Fig. 1.  The recently determined structure of SrtA covalently bound to an analog of the LPXTG sorting signal12. (A) Chemical structure of the Cbz-LPAT* peptide analog attached via a disulfide bond to the thiol of C184. (B) The first thio-acyl intermediate that forms immediately after SrtA cleavage of the sorting signal. (C) Stereo image showing the ensemble of 20 lowest energy structures of the SrtA-LPAT* complex. The protein backbone heavy atoms (blue) and the covalently linked peptide (red) are shown. Yellow spheres represent calcium ions. (D) Ribbon drawing of the structure of the SrtA-LPAT* complex (ref 12).

Inhibitor Development and Mechanistic Studies of Sortase Enzymes

Cell Surface Protein Anchoring: Mechanism and Drug Development

During an infection, bacteria use an array of surface-attached proteins to adhere to specific organ tissues, resist phagocytosis, invade host cells and acquire essential nutrients. In Gram-positive bacteria, many surface proteins are covalently anchored to the cell wall peptidylglycan by sortase enzymes (11, 20, 21). Sortases catalyze a transpeptidation reaction between a cell wall sorting signal that is located in their protein substrate and a cross-bridge peptide nucleophile that resides within the cell wall. In a collaborative research effort with Professor Mike Jung's group at UCLA we are investigating the molecular basis of sortase mediated protein anchoring reaction in S. aureus and other pathogens (4-15). We are also developing inhibitors of this process that could function as therapeutically useful anti-infective agents (13).

Fig. 2. Newly discovered pyridazinone molecules that inhibit the sortase enzymes from S. aureus and B. anthracis (ref 13). The molecules were discovered using high-throughput screening and structure activity relationship analyses. Left, the pyridazinone scaffold. Right, model of compound 2-35 bound to sortase via Induced-Fit Docking.

Mechanistic Studies:

An understanding of the molecular basis of protein anchoring is poorly understood because the intermediates of catalysis are short-lived. In recently published work, we overcame this problem by synthesizing a peptide analog of the sorting signal that forms a covalent complex with sortase (Fig. 1) (8, 12). The structure of the SrtAΔN59-LPAT* covalent complex mimics a key enzyme-protein thioacyl intermediate that sheds light onto the mechanism of transpeptidation and the role of highly conserved active site residues. It also reveals how binding of the LPXTG sorting signal triggers major changes in the structure and dynamics of the enzyme that facilitate substrate recognition and direct catalysis towards product formation. Ongoing research is using structural, computational, and biochemical approaches to investigate other aspects of the mechanism of catalysis.


Because S. aureus is a leading cause of morbidity and sortase is required for its virulence, we have used a high-throughput fluorescence assay to search for small molecule sortase inhibitors (13). This work led to several promising inhibitors that have recently been published (Fig. 2) (13). The in vivo efficacy of these molecules will be tested using a mouse model. In ongoing collaborative work with Professor Mike Jung's group we are also optimizing several other lead compounds using structure activity relationship analyses and rational design approaches.


1. Chambers, H.F. & Deleo, F.R. Waves of resistance: Staphylococcus aureus in the antibiotic era. Nat Rev Microbiol 7, 629-41 (2009).

2. Klevens, R.M. et al. Invasive methicillin-resistant Staphylococcus aureus infections in the United States. Jama 298, 1763-71 (2007).

3. Lowry, F.D. Staphylococcus aureus infections. New England Journal of Medicine 339, 520-532 (1998).

4. Comfort, D. & Clubb, R.T. A comparative genome analysis identifies distinct sorting pathways in gram-positive bacteria. Infect Immun 72, 2710-22 (2004).

5. Connolly, K.M. & Clubb, R.T. Sortase Pathways in Gram-Positive Bacteria. in Structural Biology of Bacterial Pathogenesis (eds. Waksman, G. & Caparon, M.) (Wiley, New York, 2004).

6. Connolly, K.M. et al. Sortase from Staphylococcus aureus does not contain a thiolate-imidazolium ion pair in its active site. J Biol Chem 278, 34061-5 (2003).

7. Ilangovan, U., Ton-That, H., Iwahara, J., Schneewind, O. & Clubb, R.T. Structure of sortase, the transpeptidase that anchors proteins to the cell wall of Staphylococcus aureus. Proc Natl Acad Sci U S A 98, 6056-61 (2001).

8. Jung, M.E. et al. Synthesis of (2R,3S) 3-amino-4-mercapto-2-butanol, a threonine analogue for covalent inhibition of sortases. Bioorg Med Chem Lett 15, 5076-9 (2005).

9. Liew, C.K. et al. Localization and mutagenesis of the sorting signal binding site on sortase A from Staphylococcus aureus. FEBS Lett 571, 221-6 (2004).

10. Naik, M.T. et al. Staphylococcus aureus Sortase A transpeptidase. Calcium promotes sorting signal binding by altering the mobility and structure of an active site loop. J Biol Chem 281, 1817-26 (2006).

11. Suree, N., Jung, M.E. & Clubb, R.T. Recent advances towards new anti-infective agents that inhibit cell surface protein anchoring in Staphylococcus aureus and other gram-positive pathogens. Mini Rev Med Chem 7, 991-1000 (2007).

12. Suree, N. et al. The structure of the Staphylococcus aureus sortase-substrate complex reveals how the universally conserved LPXTG sorting signal is recognized. J Biol Chem 284, 24465-77 (2009).

13. Suree, N. et al. Discovery and structure-activity relationship analysis of Staphylococcus aureus sortase A inhibitors. Bioorg Med Chem 17, 7174-85 (2009).

14. Weiner, E.M., Robson, S.A., Marohn, M. & Clubb, R.T. The sortase A enzyme that attaches proteins to the cell wall of B. anthracis contains an unusual active site architecture. J Biol Chem.

15. Yeates, T.O. & Clubb, R.T. Biochemistry. How some pili pull. Science 318, 1558-9 (2007).

16. Pilpa, R.M. et al. Solution structure of the NEAT (NEAr Transporter) domain from IsdH/HarA: the human hemoglobin receptor in Staphylococcus aureus. J Mol Biol 360, 435-47 (2006).

17. Pilpa, R.M. et al. Functionally distinct NEAT (NEAr Transporter) domains within the Staphylococcus aureus IsdH/HarA protein extract heme from methemoglobin. J Biol Chem 284, 1166-76 (2009).

18. Robson, S.A., Peterson, R., Bouchard, L.S., Villareal, V.A. & Clubb, R.T. A Heteronuclear Zero Quantum Coherence N(z)-Exchange Experiment That Resolves Resonance Overlap and Its Application To Measure the Rates of Heme Binding to the IsdC Protein. J Am Chem Soc (2010).

19. Villareal, V.A., Pilpa, R.M., Robson, S.A., Fadeev, E.A. & Clubb, R.T. The IsdC protein from Staphylococcus aureus uses a flexible binding pocket to capture heme. J Biol Chem 283, 31591-600 (2008).

20. Clancy, K.W., Melvin, J.A. & McCafferty, D.G. Sortase transpeptidases: Insights into mechanism, substrate specificity, and inhibition. Biopolymers 94, 385-96.

21. Maresso, A.W. & Schneewind, O. Sortase as a target of anti-infective therapy. Pharmacol Rev 60, 128-41 (2008).

22. Maresso, A.W. & Schneewind, O. Iron acquisition and transport in Staphylococcus aureus. Biometals 19, 193-203 (2006).

23. Mazmanian, S.K. et al. Passage of heme-iron across the envelope of Staphylococcus aureus. Science 299, 906-9 (2003).

24. Skaar, E.P. & Schneewind, O. Iron-regulated surface determinants (Isd) of Staphylococcus aureus: stealing iron from heme. Microbes Infect 6, 390-7 (2004).

25. Liu, M. et al. Direct hemin transfer from IsdA to IsdC in the iron-regulated surface determinant (Isd) heme acquisition system of Staphylococcus aureus. J Biol Chem 283, 6668-76 (2008).

26. Zhu, H. et al. Pathway for heme uptake from human methemoglobin by the iron-regulated surface determinants system of Staphylococcus aureus. J Biol Chem 283, 18450-60 (2008).