This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We are integrating x-ray crystallography, electron microscopy and 3D image reconstruction to determine pseudo-atomic resolution structures of type IV pili, which are key virulence factors in many Gram negative pathogens. Using data collected at SSRL we have solved the structures of type IV pilins from Neisseria gonorrhoeae (GC, 2.6 and Pseudomonas aeruginosa (PAK, 2.0 pilin, and most recently tcpA from Vibrio cholerae classical biotype (1.3 . Both PAK and GC pilin are remarkably similar in structure despite their lack of sequence homology in the C-terminal two-thirds of the protein. Of the type IV pilins, tcpA is the most different in sequence, and its structure reveals a new protein fold which mimics that of GC and PAK pilin fold but does not share the same connectivity. TcpA crystals are arranged as hexagonally packed helical fibers having three-start helices. The architecture of these crystallographic fibers, together with EM and mutagenic data, have allowed us to derive a model for toxin co-regulated pilus (TCP) assembly, that may be generalized for all type IV pili. We are using EM and image reconstruction to obtain a low resolution TCP structure that will be used to dock the tcpA crystal structure to generate a pseudo-atomic resolution TCP structure. We will continue to focus on structure and assembly of V. cholerae TCP by solving the crystal structure of tcpA from El Tor biotype, which is responsible for recent cholera endemics in developing countries. Our collaborator, Dr. Ronald Taylor at Dartmouth Medical School, has expressed El Tor tcpA using our classical tcpA constructs and we will purify this protein and crystallize it. Comparison of the crystal structures of classical and El Tor pilins may reveal important differences between the two biotypes and may highlight common targets for antibacterial therapeutics.
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