The mechanism of the prolyl isomerase SlyD
Final Report Abstract
SlyD from Escherichia coli is a prolyl isomerase of the FKBP type and is both an excellent catalyst of protein folding and a chaperone. The prolyl isomerase active site is located in the FKBP domain, the chaperone site resides in an extra domain of about 60 residues, termed the chaperone or IF domain. It is inserted in a loop of the FKBP domain near the catalytic site. The chaperone domain is essential for the very high folding activity of SlyD. In this project, we combined the expertise from our groups in experimental protein folding and in high resolution NMR spectroscopy to elucidate how SlyD functions at the molecular level. The prolyl isomerase domain and the chaperone domain cooperate during the catalytic cycle of SlyD such that the chaperone domain first binds to refolding protein chains with an affinity in the low micromolar range. Binding and dissociation are highly dynamic processes. This allows efficient substrate transfer to the catalytic prolyl isomerase site. Binding to the chaperone domain determines the overall KM value of catalyzed folding and overrides the inherently high sequence specificity of the prolyl isomerase site of FKBP domains. The structure of SlyD was solved by NMR spectroscopy and confirmed by x-ray crystallography. It revealed the two-domain structure of SlyD and a high flexibility of the domains. It also allowed us to identify the binding site for the protein substrates at the chaperone domain and confirmed that substrate binding is highly dynamic. The combination of the human prolyl isomerase FKBP12 with unrelated chaperone domains led to chimeric folding enzymes with high activity. The success of this design exercise emphasizes that substrate binding of folding enzymes is a generic function.
Publications
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(2009). Chaperone domains convert prolyl isomerases into generic catalysts of protein folding. Proc. Natl. Acad. Sci. USA 106, 20282-20287
Jakob, R. P., Zoldák, G., Aumüller, T., and Schmid, F. X.
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(2009). Consequences of Domain Insertion on the Stability and Folding Mechanism of a Protein. J. Mol. Biol. 386, 1138-1152
Zoldák, G., Carstensen, L., Scholz, C., and Schmid, F. X.
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(2009). NMR solution structure of SlyD from Escherichia coli: spatial separation of prolyl isomerase and chaperone function. J. Mol. Biol. 387, 295-305
Weininger, U., Haupt, C., Schweimer, K., Graubner, W., Kovermann, M., Brüser, T., Scholz, C., Schaarschmidt, P., Zoldak, G., Schmid, F. X., and Balbach, J.
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(2010). Crystal Structure Determination and Functional Characterization of the Metallochaperone SlyD from Thermus thermophilus. J. Mol. Biol., 398, 375-390
Löw, C., Neumann, P., Tidow, H., Weininger, U., Haupt, C., Friedrich-Epler, B., Scholz, C., Stubbs, M.T., Balbach, J.
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(2011). Cooperation of the prolyl isomerase and chaperone activities of the protein folding catalyst SlyD. J. Mol. Biol. 406, 176-194
Zoldák, G., and Schmid, F. X.
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(2011). Local and coupled thermodynamic stability of the two-domain and bifunctional enzyme SlyD from Escherichia coli. Biochemistry 50, 7321-7329
Haupt, C., Weininger, U., Kovermann, M., and Balbach, J.
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(2011). NMR relaxation unravels interdomain crosstalk of the two domain prolyl isomerase and chaperone SlyD. Biochim. Biophys. Acta 1814, 873-881
Kovermann, M., Zierold, R., Haupt, C., Löw, C., and Balbach, J.
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(2011). Transient enzyme-substrate recognition monitored by real-time NMR. J. Am. Chem. Soc. 133, 11154-11162
Haupt, C., Patzschke, R., Weininger, U., Gröger, S., Kovermann, M., and Balbach, J.
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(2012). Combination of the Human Prolyl Isomerase FKBP12 with Unrelated Chaperone Domains Leads to Chimeric Folding Enzymes with High Activity. J. Mol. Biol.
Geitner, A. J., and Schmid, F. X.
