Bacterial Chaperone Domain Insertions Convert Human FKBP12 into an Excellent Protein-Folding Catalyst-A Structural and Functional Analysis.

Many folding enzymes use separate domains for the binding of substrate proteins and for the catalysis of slow folding reactions such as prolyl isomerization. FKBP12 is a small prolyl isomerase without a chaperone domain. Its folding activity is low, but it could be increased by inserting the chaperone domain from the homolog SlyD of E. coli near the prolyl isomerase active site. We inserted two other chaperone domains into human FKBP12: the chaperone domain of SlpA from E. coli, and the chaperone domain of SlyD from Thermococcus sp. Both stabilized FKBP12 and greatly increased its folding activity. The insertion of these chaperone domains had no influence on the FKBP12 and the chaperone domain structure, as revealed by two crystal structures of the chimeric proteins. The relative domain orientations differ in the two crystal structures, presumably representing snapshots of a more open and a more closed conformation. Together with crystal structures from SlyD-like proteins, they suggest a path for how substrate proteins might be transferred from the chaperone domain to the prolyl isomerase domain.

Structure-Based Insights into the Dynamics and Function of Two-Domain SlpA from Escherichia coli.

SlpA (SlyD-like protein A) comprises two domains, a FK506 binding domain (FKBP fold) of moderate prolyl cis/trans-isomerase activity and an inserted in flap (IF) domain that hosts its chaperone activity. Here we present the nuclear magnetic resonance (NMR) solution structure of apo Escherichia coli SlpA determined by NMR that mirrors the structural properties seen for various SlyD homologues. Crucial structural differences in side-chain orientation arise for F37, which points directly into the hydrophobic core of the active site. It forms a prominent aromatic stacking with F15, one of the key residues for PPIase activity, thus giving a possible explanation for the inherently low PPIase activity of SlpA. The IF domain reveals the highest stability within the FKBP-IF protein family, most likely arising from an aromatic cluster formed by four phenylalanine residues. Both the thermodynamic stability and the PPIase and chaperone activity let us speculate that SlpA is a backup system for homologous bacterial systems under unfavorable conditions.

Dynamics of Aromatic Side Chains in the Active Site of FKBP12.

FKBP12, a small human enzyme, aids protein folding by catalyzing cis-trans isomerization of peptidyl-prolyl bonds, and is involved in cell signaling pathways, calcium regulation, and the immune response. The underlying molecular mechanisms are not fully understood, but it is well-known that aromatic residues in the active site and neighboring loops are important for substrate binding and catalysis. Here we report micro- to millisecond exchange dynamics of aromatic side chains in the active site region of ligand-free FKBP12, involving a minor state population of 0.5% and an exchange rate of 3600 s-1, similar to previous results for the backbone and methyl-bearing side chains. The exchange process involves tautomerization of H87. In the major state H87 is highly flexible and occupies the common HNε2 tautomer, while in the minor state it occupies the rare HNδ1 tautomer, which typically requires stabilization by specific interactions, such as hydrogen bonds. This finding suggests that the exchange process is coupled to a rearrangement of the hydrogen bond network around H87. Upon addition of the active-site inhibitor FK506 the exchange of all aromatic residues is quenched, with exception of H87. The H87 resonances are broadened beyond detection, suggesting that interconversion between tautomers prevail in the FK506-bound state. While key active-site residues undergo conformational exchange in the apo state, the exchange rate is considerably faster than the catalytic turnover, as determined herein by Michaelis-Menten type analysis of NMR line shapes and chemical shifts. We discuss alternative interpretations of this observation in terms of FKBP12 function.

Generation of a highly active folding enzyme by combining a parvulin-type prolyl isomerase from SurA with an unrelated chaperone domain.

Parvulins are small prolyl isomerases and serve as catalytic domains of folding enzymes. SurA (survival protein A) from the periplasm of Escherichia coli consists of an inactive (Par1) and an active (Par2) parvulin domain as well as a chaperone domain. In the absence of the chaperone domain, the folding activity of Par2 is virtually abolished. We created a chimeric protein by inserting the chaperone domain of SlyD, an unrelated folding enzyme from the FKBP family, into a loop of the isolated Par2 domain of SurA. This increased its folding activity 450-fold to a value higher than the activity of SurA, in which Par2 is linked with its natural chaperone domain. In the presence of both the natural and the foreign chaperone domain, the folding activity of Par2 was 1500-fold increased. Related and unrelated chaperone domains thus are similarly efficient in enhancing the folding activity of the prolyl isomerase Par2. A sequence analysis of various chaperone domains suggests that clusters of exposed methionine residues in mobile chain regions might be important for a generic interaction with unfolded protein chains. This binding is highly dynamic to allow frequent transfer of folding protein chains between chaperone and catalytic domains.

The prolyl isomerase SlyD is a highly efficient enzyme but decelerates the conformational folding of a client protein.

Folding enzymes often use distinct domains for the interaction with a folding protein chain and for the catalysis of intrinsically slow reactions such as prolyl cis/trans isomerization. Here, we investigated the refolding reaction of ribonuclease T1 in the presence of the prolyl isomerase SlyD from Escherichia coli to examine how this enzyme catalyzes the folding of molecules with an incorrect trans proline isomer and how it modulates the conformational folding of the molecules with the correct cis proline. The kinetic analysis suggests that prolyl cis → trans isomerization in the SlyD-bound state shows a rate near 100 s(-1) and is thus more than 10(4)-fold accelerated, relative to the uncatalyzed reaction. As a consequence of its fast binding and efficient catalysis, SlyD retards the conformational folding of the protein molecules with the correct cis isomer, because it promotes the formation of the species with the incorrect trans isomer. In the presence of ≥1 µM SlyD, protein molecules with cis and trans prolyl isomers refold with identical rates, because SlyD-catalyzed cis/trans equilibration is faster than conformational folding. The cis or trans state of a particular proline is thus no longer a determinant for the rate of folding.

Structural and energetic basis of infection by the filamentous bacteriophage IKe.

Filamentous phage use the two N-terminal domains of their gene-3-proteins to initiate infection of Escherichia coli. One domain interacts with a pilus, and then the other domain binds to TolA at the cell surface. In phage fd, these two domains are tightly associated with each other, which renders the phage robust but non-infectious, because the TolA binding site is inaccessible. Activation for infection requires partial unfolding, domain disassembly and prolyl isomerization. Phage IKe infects E. coli less efficiently than phage fd. Unlike in phage fd, the pilus- and TolA-binding domains of phage IKe are independent of each other in stability and folding. The site for TolA binding is thus always accessible, but the affinity is very low. The structures of the two domains, analysed by X-ray crystallography and by NMR spectroscopy, revealed a unique fold for the N-pilus-binding domain and a conserved fold for the TolA-binding domain. The absence of an activation mechanism as in phage fd and the low affinity for TolA probably explain the low infectivity of phage IKe. They also explain why, in a previous co-evolution experiment with a mixture of phage fd and phage IKe, all hybrid phage adopted the superior infection mechanism of phage fd.

Combination of the human prolyl isomerase FKBP12 with unrelated chaperone domains leads to chimeric folding enzymes with high activity.

Folding enzymes often use distinct domains for the binding of substrate proteins ("chaperone domains") and for the catalysis of slow folding reactions such as disulfide formation or prolyl isomerization. The human prolyl isomerase FKBP12 is a small single-domain protein without a chaperone domain. Its very low folding activity could previously be increased by inserting the chaperone domain from the homolog SlyD (sensitive-to-lysis protein D) of Escherichia coli. We now inserted three unrelated chaperone domains into human FKBP12: the apical domain of the chaperonin GroEL from E. coli, the chaperone domain of protein disulfide isomerase from yeast, or the chaperone domain of SurA from the periplasm of E. coli. All three conveyed FKBP12 with a high affinity for unfolded proteins and increased its folding activity. Substrate binding and release of the chimeric folding enzymes were found to be very fast. This allows rapid substrate transfer from the chaperone domain to the catalytic domain and ensures efficient rebinding of protein chains that were unable to complete folding. The advantage of having separate sites, first for generic protein binding and then for specific catalysis, explains why our construction of the artificial folding enzymes with foreign chaperone domains was successful.

Prolyl isomerases show low sequence specificity toward the residue following the proline.

Prolyl isomerases catalyze the cis/trans isomerization of peptide bonds preceding proline. Previously, we had determined the specificity toward the residue before the proline for cyclophilin-, FKBP-, and parvulin-type prolyl isomerases by using proline-containing oligopeptides and refolding proteins as model substrates. Here, we report the specificities of members of these three prolyl isomerase families for the residue following the proline, again in short peptide and in refolding protein chains. Human cyclophilin 18 and parvulin 10 from Escherichia coli show high activity, but low specificity, with respect to the residue following the proline. Human FKBP12 prefers hydrophobic residues at this position in the peptide assays and shows a very low activity in the protein folding assays. This activity was strongly improved, and the sequence specificity was virtually eliminated after the insertion of a chaperone domain into the prolyl isomerase domain of human FKBP12.