclase. Furthermore, disruption of this complex has been shown to be enough to augment CFTR activity in vivo. And thus, the determining the molecular basis by means both NHERF1 and NHERF2 scaffolding with its respective targets could lead to therapeutic targeting in a variety of human diseases. In this dissertation, we solved the co-crystal complexes between both NHERF1 PDZ2 with the C-terminal peptide of CXCR2 in addition to solving the co- crystal complex between NHERF2 PDZ1 with the C-terminal LPA2 peptide. Both structures reveal an expected mode of PDZ binding to their respective substrates with various ligand specific interactions. However, both structures also reveal unexpected findings which may unveil additional therapeutic strategies other than targeting the PDZ substrate binding site.
The Nogo-B receptor is a recently identified type one transmembrane receptor with little known function besides its role in binding Nogo-B and promoting angiogenesis by chemotaxis in vivo. However, its most recently identified role is that of binding and facilitating the oncoprotein Ras to the plasma membrane for activation. No X-ray crystallographic structure currently exists for NgBR but previous studies have confirmed to be composed of three primary domains including a cytosolic and extracellular domain with one transmembrane region. However, successive data have suggested that NgBR may exist in more than one topological orientation. Because previous attempts to crystallize NgBR have failed due to solubility issues and the possibility that it might possess various topological orientations, we chose to exploit NgBR’s structural properties using a complimentary approach to X-ray crystallography known as small angle x-ray scattering (SAXS). By incorporation of residues our expression construct in which share sequence identity to the cis-IPTase UPPs, we are able to achieve good solubility and purification of our NgBR construct. Additionally, SAXS analysis reveals that our NgBR construct exists as a globular macromolecule in solution which may pave the way for future high resolution structural studies.
Altogether, X-ray crystallography is a powerful technique for the determination of protein structure. The only bottleneck of this approach is the obtainment of a protein crystal. The final chapter in this thesis overviews well known and leading techniques in the evasion of this rate limiting step in the pursuit of solving molecular structure. And although there is still no one method for guaranteeing the formation of a protein crystal, as can be seen in our studies for the PDZ domains in NHERF1 and NHERF2, X-ray crystallography continues to unveil new structural information and potential therapeutic targets which can be implicated in the advancement of molecular medicine.
Holcomb, Joshua, "Structural Basis Of Membrane Protein Scaffolding And Signaling In Human Disease" (2017). Wayne State University Dissertations. 1809.