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NMR Solution Structure of Opa60: A Neisserial Membrane Protein That Mediates Host Phagocytosis

Fox, Daniel Andrew
Thesis/Dissertation; Online
Fox, Daniel Andrew
Columbus, Linda
The family of Opa proteins from Neisseria gonorrhoeae and N. meningitidis are eight-stranded β-barrel outer membrane proteins that induce human cells to engulf the bacterium by engaging three different host receptors: carcinoembryonic antigen cellular adhesion molecules (CEACAM), heparansulfate proteoglycans (HSPG), or integrins via HSPG and fibronectin or vitronectin. The receptor engaged depends on the sequence of two of the extracellular loops (termed hypervariable (HV) loop 1 and 2), which are highly variable between isolates. The sequence variability is generated by multiple mechanisms including recombination among opa alleles (there are 11 opa alleles in N. gonorrhoeae), single point mutations, insertions and deletions, and insertion of opa genes from coinfecting species. There are multitudes of HV sequences identified; however, only approximately 25 Opa protein sequences have been characterized in terms of receptor engagement. Multiple sequence alignment of the HV loops does not reveal specificity motifs among the family of Opa proteins due to the extreme variability in the amino acid sequences. To investigate the determinants of Opa-receptor interactions, the NMR solution structure was determined. In order to solve the structure, a suite of three dimension NMR experiments were performed to obtain an assignment, each optimized for the different domains of the protein. A variety of isotopic labeling techniques were also implemented to resolve crowded regions of the spectra. Opa 60 was cleaved using trypsin to isolate the micelle embedded β-barrel. NMR spectra for the dynamic extracellular iv loops were greatly improved at lower temperatures. Peptides corresponding to the most intrinsically dynamic regions of the hypervariable loops were synthesized to aid in assignment. The structure of Opa 60 that was solved with these restraints was then refined using molecular dynamics simulations. With the aid of all these assignment strategies, the solution NMR structure was determined and reveals that Opa 60 is a canonical eight-stranded β-barrel and the HV loops are long, disordered, and highly dynamic that loosely associate with each other, displaying latent helical content in the hypervariable regions. Note: Abstract extracted from PDF text
University of Virginia, Department of Chemistry, PHD, 2013
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