The enzymatic activity of FKBPs has been studied in several ways, with the most popular method being a spectrophotometric assay that utilizes the modified tetrapeptide substrate analogue succinyl-Ala-Leu-Pro-Phe-4-nitroanilide (suc-ALPF-pNA) or variants thereof. This protein belongs to a prokaryotic subfamily, characterized by having an insert-in-flap (IF) chaperone domain inserted into the FKBP domain in place of the so-called flap loop (also known as the 80’s loop) found in FKBP12 and many other FKBPs, which both enables it to function as an efficient chaperone and increases its PPIase activity towards partially folded protein substrates by as much as 100–200 fold. A particularly well-studied example is SlyD. FKBPs often have additional chaperone or protein–protein interaction domains. Since then, it has become clear that FKBPs are widespread in all branches of life. The first FKBP to be discovered was human FKBP12, which was identified as a binding partner of the immunosuppressive macrolide lactone FK506, hence the name of the family. These enzymes presumably all function by stabilizing the transition state, resulting in an effective rate constant for the catalyzed reaction of up to 10 8 M −1s −1, but their mechanisms are not well understood. Nature has therefore evolved three families of peptidyl-prolyl isomerases (PPIases) to facilitate cis/ trans isomerization: FK506-binding proteins (FKBPs), cyclophilins, and parvulins. Indeed, the isomerization correlation time typically falls in the seconds to minutes time regime. However, spontaneous cis/ trans isomerization occurs very slowly due to the high energy barrier imposed by the partial double bond character of the peptide bond. Protein folding requires that each proline in the sequence adopts the isoform compatible with the native fold. Although both isoforms can be found in type IV β-turns (a category with lax geometry requirements), the trans form specifically favors more narrowly defined types of turns, for example, I, II, and VIII, whereas the cis form is required for types VIa1, VIa2, and VIb. In folded proteins, prolines are predominantly found in β-turns and other loop elements, where the cis and trans isoforms have different effects on the structure. The cis form is therefore much more commonly observed for prolines than for any other residues. Due to unfavorable steric and electronic effects, the cis form is by far the least favored, except for peptidyl-prolyl bonds where the unique N-alkylation of proline markedly reduces the energy difference between the two conformations. Peptide bonds are planar with ω dihedral angles of either ~0° ( cis form) or ~180° ( trans form). 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. We found that 15-residue-long unmodified peptides can serve as better substrate mimics for the IF and FKBP domains than chemically modified tetrapeptides. Our results furthermore provide important clues to the catalytic mechanism and support the notion of inter-domain cross talk. 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. 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. We 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. 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. SlyD is a two-domain enzyme containing both a PPIase FK506-binding protein (FKBP) domain and an insert-in-flap (IF) chaperone domain. Peptidyl-prolyl isomerases (PPIases) catalyze cis/trans isomerization of peptidyl-prolyl bonds, which is often rate-limiting for protein folding.
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