W0241

X-Ray Crystal Structures of Type III Effector Proteins From Phytopathogenic Bacteria: A Mini Structural Genomics Program. Alex Singer¹, Darrell Desveaux², Jeff Chang², Zachary Nimchuk², Sarah Grant², Jeff Dangl², John Sondek¹, Laurie Betts¹, Depts. of Biology² and Pharmacology¹, Univ. of North Carolina Chapel Hill, Chapel Hill, NC 27599.

We have targeted for X-ray crystal structure determination a diverse suite of 30 proteins involved in infection plants by the bacterium Pseudomonas syringae. Most target proteins are virulence factors, delivered into plant cells using the evolutionarily conserved Type III secretion system, also used by animal pathogens including Salmonella spp., Yersinia spp., and enteropathic E. coli. Specific biochemical functions, host target molecules, and structural classes of P. syringae virulence factors are not apparent from their sequence and are almost completely uncharacterized. Our structures will generate testable hypotheses as to the function of these virulence proteins, for example by their structural homology to known classes of signaling molecules or enzymes. Our collaboration with the established and highly successful Dangl/Grant laboratory at UNC will allow detailed testing of putative functions of these proteins in planta and in plant cell culture.

We have thus far solved the structure of two open reading frame gene products encoded by the avrpPhF gene, a virulence factor involved in halo-blight disease of beans. One of these, orf2, has some structural homology to ADP-ribosylating toxins such as Diptheria toxin and Exotoxin A from Pseudomonas aeruginosa. However, in vitro testing for NAD glycohydrolase activity and in vitro labeling of plant extracts with radioactive substrate in the presence of orf 2 protein did not indicate ADP ribosylase activity for the protein. An essential glutamate residue is missing from the putative active site in orf2 that is necessary for this activity. Studies are ongoing to determine a possible protease function for this protein. The second structure, orf1, was predicted by threading algorithms (not by sequence homology) with high probability to be a chaperone homologous to a Yersinia pestis Type III chaperone called SycE which is necessary for the proper delivery of the YopE gene product, a protein with GTPase activating activity for the small G-protein RhoA. We will present these structures and progress on determining their function. We will also present our strategies for rapidly cloning, expressing, and testing these proteins for crystallizability. Our hypothesis that orf1 & orf2 form a complex in which orf1 acts as a chaperone is supported by the in vitro formation of a complex which may be stably isolated by gel filtration chromatography. This complex is being screened for crystallization conditions and its structure will be determined when such crystals are obtained.