Characterization of Multimethylation in Ricksettsia Outer Membrane Protein B (OMPB) Catalyzed by Lysine Methyl Transferases

Abstract

Rickettsiae are causative agents of typhus and spotted fever. Early detection and vaccines are crucially needed. The observation of correlation between methylation of lysine residues in rickettsial outer membrane protein B (OmpB) and bacterial virulence has suggested the importance of an enzymatic system for OmpB methylation. However no OmpB lysine methyltransferase has been functionally or structurally characterized. Bioinformatic analysis of rickettsial genomic sequences revealed DNA sequences of putative protein methyltransferases. Genes of five putative methyltransferases were synthesized, expressed in E. coli. Two types of protein lysine methyltransferases were discovered: PKMT1 and PKMT2. PKMT1 catalyzes primarily monomethylation and PKMT2 functions as trimethyltransferase as characterized using radioactivity assay, immunoblotting and mass spectrometry. RP789 from R. prowazekii, and RT0776 from R. typhi were found to be PKMT1, and RP027-028 from R prowazekii, and RT0101 from R. typhi as PKMT2. Semiquantitative integrated liquid chromatography-tandem mass spectrometry was used to characterize the location, state and level of methylation of enzymatically methylated recombinant OmpB fragments and native OmpB purified from Rickettsia. In vitro trimethylation occurs at relatively specific locations in OmpB, while monomethylation appears at over thirty lysine residues in OmpB. Native OmpB from R. typhi contains mono- and trimethyllysines at locations correlated with those catalyzed by the lysine methyltransferases in vitro. Clusters of highly trimethylated lysines were found in OmpB from virulent strains but not avirulent strain, and the number of clusters of highly trimethylated lysines correlates with the rickettsial virulence. The three dimensional structures of RP789 and RT0101, and their complexes with S-adenosylmethionine or S-adenosylhomocysteine were determined using protein X-ray crystallography. Both enzymes are dimeric, and each monomer has a novel open cleft. Mutation of selected amino acid residues followed by steady-state kinetic analysis supports that the open cleft is the OmpB binding pocket. The overall protein fold reveals the structural basis of the unusual multisite methylation. The first biochemical characterization of methylation of an OMP from Gram-negative bacteria in this thesis may lead to the elucidation of the molecular mechanism of multisite methylation, the discovery of additional links between OmpB methylation and rickettsial virulence, and development of diagnostic reagents and vaccine candidates.