Structural analysis and interaction studies of acyl-carrier protein (acpP) of Staphylococcus aureus, an extraordinarily thermally stable protein

Acyl-carrier-protein (acpP) is an essential protein in fatty acid biosynthesis of Staphylococcus aureus [Cronan, J.E. and Thomas, J. (2009). Complex enzymes in microbial natural product biosynthesis, part B: polyketides, aminocoumarins and carbohydrates. Method. Enzymol. 459, 395–433; Halavaty, A.S., Kim, Y., Minasov, G., Shuvalova, L., Dubrovska, I., Winsor, J., Zhou, M., Onopriyenko, O., Skarina, T., Papazisi, L., et al. (2012). Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria. Acta Crystallogr. Sect. D Biol. Crystallogr. 68, 1359–1370]. The inactive apo-form is converted to the active holo-enzyme by acyl-carrier protein synthase (acpS) through addition of a 4′-phosphopantetheine group from coenzyme A to a conserved serine residue of acpP [Flugel, R.S., Hwangbo, Y., Lambalot, R.H., Cronan, J.E., and Walsh, C.T. (2000). Holo-(acyl-carrier protein) synthase and phosphopantetheinyl transfer in Escherichia coli. J. Biol. Chem. 275, 959–968; Lambalot, R.H. and Walsh, C.T. (1995). Cloning, overproduction, and characterization of the Escherichia coli holo-acyl-carrier protein synthase. J. Biol. Chem. 270, 24658–24661]. Once activated, acpP acts as an anchor for the growing fatty acid chain. Structural data from X-ray crystallographic analysis reveals that, despite its small size (8 kDa), acpP adopts a distinct, mostly α-helical structure when complexed with acpS [Halavaty, A.S., Kim, Y., Minasov, G., Shuvalova, L., Dubrovska, I., Winsor, J., Zhou, M., Onopriyenko, O., Skarina, T., Papazisi, L., et al. (2012). Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria. Acta Crystallogr. Sect. D Biol. Crystallogr. 68, 1359–1370; Byers, D.M. and Gong, H. (2007). Acyl carrier protein: structure–function relationships in a conserved multifunctional protein family. Biochem. Cell Biol. 85, 649–662]. We expressed and purified recombinant, active S. aureus acpP from Escherichia coli and mimicked the beginning of fatty acid biosynthesis by employing an [14C]-acp loading assay. Surprisingly, acpP remained functional even after heat treatment at 95°C for up to 10 min. NMR data from 2D-HSQC experiments as well as interaction studies with acpS confirmed that acpP is structured and active both before and after heat treatment, with no significant differences between the two. Thus, our data suggest that S. aureus acpP is a highly stable protein capable of maintaining its structure at high temperatures.