Structural Insights into Substrate Recognition and Catalysis in Outer Membrane Protein B (OmpB) by Protein-lysine Methyltransferases from Rickettsia*

  1. David C. H. Yang‡4
  1. From the ‡Department of Chemistry, Georgetown University, Washington, D. C. 20057,
  2. §Markey Center for Structural Biology, Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907,
  3. Laboratory of Biochemistry and
  4. **Proteomics Core Facility, NHLBI and
  5. ‡‡Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, and
  6. ‖Viral and Rickettsial Diseases Department, Infectious Diseases Directorate, Naval Medical Research Center, Silver Spring, Maryland 20910
  1. ↵3 To whom correspondence may be addressed. E-mail: nnoinaj{at}purdue.edu.
  2. ↵4 To whom correspondence may be addressed. E-mail: yangdc{at}georgetown.edu.

  1. ↵1 Both authors contributed equally to this work.

Abstract

Rickettsia belong to a family of Gram-negative obligate intracellular infectious bacteria that are the causative agents of typhus and spotted fever. Outer membrane protein B (OmpB) occurs in all rickettsial species, serves as a protective envelope, mediates host cell adhesion and invasion, and is a major immunodominant antigen. OmpBs from virulent strains contain multiple trimethylated lysine residues, whereas the avirulent strain contains mainly monomethyllysine. Two protein-lysine methyltransferases (PKMTs) that catalyze methylation of recombinant OmpB at multiple sites with varying sequences have been identified and overexpressed. PKMT1 catalyzes predominantly monomethylation, whereas PKMT2 catalyzes mainly trimethylation. Rickettsial PKMT1 and PKMT2 are unusual in that their primary substrate appears to be limited to OmpB, and both are capable of methylating multiple lysyl residues with broad sequence specificity. Here we report the crystal structures of PKMT1 from Rickettsia prowazekii and PKMT2 from Rickettsia typhi, both the apo form and in complex with its cofactor S-adenosylmethionine or S-adenosylhomocysteine. The structure of PKMT1 in complex with S-adenosylhomocysteine is solved to a resolution of 1.9 Å. Both enzymes are dimeric with each monomer containing an S-adenosylmethionine binding domain with a core Rossmann fold, a dimerization domain, a middle domain, a C-terminal domain, and a centrally located open cavity. Based on the crystal structures, residues involved in catalysis, cofactor binding, and substrate interactions were examined using site-directed mutagenesis followed by steady state kinetic analysis to ascertain their catalytic functions in solution. Together, our data reveal new structural and mechanistic insights into how rickettsial methyltransferases catalyze OmpB methylation.

Footnotes

  • ↵2 Supported by the Intramural Research Program of the NIDDK, National Institutes of Health.

  • ↵* The work was supported in part by Naval Medical Logistic Command Award N62645 (to D. C. H. Y.) and Work Unit Number 6000.RAD1.J.A0310 (to the Naval Medical Research Center). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

  • Received February 23, 2016.
  • Revision received July 27, 2016.
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  1. First Published on July 29, 2016 doi: 10.1074/jbc.M116.723460 The Journal of Biological Chemistry 291, 19962-19974.
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