Inteins, self-splicing protein elements, interrupt genes and proteins in many microbes, including the human pathogen Mycobacterium tuberculosis. Using conserved catalytic nucleophiles at their N- and C-terminal splice junctions, inteins are able to excise out of precursor polypeptides. The splicing of the intein in the mycobacterial recombinase RecA is specifically inhibited by the widely used cancer therapeutic cisplatin, cis-[Pt(NH3)2Cl2], and this compound inhibits mycobacterial growth. Mass spectrometric and crystallographic studies of Pt(II) binding to the RecA intein revealed a complex in which two platinum atoms bind at N- and C-terminal catalytic cysteine residues. Kinetic analyses of NMR spectroscopic data support a two-step binding mechanism in which a Pt(II) first rapidly interacts reversibly at the N terminus followed by a slower, first order irreversible binding event involving both the N and C termini. Notably, the ligands of Pt(II) compounds that are required for chemotherapeutic efficacy and toxicity are no longer bound to the metal atom in the intein adduct. The lack of ammine ligands and need for phosphine represent a springboard for future design of platinum-based compounds targeting inteins. Because the intein splicing mechanism is conserved across a range of pathogenic microbes, developing these drugs could lead to novel, broad range antimicrobial agents.
inhibition mechanism
intein
metal ion-protein interaction
Mycobacterium tuberculosis
protein drug interaction
protein splicing
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The atomic coordinates and structure factors (codes5I0A and 5K08) have been deposited in the Protein Data Bank (http://wwpdb.org/).
This work was supported by National Institutes of Health Grants GM085006 (to A. S.) and GM39422 and GM44844 (to M. B.). 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.
