Abstract
BackgroundPeptidyl-prolyl isomerases (PPIases) catalyze cis/trans isomerization of peptidyl-prolyl bonds, which is often rate-limiting for protein folding. SlyD is a two-domain enzyme containing both a PPIase FK506-binding protein (FKBP) domain and an insert-in-flap (IF) chaperone domain. To date, the interactions of these domains with unfolded proteins have remained rather obscure, with structural information on binding to the FKBP domain being limited to complexes involving various inhibitor compounds or a chemically modified tetrapeptide.ResultsWe have characterized the binding of 15-residue-long unmodified peptides to SlyD from Thermus thermophilus (TtSlyD) in terms of binding thermodynamics and enzyme kinetics through the use of isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, and site-directed mutagenesis. We show that the affinities and enzymatic activity of TtSlyD towards these peptides are much higher than for the chemically modified tetrapeptides that are typically used for activity measurements on FKBPs. In addition, we present a series of crystal structures of TtSlyD with the inhibitor FK506 bound to the FKBP domain, and with 15-residue-long peptides bound to either one or both domains, which reveals that substrates bind in a highly adaptable fashion to the IF domain through β-strand augmentation, and can bind to the FKBP domain as both types VIa1 and VIb-like cis-proline β-turns. Our results furthermore provide important clues to the catalytic mechanism and support the notion of inter-domain cross talk.ConclusionsWe found that 15-residue-long unmodified peptides can serve as better substrate mimics for the IF and FKBP domains than chemically modified tetrapeptides. We furthermore show how such peptides are recognized by each of these domains in TtSlyD, and propose a novel general model for the catalytic mechanism of FKBPs that involves C-terminal rotation around the peptidyl-prolyl bond mediated by stabilization of the twisted transition state in the hydrophobic binding site.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0300-3) contains supplementary material, which is available to authorized users.
Highlights
Peptidyl-prolyl isomerases (PPIases) catalyze cis/trans isomerization of peptidyl-prolyl bonds, which is often rate-limiting for protein folding
We have shown that 15-residue-long unmodified peptides bind to SlyD from Thermus thermophilus (TtSlyD) with affinities that are similar to those of partially folded proteins, but considerably higher than estimated for the chemically modified tetrapeptides that are typically used for functional studies on FK506-binding protein (FKBP)
We have shown that the enzymatic activity towards the 15-residue-long S2-P25A peptide is much higher than for both tetrapeptides and partially folded proteins, implying that TtSlyD is most efficient when acting on unfolded proteins
Summary
Peptidyl-prolyl isomerases (PPIases) catalyze cis/trans isomerization of peptidyl-prolyl bonds, which is often rate-limiting for protein folding. Prolines are predominantly found in βturns and other loop elements [3, 4], where the cis and trans isoforms have different effects on the structure. Nature has evolved three families of peptidyl-prolyl isomerases (PPIases) to facilitate cis/trans isomerization: FK506-binding proteins (FKBPs), cyclophilins, and parvulins [8, 9]. These enzymes presumably all function by stabilizing the transition state, resulting in an effective rate constant for the catalyzed reaction of up to 108 M−1s−1 [9], but their mechanisms are not well understood [9, 10]
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