K-127. Identification of Multiprotein Complexes in Helicobacter pylori Using the Tandem Affinity Purification (TAP) Technology

K. Stingl1,2, D. Leduc1, K. Schauer1, C. Ecobichon1, A. Labigne1, P. Lenormand1, J-C. Rousselle1, A. Namane1, H. De Reuse1;
1Inst. Pasteur, Paris, FRANCE, 2Inst. fuer Allgemeine Zoologie und Genetik, Muenster, GERMANY.

Most of the cellular processes involve protein complexes. The tandem affinity purification (TAP) technology was adapted to monitor protein interactions in the gastric pathogen Helicobacter pylori. TAP allows the purification of multiprotein complexes expressed from the original organism (H. pylori) under native conditions and physiological expression levels of a bait protein. Two systems were explored in H. pylori (i) the essential glutaminyl-tRNA amidotransferase (AdT) and (ii) the nickel metallocenter assembly complexes of hydrogenase and urease, two enzymes required for in vivo colonization. The AdT system is necessary for Asparagine (Asn) and Glutamine (Gln) formation in H. pylori by catalyzing the amidation of Aspartate and Glutamate, respectively mischarged on tRNAAsn and tRNAGln. The Adt activity is supported by the essential heterotrimeric GatCAB enzyme. This system was chosen to validate the TAP in H. pylori, to evaluate its specificity and its potential for the study of essential functions. Tagged chromosomal copies of the gatA and gatC genes did not affect H. pylori viability. A complex composed of GatA, GatB and GatC was purified in large amounts with both baits, no additional partner was found. Assembly of metallocenters in enzymes is a complex process that requires the activities of multiple proteins. Taking as baits the UreG and HypB accessory proteins, we studied the nickel incorporation complexes of urease and hydrogenase, respectively. A complete urease incorporation complex was isolated, composed of the four accessory proteins UreG-E-F-H in association with the UreA-B structural subunits. Urease interactome partially overlapped with the hydrogenase nickel incorporation complex which contained SlyD (involved in hydrogenase maturation in E. coli) and proteins implicated in the formation of Fe-S clusters, present in the small subunit of hydrogenase. In conclusion, we showed that TAP is functional to study "biomolecular factories" in H. pylori and hypothesize the existence of a hydrogenase "metal incorposome" consisting of both nickel-incorporating and Fe-S cluster-forming proteins.