BACKGROUND
peptide bonds are planar with ω dihedral angles of either ~0°  or ~180° .
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  <cit> .
the cis form is therefore much more commonly observed for prolines than for any other residues  <cit> .
in folded proteins, prolines are predominantly found in β-turns and other loop elements  <cit> , where the cis and trans isoforms have different effects on the structure.
although both isoforms can be found in type iv β-turns , 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 via <dig>  via <dig>  and vib  <cit> .
protein folding requires that each proline in the sequence adopts the isoform compatible with the native fold  <cit> .
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.
indeed, the isomerization correlation time typically falls in the seconds to minutes time regime  <cit> .
nature has therefore evolved three families of peptidyl-prolyl isomerases  to facilitate cis/trans isomerization: fk506-binding proteins , cyclophilins, and parvulins  <cit> .
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− <dig>  <cit> , but their mechanisms are not well understood  <cit> .
the first fkbp to be discovered was human fkbp <dig>  which was identified as a binding partner of the immunosuppressive macrolide lactone fk <dig>  hence the name of the family  <cit> .
since then, it has become clear that fkbps are widespread in all branches of life  <cit> .
fkbps often have additional chaperone or protein–protein interaction domains  <cit> .
a particularly well-studied example is slyd  <cit> .
this protein belongs to a prokaryotic subfamily, characterized by having an insert-in-flap  chaperone domain inserted into the fkbp domain in place of the so-called flap loop  found in fkbp <dig> and many other fkbps  <cit> , 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– <dig> fold  <cit> .
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  or variants thereof  <cit> .
structures have been determined for numerous fkbps in both the apo and inhibitor-bound forms.
however, to the best of our knowledge, only two structures have been obtained with a bound peptide, which in both cases is suc-alpf-pna  <cit> .
while chemically modified tetrapeptides are well suited for studying the effects of the residues neighboring the proline, they are not ideal substrate mimics, because they bind to escherichia coli slyd with much lower affinity than refolding protein substrates  <cit> .
the low affinity of these peptides probably relates to the smaller interaction surface compared to protein substrates, but could also relate to their limited capacity to form naturally occurring structural elements, such as β-turns.
structural insights into how substrates interact with the if domain have so far been based on a single structure of the slyd homologue slpa from e. coli, in which an uncleaved purification tag is bound at the substrate binding site of the if domain  <cit> .
in order to improve our understanding of the mechanism of slyd and of fkbps in general, we set out to analyze the kinetics, energetics, and structural basis for substrate binding and inhibition of slyd from thermus thermophilus  using 15-residue-long unmodified peptides, which we reasoned would be better mimics of natural unfolded protein substrates than the traditionally used 4-nitroanilide tetrapeptides.
indeed, these long peptides display much improved binding affinity and enzymatic turnover compared to the tetrapeptides.
the enzyme peptide complexes are fairly heterogeneous in their structural and energetic aspects, but common principles could be identified for both the if and the fkbp domain.
our results shed new light on how substrates are recognized, and have enabled us to propose a model for the catalytic mechanism.
RESULTS
to investigate the mechanism of ttslyd, we characterized its substrate binding and catalytic properties, as well as the three-dimensional structures of a number of ttslyd:peptide complexes, through the use of isothermal titration calorimetry , nuclear magnetic resonance  spectroscopy, x-ray crystallography, and site-specific mutagenesis.
peptide binding studies
to identify peptides that overcome the limitations of currently used substrate mimics and are suitable for structural studies, we used itc to characterize the binding of several different peptides to ttslyd, as exemplified in fig.
1a–d.
we mainly used 15-residue-long proline-containing segments from proteins that have previously been shown to bind to ttslyd and/or other proteins from the slyd family, namely rnase t <dig>  which is a model protein for folding studies, and the ribosomal proteins s <dig> and s <dig>  <cit> ; see table  <dig> for the complete list of peptide sequences.
table  <dig> summarizes the results of the binding studies.
interestingly, peptides derived from s <dig> and s <dig> display a dual binding mode with both a high- and a low-affinity binding site .
in order to identify the binding sites we next monitored peptide binding by heteronuclear nmr spectroscopy using a variant of the s <dig> peptide .
chemical shift perturbations on full-length ttslyd  upon addition of peptide clearly show binding to both the if and fkbp domains .
quantitative analyses of chemical shift changes as a function of added peptide enabled us to assign the stronger binding event to the if domain and the weaker one to the fkbp domain .
the highest affinities were obtained for the s <dig> peptide with kd1 =  <dig>  μm and kd2 =  <dig>  μm, followed by the s <dig> peptide with kd1 =  <dig>  μm and kd2 =  <dig>  μm .
addition of 5 % dimethyl sulfoxide , which was required for dissolving some peptides at high concentration, histidine tag cleavage, or a change in temperature , had only minor effects on the affinity of the s <dig> peptide .
in general, peptide binding to ttslydfl is driven by a favorable change in enthalpy .
a comparison of the thermodynamic fingerprints of the s <dig> and s <dig> peptides revealed that the slightly lower affinity of the s <dig> peptide to the if domain is caused by a reduction in binding enthalpy , while the weaker binding of the s <dig> peptide to the fkbp domain is due to entropic effects .
for the t <dig> peptide, a single-site model was sufficient to describe binding to ttslydfl, and the affinity was found to be much lower  than for the s <dig> and s <dig> peptides, which is explained primarily by an increase in unfavorable binding entropy.
notably, the affinities observed for the s <dig> and s <dig> peptides are in the same range or higher than for binding of a refolding protein substrate at high salt concentration to e. coli slyd   <cit> , and significantly higher than the reported kd of ~44 μm estimated for binding of the suc-alpf-pna peptide to e. coli slyd at high salt concentration  <cit> .
we therefore conclude that the s <dig> and s <dig> peptides can serve as improved substrate mimics for functional and structural studies of ttslyd.fig.
<dig> isothermal titration calorimetry  and nuclear magnetic resonance  binding studies.
typical itc data are shown for binding of peptides to ttslyd: a binding of the s <dig> peptide to full-length ttslyd .
b binding of the s2-long <dig> peptide to ttslydfl.
c binding of the s2-short <dig> peptide to ttslydfl.
d binding of the s2-w23a peptide to a ttslyd construct in which the insert-in-flap  domain is replaced by the flap loop from human fkbp <dig> .
upper panels: raw heat pulse data.
lower panels: integrated heat changes upon binding plotted against the peptide/protein concentration ratio resulting in differential binding isotherms that can be adequately described by a two-site  or a one-site  binding model.
resulting binding parameters are summarized in table  <dig>  e nmr-titration of 15n-labeled ttslydfl with the s2-p25a peptide at 25 °c.
the relative change in chemical shift is plotted versus the total concentration of added peptide, with red dots corresponding to residues in the if domain  and blue dots to the fkbp domain .
the error bars indicate the standard error.
the black solid curve represents the theoretical binding isotherm calculated using kd1 =  <dig>  μm and the dashed curve represents the theoretical binding isotherm calculated using kd2 =  <dig>  μm, as obtained from the itc measurements .
f mean weighted chemical shift changes are plotted against residue number.
resonances completely broadened by peptide binding are indicated by gray bars covering the full vertical scale.
the cut-off of  <dig>  ppm is shown as a red dotted line.
g residues with a shift difference > <dig>  ppm or completely broadened are highlighted in red on the structure of the ttslydfl:s <dig> complex , with the peptide shown in blue
trywnpkmkpfifga
rywnpkmkpfifg
ywnpkmkpfif
wnpkmkpfi
wnpkmkp
for peptides derived from the s <dig> protein, parts overlapping the original s <dig> peptide are underlined.
the expected charges at neutral ph are listed.
these were calculated using − <dig> for glu and asp, + <dig> for lys and arg, and  <dig> or + <dig> for his .
in addition to the peptides listed here, we also used the following five mutants of the s <dig> peptide: p25a, p29e, p25a/p29e, p25n/p29n, and w23a
ttslydfl
ttslydΔif
in most cases, the isothermal titration calorimetry data clearly supported the presence of two binding sites.
where data could be described by only a single binding site, the results are given in the columns for binding site  <dig>  note that the binding experiments with the proline double mutant peptides were carried out in 5 % dimethyl sulfoxide  due to solubility issues.
however, this is not expected to appreciably affect the experiments, as the s <dig> peptide was found to bind with similar affinities in both the presence and absence of 5 % dmso
in order to characterize the requirements for binding, we next analyzed binding of a series of variants of the s <dig> peptide  to ttslydfl.
to test the sequence dependency, we used a set of peptides corresponding to different segments of the s <dig> protein: three peptides covered sequences shifted n-terminally by three, six, or nine residues, compared to the original s <dig> peptide , and two peptides were shifted c-terminally by three or six residues .
these peptides all retained the capacity for binding to both sites, though the affinities were most often reduced when compared to the original s <dig> peptide .
in most cases, enthalpy losses were to some extent compensated by reduced entropy penalties and, in some cases, even gains in entropy .
to study the length dependency, we used variants of the s <dig> peptide extended or truncated with up to eight residues.
extension of the peptide had minor to moderate effects on the affinity for the if domain  and the fkbp domain  .
removing one or two residues from each side  reduced the affinity for the if domain about twofold and sevenfold, respectively, while the affinities for the fkbp domain remained relatively unperturbed.
after removing an additional two to four residues , the affinities were substantially reduced, and it was no longer possible to resolve the separate binding events .
the stoichiometries were, however, significantly higher than  <dig> after fitting to a single binding site model, indicating that these peptides may still engage both binding sites .
we also measured binding of an eight-residue-long peptide representing the plasmid-derived linker sequence bound to the if domain in the crystal structure of slpa , which was found to bind with an affinity similar to that of the seven-residue-long s2-short <dig> peptide.
we conclude that the sequence requirements for peptide binding to ttslydfl are rather lax, which is in part because the enthalpic losses incurred by apparent sequence mismatching are readily compensated by gains in entropy, and that the affinity of binding to both domains is sensitive to the length of the peptide .
next we tested the contribution of proline residues to the binding events.
there are two proline residues in the s <dig> peptide, p <dig> and p <dig>  neither single mutations , nor double mutations  abrogated binding to any of the two domains, though the affinities were moderately reduced in most cases, and strongly so for the p25a/p29e double mutant .
furthermore, mutating the single proline residue in the slpa linker peptide to threonine did not appreciably alter the affinity of this peptide for ttslydfl .
we therefore conclude that binding of substrates to ttslydfl does not strictly require the presence of proline residues.
next, we tested binding of an s <dig> mutant peptide where the sole tryptophan residue was replaced by alanine .
this peptide retained the ability to bind to both domains of ttslydfl, albeit with moderately reduced affinities .
the results using point-mutated peptide variants thus further underline the lax sequence specificity of ttslyd.
the thermodynamic parameters of binding to the if domain are similar for the wild-type and point-mutated peptides, but the enthalpies of binding to the fkbp domain are significantly reduced for the p29e, p25a/p29e, p25n/p29n, and w23a variants, which is again partly compensated for by gains in entropy .
finally, we also analyzed binding of the s <dig>  s <dig>  and mutated variants of the s <dig> peptide to a chimeric ttslyd construct, in which the if domain is replaced by the flap loop from human fkbp <dig> .
as expected, only one binding event was observed in this case .
furthermore, the affinities for the fkbp domain were found to be  <dig> – <dig> -times lower in the absence of the if domain , which is primarily due to a more unfavorable binding entropy .
overall structure of ttslyd in complex with peptides and fk506
three structures of ttslydfl were known prior to this study : two apo structures  and one with the modified tetrapeptide suc-alpf-pna bound to the fkbp domain   <cit> .
however, no structures had been determined for ttslyd, or any other member of the slyd family, in complex with longer, unmodified peptides or with inhibitors.
we therefore co-crystallized the full-length and Δif constructs of ttslyd with several of the peptides identified as substrates in the binding studies, namely the s <dig>  s <dig>  t <dig>  s2-plus <dig>  and s2-w23a peptides.
in addition, we carried out co-crystallization with the fkbp inhibitor fk <dig>  for phasing, we used both molecular replacement and single-wavelength anomalous diffraction , as detailed in the “methods.” in total, we obtained five structures of ttslydfl and three of ttslydΔif at a maximum resolution of up to  <dig>  Å .
furthermore, there is more than one molecule of ttslyd in the asymmetric unit in several cases , which for ttslydfl:s <dig> and ttslydfl:s2-w23a display substantial differences in substrate binding .
in most of the ttslydfl complexes, a peptide is bound to each of the if and fkbp domains .
notably, the positions of the binding sites in the crystal structures are the same as in solution, as confirmed by mapping of the chemical shift perturbations .fig.
<dig> overview of structures.
the structures of all five full-length ttslyd  and three ttslyd constructs with the insert-in-flap  domain replaced by the flap loop from human fkbp <dig>  are shown in ribbon representation with the fk506-binding protein  domain in white, if domain in blue, inter-domain loops in orange , and flap loop in black .
spheres designate bound anions , and pink sticks represent the bound peptides and fk <dig>  all structures are shown in the same orientation and are labeled according to which substrate is bound.
note that the ttslydfl:s <dig> and ttslydfl:s2w23a structures display different peptide binding modes for the different ttslydfl copies in their asymmetric units.
ttslydfl:s <dig> thus presents two different binding modes at the if domains of ttslydfl molecules a and b , while ttslydfl:s2w23a displays two different binding modes at the fkbp domain of molecules a and c contra molecules b and d .
additional file  <dig> shows the binding site for the chloride ion in detail, and additional file  <dig> shows a metal binding site that was omitted from the main figure for clarity
<dig>
<dig>
<dig>
<dig>
<dig>
<dig>
<dig>
<dig>
<dig>
90
<dig>
<dig>
<dig>
90
90
90
r
sym
i/σi
r
work / r
free
all three ttslyd∆if data sets were processed anomalously, while the full-length ttslyd data sets were not.
numbers in parentheses refer to statistics for the outer shell.
the ramachandran statistics and clash scores were determined using the molprobity validation tool
apart from the differences in substrate binding, the if and fkbp domains show only small structural variations except in the β8–β <dig> hairpin  of the if domain  and the c-terminal tail of the fkbp domain , which is in line with previous results on ttslyd and other members of the slyd family .
the if and fkbp domains are connected via loop65− <dig> and loop118− <dig>  which adopt the same conformations as previously described for other members of the slyd family  <cit> .
however, in spite of the loops being structurally well defined, some degree of bending must take place in these regions, because the relative orientation of the two domains is quite variable .
a computational analysis suggests that there is a key pivot point in the hinge region comprising residues 62– <dig> , which can be considered part of both the fkbp domain and the inter-domain connectors, and which is also conserved in many other fkbps where it forms the n-terminal base of the flap loop  <cit> .
a high mobility of the y <dig> side chain is furthermore confirmed by aromatic 1h–13c nmr spectra of apo ttslydfl, where y <dig> is the only aromatic residue completely broadened beyond detection due to conformational exchange dynamics affecting both the δ and ε positions .
as discussed further below, this variability is intimately connected with interactions between the side chain of y <dig> and the peptides bound to the fkbp domain.
the fkbp <dig> flap loop inserted into ttslydΔif in place of the if domain adopts essentially the same conformation as in full-length fkbp <dig> in the case of ttslydΔif:fk <dig>  while it is partially disordered in ttslydΔif:s2-w23a and ttslydΔif:s <dig> .fig.
<dig> structural flexibility.
a superimposition of the insert-in-flap  domain for all molecules of the five full-length ttslyd  structures.
colored as in fig.
<dig> except that the structurally most variable region is red .
b superimposition of the fk506-binding protein  domain for all eight ttslydfl structures and ttslyd constructs with the insert-in-flap  domain replaced by the flap loop from human fkbp <dig> .
colored as in fig.
<dig>  except that residues 62– <dig>  which can be considered as part of both the fkbp domain and the connector loops, are violet, and that the structurally most variable region is red .
c all ttslydfl molecules of the five ttslydfl structures were superimposed on the inter-domain loops .
d a dyndom computational analysis was carried out to identify putative hinge regions between the domains based on pair-wise superimpositions of all possible combinations of ttslydfl molecules.
the results are displayed in a “putty” representation: the more commonly a given residue was found to be part of a hinge region, the thicker the putty trace.
for loop65– <dig>  the thick part clearly converges around residues 62– <dig> at the n-terminal junction, while it is more diffusely distributed for loop118– <dig>  additional file  <dig> shows 1h–13c transverse relaxation optimized spectroscopy hetero single quantum coherence  spectra of the fδ,ε/yδ and yε region data that corroborate the results from the dyndom analysis
a previously unrecognized chloride anion is bound near the binding site of the fkbp domain in all of the new structures, except ttslydfl:s2-plus <dig> where it is replaced by sulfate , and a metal ion is bound in a previously identified binding site near the c-terminus  <cit>  in the ttslydΔif structures, but not the ttslydfl structures .
the role of the chloride ion is unknown, whereas the metal binding site could be important for facilitating metal insertion and folding of metalloproteins  <cit> .
peptide binding to the if domain
a peptide is bound to the if domain in four structures: ttslydfl:s <dig>  ttslydfl:s2-w23a, ttslydfl:s2-plus <dig>  and ttslydfl:t <dig> .
indeed, the only structure with a substrate-free if domain is ttslydfl:fk <dig> , which was crystallized in the absence of a peptide.
the binding site consists of a highly hydrophobic groove comprising v <dig>  f <dig>  f <dig>  l <dig>  v <dig>  and f <dig>  as well as the edge of the β8–β <dig> hairpin .
the binding mode is highly variable among the structures.
moreover, in the case of ttslydfl:s <dig>  there are two ttslydfl molecules in the asymmetric unit , which bind the peptides in very different ways .
the resolution of this structure is, however, rather low , and although the electron density map clearly shows that the peptide binds differently to the if domains of molecules a and b, the side chains are poorly defined for the peptide bound to molecule b .
the s <dig> peptide inserts w23s <dig> and i31s <dig> into the binding groove of molecule a , whereas it appears to insert p29s <dig> and f32s <dig> in molecule b .
we therefore expected that a peptide where w23s <dig> is mutated to alanine would bind similarly to the s <dig> peptide bound to molecule b in the ttslydfl:s <dig> structure.
surprisingly, the structure of ttslydfl:s2-w23a revealed instead a third binding mode where f30s <dig> and i31s <dig> are inserted into the binding groove .
furthermore, although the s2-plus <dig> peptide encompasses the same residues that form most of the intermolecular contacts in both the ttslydfl:s <dig> and ttslydfl:s2-w23a structures, including w23s <dig>  p29s <dig>  i31s <dig>  and f32s <dig> , it was found to bind in yet a fourth mode with v38s <dig> and i40s <dig> inserted into the binding groove .
interestingly, although the binding mode is variable, three aspects are shared for all structures:  the peptides bind to the β8–β <dig> hairpin through β-strand augmentation with two to four hydrogen bonds ,  the peptides generally insert two hydrophobic side chains into the binding groove as detailed above , and  peptide binding does not perturb the hydrophobic groove .
the only exception to rule  is the t <dig> peptide, which inserts only one hydrophobic residue , but this peptide also binds with much lower affinity than the s <dig>  s2-w23a, and s2-plus <dig> peptides .
in addition to the shared core interactions, a number of highly variable peripheral interactions are also formed.
these mainly include van der waal interactions , but also some hydrogen bonds, as well as a single salt bridge in the case of ttslydfl:s2-plus <dig> .
the fact that rather extensive interactions are formed between the if domain and the peptides is also reflected in the favorable binding enthalpy measured by itc.
interestingly, the proline residues of the peptides bound to the if domains are all in the trans conformer—with the possible exception of the s <dig> peptide bound to molecule b in the ttslydfl:s <dig> structure, where p29s <dig> could be in cis configuration —and in most cases do not form direct interactions with the if domain.
this is in good agreement with the binding studies, which showed that the if domain is capable of binding peptides both with and without proline residues with high affinity.fig.
<dig> binding of peptides to the insert-in-flap  domain.
a the substrate-free if domain of ttslydfl:fk <dig>  is depicted as in fig.
<dig>  except that the backbone of β <dig> is shown in sticks, and the side chains of residues forming a hydrophobic binding groove are shown in green sticks and semi-transparent spheres.
b binding of the s <dig> peptide to the if domain of molecule a in the ttslydfl:s <dig> structure.
the backbone of the peptide is shown in pink sticks, and the hydrophobic side chains that are sequestered in the hydrophobic binding groove are shown in sticks and semi-transparent spheres.
dashes indicate main chain hydrogen bonds between the peptide and β <dig>  c peptide binding in ttslydfl:s <dig> .
d peptide binding in ttslydfl:s2-w23a .
e peptide binding in ttslydfl:s2-plus <dig>  f peptide binding in ttslydfl:t <dig>  additional file  <dig> shows the electron density map for the s <dig> peptides bound to the if domain, additional file  <dig> shows an analysis of the structural changes in the if domain induced by substrate binding, and additional file  <dig> shows the peripheral substrate:if domain interactions
the binding site of the fkbp domain
we determined several crystal structures of ttslydfl and ttslydΔif with either a long peptide or fk <dig> bound to the fkbp domain, namely ttslydfl:s <dig>  ttslydfl:s2-w23a, ttslydfl:t <dig>  ttslydfl:fk <dig>  ttslydΔif:s2-w23a, ttslydΔif:s <dig>  and ttslydΔif:fk <dig>  whereas the ttslydfl:s2-plus <dig> structure displayed a substrate-free fkbp domain .
to the best of our knowledge, these represent the first structures of an fkbp domain in complex with long, unmodified peptides, and the first structures of a member of the slyd family in complex with a macrolide inhibitor.
both fk <dig> and the peptides bind in the hydrophobic pocket, which is composed of numerous hydrophobic and aromatic residues, including y <dig>  l <dig>  l <dig>  y <dig>  l <dig>  i <dig>  l <dig>  l <dig>  and f <dig>  and is flanked by d <dig>  y <dig>  and h <dig> as well as the bound anion .
binding of fk <dig> or peptides strongly affect the position of y <dig>  as further described below, and the loop encompassing l <dig> and i <dig> also moves slightly, but otherwise the binding site remains relatively unperturbed .fig.
<dig> binding of peptides and fk <dig> to the fk506-binding protein  domain.
a the substrate-free fkbp domain of ttslydfl:s2-plus <dig>  the color scheme is the same as used in fig.
<dig>  except that residues 62– <dig> are violet, and the residues forming the hydrophobic binding pocket are green.
residues involved in binding are shown in sticks.
b binding of fk <dig> to full-length ttslyd .
dashes indicate distances up to  <dig>  Å between conventional hydrogen bond donors and acceptors .
the pipecolinyl ring and selected atoms of fk <dig> are labeled.
c binding of the s2-w23a peptide to molecule a in ttslydfl:s2-w23a.
a similar binding mode is also seen for molecule c and ttslydfl:s <dig>  for clarity, side chains are only shown for residues 27– <dig> of the peptide.
the residues of the peptide are labeled.
d binding of the s2-w23a peptide to molecule d in ttslydfl:s2-w23a.
a similar binding mode is also seen for molecule b and ttslydΔif:s2-w23a.
e ramachandran plots for residues k28s <dig> and p29s <dig> of ttslydfl:s <dig> as well as ttslydfl:s2-w23a molecules a and c compared to the standard values for i +  <dig> and i +  <dig> residues of a type via <dig> β-turn.
there is a clear match.
f similar ramachandran plots for residues k28s <dig> and p29s <dig> of ttslydfl:s2-w23a molecules b and d as well as ttslydΔif:s2w23-a compared to the standard values of a type vib β-turn.
there is a partial match, but the phi angle of k28s <dig>  is off by 40–60°.
additional file  <dig> shows an analysis of the structural changes in the fkbp domain induced by substrate binding, additional file  <dig> illustrates the non-canonical binding modes of the t <dig> and s <dig> peptides, and additional file  <dig> shows the peripheral substrate:fkbp domain interactions
binding of fk <dig> to the fkbp domain
fk <dig> binds in a similar way to all three copies of ttslyd in the asymmetric unit of ttslydfl:fk <dig> and in both copies in ttslydΔif:fk <dig> , but exhibits some structural variation in parts of the molecule that are more distal to the binding pocket.
the binding mode is similar to that observed in the fk506–fkbp <dig> complex  <cit> .
specifically, the pipecolinyl ring, which mimics a proline side chain, is inserted into the center of the hydrophobic pocket, and four hydrogen bonds are formed: two to the backbone of n <dig> and i <dig> , and one each to the side chains of d <dig> and y <dig>  .
apart from these conventional hydrogen bonds, a number of weaker ch–o hydrogen bonds are also present.
most noteworthy are three potential interactions between the c <dig> carbonyl oxygen of fk <dig> and ch groups of y <dig>  l <dig>  and f <dig>  which are reminiscent of three putative ch–o interactions observed between fk <dig> and fkbp <dig> residues y <dig>  f <dig>  and f <dig>  <cit> .
indeed, y <dig> and f <dig> are equivalent to ttslyd residues y <dig> and f <dig>  respectively.
peptide binding to the fkbp domain
the s <dig> and s2-w23a peptides both bind by inserting the side chain of p29s <dig> in cis form into the center of the binding pocket .
the binding mode is the same for both ttslydfl molecules in the ttslydfl:s <dig> structure , whereas the s2-w23a peptide binds in two different ways to the four molecules in the ttslydfl:s2-w23a structure: one similar to the s <dig> peptide , and one in a different but partially overlapping fashion .
this latter binding mode was also observed in the ttslydΔif:s2-w23a structure.
the ttslydfl:t <dig> and ttslydΔif:s <dig> structures both show alternative binding modes.
the t <dig> peptide adopts a reverse orientation as compared to the s <dig> and s2-w23a peptides, and forms only few interactions with the fkbp domain .
the s <dig> peptide binds to ttslydΔif by inserting a valine instead of a proline residue into the binding pocket , which supports the conclusion that in vitro binding to the fkbp domain does not strictly require the presence of proline residues.
the non-canonical binding modes agree well with the weaker affinities of these peptides, but it is unclear if they mimic any physiologically relevant interactions.
the two different binding modes observed for the s <dig> and s2-w23a peptides are characterized by different β-turn conformations.
β-turns consist by definition of four residues with a distance between the cα atoms of residues i and i +  <dig> of 7 Å or less, and are divided into nine types according to the phi and psi torsion angles of residues i +  <dig> and i +  <dig>  with the additional requirement for types via <dig>  via <dig>  and vib that i +  <dig> must be a cis-proline  <cit> .
we found that the two binding modes observed for the peptides bound to both ttslydfl molecules in the ttslydfl:s <dig> crystal, and for the peptide bound to ttslyd molecules a or c in the ttslydfl:s2-w23a crystal , conform to a type via <dig> β-turn with cis-p29s <dig> in position i +  <dig> , whereas the conformation of the peptide bound to ttslyd molecules b or d in the ttslydfl:s2-w23a crystal, as well as in ttslydΔif:s2-w23a , conforms to a type vib-like β-turn with a distorted i +  <dig> phi angle .
a number of interactions are shared between the via <dig> and vib-like binding modes :  two β-strand type interactions are formed between k28s <dig> and n <dig> and i <dig> of ttslyd,  the backbone nitrogen of p29s <dig> is within potential hydrogen-bonding distance  of the hydroxyl group of y <dig> from ttslyd,  the side chains of m27s <dig>  k28s <dig>  and p29s <dig> interact in a similar way with the binding pocket via van der waal and hydrophobic interactions, and  k28s <dig> interacts electrostatically with the bound chloride ion.
note that although k28s <dig> is well accommodated, the binding pocket clearly has room for larger side chains.
indeed, when the activity of slyd from e. coli was screened with an ala-x-pro-phe tetrapeptide with each of the  <dig> proteinogenic residues in the “x” position, aromatic residues were found to yield the highest kcat/km values, while lysine was in the middle range  <cit> .
notwithstanding the listed similarities, there are, however, a number of differences between the two binding modes.
most notably, y <dig> forms different interactions in the two forms apart from the shared potential hydrogen bond with the backbone nitrogen of p29s2: in the via <dig> form, the hydroxyl group of the y <dig> side chain is within hydrogen-bonding distance of the backbone nitrogen of f30s <dig> and the carbonyls of m27s <dig> and i31s <dig> , while in the vib-like form, it is instead within hydrogen-bonding distance of the backbone carbonyl of p29s <dig> .
in addition, a hydrogen bond is formed between f30s <dig> and h <dig> for the vib-like form, but not the via <dig> form, and several differences are also observed in peripheral interactions .
notably, k26s <dig>  m27s <dig>  and k28s <dig> adopt almost the same conformations in both binding modes.
the differences in binding mode of the via <dig> and vib-like forms thus lie mainly in the residues that are found in the c-terminal direction from p29s <dig> .
comparison of the binding modes of fk <dig> and the peptides
a comparison of the fk <dig> and peptide binding modes reveals that the interactions between fk <dig> and the backbone atoms of n <dig> and i <dig>  which are mediated by the o <dig> carbonyl oxygen atom at c <dig> and the o <dig> hydroxyl group at c <dig>  respectively, overlap with the two β-strand type interactions formed by k28s <dig> in the s <dig> and s2-w23a peptides .
it furthermore shows that the pipecolinyl ring partially overlaps with the side chain of cis-p29s <dig> in both peptide binding modes, but is in a roughly orthogonal orientation relative to these , which enables it to reach considerably deeper into the pocket.
moreover, there is a partial overlap between the large appendage at the c <dig> atom of fk <dig> and the side chain of m27s <dig> .
the pipecolinyl ring is flanked by a dicarbonyl moiety encompassing both an amide carbonyl at c <dig> and an α-keto carbonyl at c <dig> , which are both candidates for mimicking the carbonyl group of a bound proline residue  <cit> .
the carbonyl at c <dig> is in the trans form, as was also observed in the fkbp12:fk <dig> complex  <cit> .
yet, it still hydrogen bonds with y <dig> similarly to the cis-p29s <dig> residue of the type vib-like peptide, though at a different angle .
this is made possible through the rotation of the pipecolinyl ring described above combined with a difference in the proline phi and psi angles relative to the equivalent angles in fk <dig> and a slight change in the position of y <dig> .
the carbonyl group at c <dig> is almost orthogonal to the carbonyl at c <dig> , which has been suggested to enable fk <dig> to mimic the twisted transition state  <cit> .
it does not form conventional hydrogen bonds, but points directly into a sub-pocket of the binding site formed by y <dig>  l <dig>  and f <dig>  with which it forms ch–o hydrogen bonds, as described above.
as hinted above, the position of y <dig> differs depending on which substrate is bound.
indeed, it does not only differ between fk <dig> and peptides, but also between peptides adopting different binding modes .
this may suggest that the flexibility of the hinge region encompassing y <dig> is important for enabling the fkbp domain to adapt to different substrates.
furthermore, the different positions of y <dig> also translate into different positions of the if and fkbp domains relative to each other , which may be relevant in relation to the reported cross talk between them  <cit> .fig.
<dig> comparison of binding modes for the fk506-binding protein  domain.
a superimposition of the fkbp domains of ttslydfl:s2-w23a molecule b , ttslydfl:s2-w23a molecules d , and ttslydfl:fk <dig> , showing the variation in interactions with the backbone of n35–i <dig>  selected amino acid residues and fk <dig> atoms are labeled.
b same overlay but showing instead the interaction with y <dig>  c superimposition of the fkbp domains of all full-length ttslyd  molecules in the ttslydfl:s2-w23a, ttslydfl:s <dig>  ttslydfl:fk <dig>  and ttslydfl:s2-plus <dig> structures, showing how the orientation of the insert-in-flap  domain varies with the position of y <dig>  and thus with which substrate is bound.
ttslydfl molecules binding the peptides in the type via <dig> or vib-like modes are purple and light blue, respectively, ttslydfl:fk <dig> is yellow, and ttslydfl:s2-plus <dig>  is gray
enzymatic activity
in order to accurately measure the catalytic activity of ttslydfl on peptidyl-prolyl cis/trans isomerization, we carried out michaelis-menten studies under equilibrium conditions using nmr lineshape analysis, which is capable of monitoring the rate of exchange between cis and trans conformations of the peptide substrate.
initially, we benchmarked the method against literature data using the standard suc-alpf-pna tetrapeptide .
we measured a kcat/km value of  <dig>  ±  <dig>  μm−1s− <dig> for the wild-type ttslydfl, which is in good agreement with previously published data obtained using other methods  <cit> .
for the ttslydΔif construct, we obtained kcat/km =  <dig>  ±  <dig>  μm−1s− <dig>  showing a minor influence of the if domain.
interestingly, no such effect of the if domain was detected for e. coli slyd when isomerization of suc-alpf-pna was monitored using uv/vis spectroscopy rather than nmr  <cit> .fig.
<dig> michaelis-menten analysis of ttslyd at 25 °c.
the data for full-length ttslyd  domain replaced by the flap loop from human fkbp <dig>  are shown as gray open circles.
a determination of kcat/km using the suc-alpf-pna tetrapeptide as substrate.
the enzyme concentrations were varied, while 500 μm suc-alpf-pna  was used in all experiments.
we determined kcat/km to be  <dig>  ±  <dig>  • 106 m−1s− <dig> for ttslydfl and  <dig>  ±  <dig>  • 106 m−1s− <dig> for ttslydΔif, using linear regression analysis.
b full michaelis-menten analysis using the s2-p25a peptide as substrate.
a quantity of  <dig>  μm of enzyme was used in all experiments, while the peptide concentration was varied.
by using the previously determined cis content of  <dig>  %, these concentrations were transformed into concentrations of s2-p25a cis.
michaelis-menten fitting of ttslydfl resulted in a kcat/km of  <dig>  ±  <dig>  • 108 m−1s− <dig> with kcat =  <dig>  ±  <dig>  s− <dig> and km = 2000 ± 410 μm.
because less curvature and precision was obtained for ttslydΔif, only the kcat/km of  <dig>  ±  <dig>  • 108 m−1s− <dig> is reported
having validated the method, we next turned to the longer unmodified peptides of interest here.
we opted to use the s2-p25a peptide for these studies because it has the advantage over the s <dig> peptide that it contains only one proline residue , making data interpretation more straightforward, while it still binds well to both domains .
the 13c shifts of proline in the s2-p25a peptide show the same characteristics as an isolated proline amino acid.
by comparing intensities between the cis and trans forms, the relative population of the cis form was found to be  <dig>  ± 1 %.
we determined apparent rate constants for ttslydfl and ttslydΔif as a function of peptide concentration .
the resulting kcat/km value was 368 ± 4 μm–1s– <dig> for ttslydfl, which is a factor of 100– <dig> higher compared to results obtained previously for various members of the slyd family using tetrapeptides or unfolded proteins  <cit> , and even exceeds those generally reported for fkbps and other ppiases  <cit> .
for ttslydΔif the obtained kcat/km was 248 ± 1 μm–1s– <dig>  clearly indicating that the if domain affects the activity of the fkbp domain, similarly to what was observed using tetrapeptide substrates.
however, the difference in kcat/km between ttslydfl and ttslydΔif is much smaller than what has been observed using protein substrates  <cit> , and may simply relate to the lower binding affinity of ttslydΔif relative to ttslydfl .
in the case of ttslydfl we could separate the two parameters, yielding kcat =  106 s– <dig> and km =  <dig>  ±  <dig>  mm.
neither value is very precisely determined, but it is obvious from the modest curvature of the michaelis-menten plot  and the substrate concentrations used here that kcat > >  <dig>  s− <dig> and km > > 100 μm, where the latter value is significantly higher than kd.
taken together, these results underline the high catalytic efficiency of ttslyd when acting on unfolded substrates.
mutational probing of catalytically important residues
to better understand which residues play an important role for binding and catalysis, we generated a number of mutations in ttslydfl and tested their ability to bind the wild-type s <dig> peptide and catalyze cis/trans isomerization .
specifically, we mutated y <dig> and h <dig> of the inter-domain loops; y <dig>  d <dig>  n <dig>  i <dig>  and f <dig> of the fkbp domain; and a <dig>  y <dig>  q <dig>  and m <dig> of the if domain .
the affinities for binding of the s <dig> peptide to these mutants were measured by itc and the activity determined by nmr using the standard suc-alpf-pna tetrapeptide, in order to generate results comparable to previously published activity data.
to separate contributions to binding from contributions to catalysis, the nmr-derived kcat/km values were plotted against the fkbp domain-specific kd values of the s <dig> peptide determined by itc, and compared to the results expected when a mutation affects only binding or only the turnover rate .
although the kcat/km and kd values refer to different substrates, this analysis should provide valuable insights into the relative impact of a given mutation on binding and turnover.fig.
<dig> mutational studies.
a mapping of mutated residues on the structure.
the side chains of mutated residues are shown in sticks and semi-transparent spheres for ttslydfl:s <dig> molecule a and colored according to activity relative to that of the wild type : dark red, 0–30 %; pink, 31–40 %; pale pink, 41–50 %; pale cyan, 50–60 %; bright blue, 61–80 %; dark blue, 81–100 %.
note: y <dig> was also mutated to phe, which caused a reduction to 76 % of that of the wild type.
b sequence conservation.
same as in panel a, except that the structure is colored by increasing level of conservation, ramped from teal  over cyan, white, and pink to purple .
c effect of mutations on activity and binding.
kcat/km values from the suc-alpf-pna tetrapeptide assay plotted against fk506-binding protein  domain-specific kd values of the s <dig> peptide.
full-length ttslyd  is shown in black , ttslyd constructs with the insert-in-flap  domain replaced by the flap loop from human fkbp <dig>  are shown in gray , if domain mutants are shown in red, fkbp-domain mutants in blue, and linker mutants in green.
the solid gray curve crossing the ttslydfl wild-type data point shows the result of varying km  only.
the dashed gray curve was generated with kcat =  <dig>  · kcat .
the effect of all mutations close to the solid curve  can be explained primarily by binding, assuming that the effects on kd and km are the same.
mutations with data points falling on the dashed curve  can be seen to reduce kcat by a factor of two compared to the wild type, while showing variable binding strength.
f128a and y63a have apparently greater effects on kcat, highlighting the importance of these residues
ttslydfl
ttslydΔif
the affinities and thermodynamic parameters were determined at 20 °c for the wild type s <dig> peptide using isothermal titration calorimetry, while the activities were determined for the suc-alpf-pna tetrapeptide using nuclear magnetic resonance spectroscopy.
ttslyd
Δif ttslyd constructs with the insert-in-flap domain replaced by the flap loop from human fkbp <dig>  ttslyd
fl full-length ttslyd
the changes in affinities are about twofold or less in all cases, except for mutations of y <dig>  where a fivefold increase in the affinity of the if domain was observed , thus supporting the notion of inter-domain cross talk.
the mutation with the strongest effect on ppiase activity was y63a, which retained only 21 % activity .
it is therefore likely that this residue plays an important role in the catalytic mechanism, in keeping with its ideal position for forming hydrogen bonds with the substrate .
this interpretation is further supported by its high degree of sequence conservation  as well as the kcat/km versus kd plot .
however, the effect on activity is clearly not exclusively due to hydrogen bonds formed by the hydroxyl group, because the y63f mutant retained as much as 76 % activity .
interestingly, the affinity of the fkbp domain was only negligibly decreased for y63a, but was  <dig> -times higher for y63f with a significantly greater enthalpy and largely unperturbed entropy of binding as compared to the wild type .
this is similar to what has been observed for binding of fk <dig> or rapamycin to the equivalent y82f mutant of fkbp <dig>  where detailed analysis of the binding thermodynamics indicate that the effect of the mutation is due to altered solvation  <cit> .
the second-most detrimental mutations were y92a, m96a, and f128a, which each retained 30–40 % activity .
f <dig> forms part of the hydrophobic binding site  and is highly conserved , whereas y <dig> and m <dig> are found in the dynamic β8–β <dig> hairpin of the if domain and are rather poorly conserved .
considering the effect of the binding affinity on kcat/km , f <dig> seems to be as important for catalysis as y <dig>  interestingly, while the absence of the if domain has a positive effect on kcat, the y92a and m96a mutations seem instead to have a negative effect .
we hypothesize that the effect of these two mutations on the catalytic activity is due to interference with inter-domain cross talk .
discussion
substrate selectivity of the if domain
itc experiments revealed that the if domain binds long peptides with up to nanomolar affinity, and that substantial sequence variation of the substrate can be accommodated through enthalpy-entropy compensation.
notably, we also found that peptides without proline residues can bind to the if domain, which is in line with a previous study showing that proline-free substrates can inhibit binding of proline-containing substrates to e. coli slyd  <cit> .
four structures were obtained with 15-residue-long peptides bound to the if domain .
the peptides bind in the large hydrophobic groove delineated by the 310-helix and β8–β <dig> hairpin through β-strand augmentation, as was also previously observed for the linker of the uncleaved purification tag in the structure of e. coli slpa  <cit> , and originally predicted based on structural analysis of apo ttslydfl  <cit> .
the binding modes are highly variable , but seem to nonetheless be governed by common recognition principles.
it thus appears that the flexible β8–β <dig> hairpin and the likewise flexible unfolded polypeptide stretch of the substrate are able to structurally adapt to each other, such that one or more  hydrophobic side chains of the substrate can be sequestered in the quite rigid hydrophobic groove.
this highly adaptable binding strategy explains how the if domain is able to bind extended/flexible polypeptide stretches containing hydrophobic residues in various different sequence contexts.
notably, such stretches are a hallmark of unfolded proteins, and the if domain therefore seems ideally suited for its function as a folding chaperone.
substrate selectivity of the fkbp domain
most of the peptides tested in the itc binding studies were found to bind to the fkbp domains of both ttslydfl and ttslydΔif, though the affinities were almost invariably highest for the former.
interestingly, as was also found to be the case for the if domain, the presence of proline residues in the substrate is not a requirement for binding to the fkbp domain in vitro.
it is questionable whether such proline-independent binding is physiologically relevant, but this could potentially be the case if the fkbp domain can serve as an auxiliary binding site for the chaperone domain, such as has been suggested for trigger factor  <cit> , or if it can engage non-proline dimerization motifs of cognate interaction partners, such as has been described for human fkbp <dig>  <cit> .
five structures were obtained of ttslydfl or ttslydΔif with a 15-residue-long peptide bound to the fkbp domain .
the peptide inserts a cis-proline residue into the hydrophobic binding pocket in all cases, except in the ttslydΔif:s <dig> structure, where a trans-valine residue is inserted instead, thus further supporting the conclusion that proline residues are not essential for binding to the fkbp domain.
two peptides, s <dig> and s2-w23a, adopt β-turn structures with a cis-proline in the i +  <dig> positions.
a major role of cis-prolines is to enable the formation of type via <dig>  via <dig>  and vib β-turns, which all strictly require the presence of a cis-proline in the i +  <dig> position  <cit> .
based on early computational studies, it was suggested that fkbp <dig> may be specific for type via β-turns  <cit> , whereas the crystal structure of a tetrapeptide–cyclophilin a complex showed the substrates bound as a type vib β-turn  <cit> .
taken together, this indicated that there might be a “division of labor” in the cell, with fkbps and cyclophilins acting on different types of cis-proline β-turns.
however, our results speak against such a scenario, because both type via <dig> and distorted vib β-turns are observed in our ttslydfl:s <dig>  ttslydfl:s2-w23a, and ttslydΔif:s2-w23a structures, which strongly suggests that fkbps can catalyze cis/trans isomerization of prolines present in  both these two types of β-turns.
a putative mechanism for transition state stabilization at the fkbp domain
the catalytic mechanism of ppiases does not involve any bond formation or breakage, but hinges instead on rotation around the peptidyl-prolyl bond, which is at least partially mediated by preferential stabilization of the twisted transition state  <cit> .
in the case of the cyclophilins, there has been some debate as to whether it is the part n- or c-terminal to the peptidyl-prolyl bond that rotates .
the fkbp field has not seen a similar debate, which is probably mainly due to a dearth of informative substrate complexes.
however, on the basis of the structures presented here, we find it most likely that fkbps operate with c-terminal rotation , though we concede that a definitive conclusion regarding this question will require that structures representing the trans form are also obtained.
the basis for our assertion is that the two residues n-terminal to the peptidyl-prolyl bond are anchored in the same way in both the type via <dig> and vib-like binding modes through two β-strand type hydrogen bonds supplemented by side chain interactions with the hydrophobic binding pocket , whereas the residues found c-terminal to the peptidyl-prolyl bond adopt very different positions in the via <dig> and vib-like β-turns, suggesting that this part would have more freedom to rotate during catalysis .
in line with this, it has been shown that fkbp <dig> exhibits higher sequence specificity towards the residue that immediately precedes the proline than the one that immediately follows it  <cit> .
in addition, we also found that fk <dig> forms two hydrogen bonds that mimic the β-strand type hydrogen bonds formed by the peptides .
moreover, this is not unique to ttslyd:fk <dig>  but is also commonly observed in structures of fkbp12:inhibitor complexes  <cit> .
indeed, based on such structures, it was already predicted that peptides would bind via two β-strand type hydrogen bonds in the same manner, as we have now observed for ttslyd  <cit> .
furthermore, the large appendage at the c <dig> position of fk <dig> overlaps with the side chain of m27s <dig>  that is, the residue that precedes the proline by two positions.
we therefore conclude that binding of fk <dig> involves several interactions that mimic those of the n-terminal part of a bound polypeptide, which supports the notion that this is the part that is kept anchored during catalysis.fig.
<dig> model for fk506-binding protein -mediated rotation around the peptidyl-prolyl bond.
a s2-w23a peptide bound to full-length ttslyd  molecule d .
the color scheme is the same as for fig.
<dig>  except that the colors are more subdued.
the hypothesized rotations of the p29s <dig> proline residue are indicated.
we suggest that the residue found immediately n-terminal to the peptidyl-prolyl bond are kept anchored during catalysis through β-strand type hydrogen bonds and side chain interactions with the hydrophobic pocket .
the c-terminal part, including the side chain of the proline, then rotates from the trans to the cis form via the twisted transition state  or vice versa, as indicated.
notably, the model predicts that the proline side chain penetrates deepest into the pocket in the twisted transition state, which may confer preferential stabilization of this form over the ground states.
b binding of fk <dig> to ttslydfl.
the orientation and color scheme are the same as in panel a. the pipecolinyl ring is in the same position as expected for the proline side chain when in the syn form, which supports the notion that fk <dig> can be viewed as a mimic of the twisted transition state
interestingly, a consequence of c-terminal rotation is that the proline side chain would reach considerably deeper into the binding pocket and interact more extensively with it in the syn form  relative to the cis and trans forms , which could be a key factor in conferring preferential stabilization of the twisted transition state.
in keeping with this notion, it is well established that the hydrophobic environment of the binding pocket is critically important for the catalytic mechanism , as is also supported by our finding that the f128a mutation in the bottom of the binding pocket reduced the catalytic activity to 35 % of that of the wild type.
if, on the other hand, ttslyd mediates n-terminal rotation, then the proline side chain would remain throughout catalysis in the shallow position observed in the present structures.
fk <dig> as a potential mimic of the twisted transition state
fk <dig> has been proposed to mimic the twisted transition state with the pipecolinyl ring being a surrogate of the proline side chain, and the roughly orthogonal α-keto carbonyl group at c <dig> being a surrogate of its twisted backbone carbonyl group  <cit> .
the situation is, however, complicated by the fact that a trans amide carbonyl is found at the c <dig> position between the ring and the α-keto carbonyl, which could also serve as a mimic of the proline carbonyl group.
interestingly, we found that y <dig> hydrogen bonds to both the c <dig> carbonyl oxygen atom of fk <dig> and the cis-proline carbonyl atoms of peptides bound as a type vib-like β-turn .
it may therefore be argued that fk <dig> mimics aspects of the type vib-like cis-state, though it should be pointed out that the angles of the y63-carbonyl hydrogen bonds are markedly different in the two cases, as a consequence of the different positions of the two carbonyl groups .
a probably more significant observation is that the pipecolinyl ring is roughly orthogonal to the side chains of the cis-prolines of the bound peptides regardless of their adopted binding mode , and penetrates considerably deeper into the binding pocket.
indeed, under the premise that catalysis proceeds through c-terminal rotation around the peptidyl-prolyl bond, this finding strongly supports the notion that fk <dig> mimics the twisted transition state of the substrate .
the role of tyrosine- <dig> in the catalytic mechanism
in our mutational analysis of the binding site, the y63a mutation was found to be the most severe, with a residual activity of only 21 %.
the corresponding y <dig> residue in fkbp <dig> has been proposed to aid in catalysis by forming a hydrogen bond to the proline imide nitrogen, thereby lowering the rotational barrier of the peptidyl-prolyl bond  <cit> .
in line with this, we found that the hydroxyl group of y <dig> is indeed within hydrogen-bonding distance of the imide nitrogen of cis-p29s <dig> in both the type via <dig> and vib-like peptide binding modes, if applying a generous cut-off .
the hydroxyl group of y <dig> is furthermore within hydrogen-bonding distance of the backbone nitrogen of f30s <dig> and the carbonyls of m27s <dig> and i31s <dig> in the case of the type via <dig> binding mode , and of the p29s <dig> carbonyl group in the case of the type vib-like binding mode , which might implicate it in appropriately orienting the substrate in the binding site.
the functional role of y <dig> is, however, not entirely dependent on the hydroxyl group, given that the y63f mutant retained as much as 76 % activity.
in line with this, a mutational study on human fkbp <dig> where the equivalent y <dig> residue was replaced by each of the other  <dig> proteinogenic amino acids showed that aromatic residues, arginine, and proline retained or even improved activity, whereas most other residues caused a substantial reduction  <cit> .
furthermore, similar results have also been obtained for the equivalent y <dig> residue in plasmodium vivax fkbp <dig>  except that arginine and, in particular, proline replacements were less well tolerated  <cit> .
finally, it may be noted that although y <dig> is highly conserved, a phenylalanine is found in its place in some catalytically active fkbps, for example, e. coli slpa  <cit> .
it is therefore clear that other interactions must be able to partially substitute for any functionally important hydrogen bonds formed by the hydroxyl group, for example, ch–π, ch–o, or ch–n hydrogen bonds and/or van der waal interactions.
in relation to this point, it may be noted that several putative ch–o hydrogen bonds between fkbps and bound inhibitors have been identified in fkbp:inhibitor structures  <cit> , and that an nmr analysis of aromatic ring flips has identified a hydrogen bond between the ζ hydrogen of f <dig> and rapamycin in the fkbp12:rapamycin complex  <cit> .
comparisons of the crystal structures obtained for ttslydfl indicate that y <dig> is found in a highly mobile hinge region, which we further confirmed by aromatic 1h–13c nmr studies on apo ttslyd.
furthermore, the flap loop of fkbp <dig>  which encompasses the equivalent y <dig> residue, has also been shown to be flexible or mobile .
this mobility of y <dig> can be expected to enable a certain level of dynamic remodeling of the binding site, which could be important for allowing it to optimally interact with structurally different substrates and/or for adapting to their motions during catalysis.
in conclusion, the role of y <dig> is not yet fully clarified, but likely hinges on a combination of its ideal position for interacting with the substrate and its high level of mobility.
evidence for inter-domain cross talk
it has been reported that there is cross talk between the if and fkbp domains in the sense that binding to one domain affects dynamics  <cit> , substrate affinity  <cit> , and stability  <cit>  of the other.
here we show that 15-residue-long peptides can be bound to each of the two domains at the same time, and that deletion of the if domain results in reduced affinity and activity of the fkbp domain of ttslyd.
interestingly, the reduction in affinity stems mainly from less favorable entropy, suggesting that it may be a consequence of the enhanced dynamics of the fkbp domain that reportedly results from substrate binding at the if domain  <cit> .
we furthermore show that mutating y <dig> or h <dig>  which are part of both the inter-domain connectors and the active site of the fkbp domain, increases the affinity of the if domain up to fivefold, and that y <dig> adopts different orientations depending on the substrate, which interestingly correlates with different positions of the if and fkbp domains relative to each other .
this may suggest that y <dig> and the connector loops may be important for inter-domain cross talk, but more studies will be needed to confirm this.
finally, we show that mutating y <dig> or m <dig> in the if domain to alanine reduces the ppiase activity to 36–39 % relative to the wild type without substantially affecting affinities, indicating that these mutations may affect the dynamics of catalytically important fkbp residues.
in conclusion, our results clearly support the notion of inter-domain cross talk in slyd, and highlight the need for further experiments to pinpoint the molecular mechanisms underlying this phenomenon.
catalytic activity
our nmr lineshape analysis of ttslydfl activity on the commonly used tetrapeptide  yielded kcat/km =  <dig>  106 m−1s− <dig>  which is in line with previously published values obtained for e. coli slyd using a fluorescence-based assay .
the presence of the if domain was found to have a small effect on the activity towards tetrapeptides, which contrasts with previous studies, where no effect was observed  <cit> .
this effect probably relates to the effect of the if domain on the affinity of the fkbp domain.
for partially folded proteins, similar kcat/km values  but much higher affinities have been reported  <cit> .
however, in these cases the absence of the if domain decreased the activity by a factor of 100– <dig>  <cit> .
our studies using 15-residue-long peptides paint a different picture.
these peptides bind with affinities that are similar to those of partially folded proteins, and thus much higher than those of tetrapeptides.
however, the nmr-derived activity for the s2-p25a peptide is  <dig>  108 m−1s− <dig>  and thus a factor of 100-times higher than for both tetrapeptides and partially folded proteins.
indeed, the activity is of the same order as that of super-efficient enzymes, with kcat/km in the range 108–1010 m−1s− <dig>  whose activity is considered diffusion-limited  <cit> .
this might appear surprising given that ttslyd does not have a defined optimal substrate, but rather catalyzes the isomerization of a broad range of proline-containing peptides that form variable contacts with ttslyd.
however, it can be explained on the grounds that the catalytic efficiency of ttslyd is not hindered by substrate specificity.
the if domain was found to have a definite but small effect on the activity , similar to what we observed for suc-alpf-pna.
our michaelis-menten analysis of ttslydfl with the s2-p25a peptide revealed kcat =  <dig>  s– <dig> and km = 2000 μm, a result that highlights the high efficiency of ttslyd.
for comparison, the homologous human fkbp <dig> yields a kcat above  <dig>  s− <dig> with suc-alpf-pna as a substrate , again indicating high catalytic efficiency.
this contrasts with kcat values determined for partially folded protein substrates, which have been estimated to be around 1 s– <dig>  <cit> .
our findings can be summarized as follows: a high catalytic rate constant of kcat >  <dig>  s− <dig> is obtained with both s2-p25a and the suc-alpf-pna tetrapeptide as substrates.
however, because the longer s2-p25a peptide can make more contacts with the protein , or retain more flexibility  in the bound state, it has lower kd and km values, resulting in higher activity .
by contrast, with partially folded proteins as substrates, one observes a very low kcat , a strong impact of the if domain, and a lower overall activity than what is found using peptide substrates.
taken together, the available data suggest that while slyd can isomerize various kinds of substrates, including partially folded or unfolded proteins, it is most efficient when acting on unfolded polypeptide chains.
CONCLUSIONS
we have shown that 15-residue-long unmodified peptides bind to 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 fkbps.
we therefore conclude that long unmodified peptides are better mimics of unfolded protein substrates than the classical tetrapeptides.
we have furthermore 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.
in addition, we have presented several structures of ttslyd in complex with 15-residue-long peptides, which represent the first structures of an fkbp protein in complex with long unmodified peptides.
these structures show that unfolded polypeptides bind to the if domain in a highly adaptable fashion involving β-strand augmentation and hydrophobic interactions, which agrees well with the low sequence specificity of this domain.
furthermore, they also show that substrates can bind to the fkbp domain as both types via <dig> and vib-like β-turns, indicating that fkbps are functionally more versatile than previously appreciated.
based on a comparison of our structures, we have proposed 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 partially rotated transition state in the hydrophobic binding site.
our results furthermore establish that y <dig>  is important for the catalytic activity of ttslyd.
why this is the case is still not entirely clear, but it likely relates to its ideal position for interacting with substrates bound to the fkbp domain and its high mobility.
finally, we have found further evidence for the previously reported phenomenon of inter-domain cross talk between the if and fkbp domains, and propose that y <dig> may also be important in this context.
to sum up, we show in great detail how the if and fkbp domains recognize unfolded protein mimics, and provide novel insights into the general catalytic mechanism of fkbps.

 peptide bonds are planar with ω dihedral angles of either ~0° or ~180°. 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. the cis form is therefore much more commonly observed for prolines than for any other residues. 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. although both isoforms can be found in type iv β-turns, 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 via via and vib. protein folding requires that each proline in the sequence adopts the isoform compatible with the native fold. 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. indeed, the isomerization correlation time typically falls in the seconds to minutes time regime. nature has therefore evolved three families of peptidyl-prolyl isomerases to facilitate cis/trans isomerization: fk506-binding proteins, cyclophilins, and parvulins. 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−, but their mechanisms are not well understood. the first fkbp to be discovered was human fkbp which was identified as a binding partner of the immunosuppressive macrolide lactone fk hence the name of the family. since then, it has become clear that fkbps are widespread in all branches of life. fkbps often have additional chaperone or protein–protein interaction domains. a particularly well-studied example is slyd. this protein belongs to a prokaryotic subfamily, characterized by having an insert-in-flap chaperone domain inserted into the fkbp domain in place of the so-called flap loop found in fkbp 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– fold. 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 or variants thereof. structures have been determined for numerous fkbps in both the apo and inhibitor-bound forms. however, to the best of our knowledge, only two structures have been obtained with a bound peptide, which in both cases is suc-alpf-pna. while chemically modified tetrapeptides are well suited for studying the effects of the residues neighboring the proline, they are not ideal substrate mimics, because they bind to escherichia coli slyd with much lower affinity than refolding protein substrates. the low affinity of these peptides probably relates to the smaller interaction surface compared to protein substrates, but could also relate to their limited capacity to form naturally occurring structural elements, such as β-turns. structural insights into how substrates interact with the if domain have so far been based on a single structure of the slyd homologue slpa from e. coli, in which an uncleaved purification tag is bound at the substrate binding site of the if domain. in order to improve our understanding of the mechanism of slyd and of fkbps in general, we set out to analyze the kinetics, energetics, and structural basis for substrate binding and inhibition of slyd from thermus thermophilus using 15-residue-long unmodified peptides, which we reasoned would be better mimics of natural unfolded protein substrates than the traditionally used 4-nitroanilide tetrapeptides. indeed, these long peptides display much improved binding affinity and enzymatic turnover compared to the tetrapeptides. the enzyme peptide complexes are fairly heterogeneous in their structural and energetic aspects, but common principles could be identified for both the if and the fkbp domain. our results shed new light on how substrates are recognized, and have enabled us to propose a model for the catalytic mechanism.  to investigate the mechanism of ttslyd, we characterized its substrate binding and catalytic properties, as well as the three-dimensional structures of a number of ttslyd:peptide complexes, through the use of isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, x-ray crystallography, and site-specific mutagenesis. peptide binding studies to identify peptides that overcome the limitations of currently used substrate mimics and are suitable for structural studies, we used itc to characterize the binding of several different peptides to ttslyd, as exemplified in fig. 1a–d. we mainly used 15-residue-long proline-containing segments from proteins that have previously been shown to bind to ttslyd and/or other proteins from the slyd family, namely rnase t which is a model protein for folding studies, and the ribosomal proteins s and s; see table  for the complete list of peptide sequences. table  summarizes the results of the binding studies. interestingly, peptides derived from s and s display a dual binding mode with both a high- and a low-affinity binding site. in order to identify the binding sites we next monitored peptide binding by heteronuclear nmr spectroscopy using a variant of the s peptide. chemical shift perturbations on full-length ttslyd upon addition of peptide clearly show binding to both the if and fkbp domains. quantitative analyses of chemical shift changes as a function of added peptide enabled us to assign the stronger binding event to the if domain and the weaker one to the fkbp domain. the highest affinities were obtained for the s peptide with kd1 =   μm and kd2 =   μm, followed by the s peptide with kd1 =   μm and kd2 =   μm. addition of 5 % dimethyl sulfoxide, which was required for dissolving some peptides at high concentration, histidine tag cleavage, or a change in temperature, had only minor effects on the affinity of the s peptide. in general, peptide binding to ttslydfl is driven by a favorable change in enthalpy. a comparison of the thermodynamic fingerprints of the s and s peptides revealed that the slightly lower affinity of the s peptide to the if domain is caused by a reduction in binding enthalpy, while the weaker binding of the s peptide to the fkbp domain is due to entropic effects. for the t peptide, a single-site model was sufficient to describe binding to ttslydfl, and the affinity was found to be much lower than for the s and s peptides, which is explained primarily by an increase in unfavorable binding entropy. notably, the affinities observed for the s and s peptides are in the same range or higher than for binding of a refolding protein substrate at high salt concentration to e. coli slyd, and significantly higher than the reported kd of ~44 μm estimated for binding of the suc-alpf-pna peptide to e. coli slyd at high salt concentration. we therefore conclude that the s and s peptides can serve as improved substrate mimics for functional and structural studies of ttslyd.fig. isothermal titration calorimetry and nuclear magnetic resonance binding studies. typical itc data are shown for binding of peptides to ttslyd: a binding of the s peptide to full-length ttslyd. b binding of the s2-long peptide to ttslydfl. c binding of the s2-short peptide to ttslydfl. d binding of the s2-w23a peptide to a ttslyd construct in which the insert-in-flap domain is replaced by the flap loop from human fkbp. upper panels: raw heat pulse data. lower panels: integrated heat changes upon binding plotted against the peptide/protein concentration ratio resulting in differential binding isotherms that can be adequately described by a two-site or a one-site binding model. resulting binding parameters are summarized in table  e nmr-titration of 15n-labeled ttslydfl with the s2-p25a peptide at 25 °c. the relative change in chemical shift is plotted versus the total concentration of added peptide, with red dots corresponding to residues in the if domain and blue dots to the fkbp domain. the error bars indicate the standard error. the black solid curve represents the theoretical binding isotherm calculated using kd1 =   μm and the dashed curve represents the theoretical binding isotherm calculated using kd2 =   μm, as obtained from the itc measurements. f mean weighted chemical shift changes are plotted against residue number. resonances completely broadened by peptide binding are indicated by gray bars covering the full vertical scale. the cut-off of  ppm is shown as a red dotted line. g residues with a shift difference >  ppm or completely broadened are highlighted in red on the structure of the ttslydfl:s complex, with the peptide shown in blue trywnpkmkpfifga rywnpkmkpfifg ywnpkmkpfif wnpkmkpfi wnpkmkp for peptides derived from the s protein, parts overlapping the original s peptide are underlined. the expected charges at neutral ph are listed. these were calculated using − for glu and asp, + for lys and arg, and or + for his. in addition to the peptides listed here, we also used the following five mutants of the s peptide: p25a, p29e, p25a/p29e, p25n/p29n, and w23a ttslydfl ttslydΔif in most cases, the isothermal titration calorimetry data clearly supported the presence of two binding sites. where data could be described by only a single binding site, the results are given in the columns for binding site note that the binding experiments with the proline double mutant peptides were carried out in 5 % dimethyl sulfoxide due to solubility issues. however, this is not expected to appreciably affect the experiments, as the s peptide was found to bind with similar affinities in both the presence and absence of 5 % dmso in order to characterize the requirements for binding, we next analyzed binding of a series of variants of the s peptide to ttslydfl. to test the sequence dependency, we used a set of peptides corresponding to different segments of the s protein: three peptides covered sequences shifted n-terminally by three, six, or nine residues, compared to the original s peptide, and two peptides were shifted c-terminally by three or six residues. these peptides all retained the capacity for binding to both sites, though the affinities were most often reduced when compared to the original s peptide. in most cases, enthalpy losses were to some extent compensated by reduced entropy penalties and, in some cases, even gains in entropy. to study the length dependency, we used variants of the s peptide extended or truncated with up to eight residues. extension of the peptide had minor to moderate effects on the affinity for the if domain and the fkbp domain. removing one or two residues from each side reduced the affinity for the if domain about twofold and sevenfold, respectively, while the affinities for the fkbp domain remained relatively unperturbed. after removing an additional two to four residues, the affinities were substantially reduced, and it was no longer possible to resolve the separate binding events. the stoichiometries were, however, significantly higher than after fitting to a single binding site model, indicating that these peptides may still engage both binding sites. we also measured binding of an eight-residue-long peptide representing the plasmid-derived linker sequence bound to the if domain in the crystal structure of slpa, which was found to bind with an affinity similar to that of the seven-residue-long s2-short peptide. we conclude that the sequence requirements for peptide binding to ttslydfl are rather lax, which is in part because the enthalpic losses incurred by apparent sequence mismatching are readily compensated by gains in entropy, and that the affinity of binding to both domains is sensitive to the length of the peptide. next we tested the contribution of proline residues to the binding events. there are two proline residues in the s peptide, p and p neither single mutations, nor double mutations abrogated binding to any of the two domains, though the affinities were moderately reduced in most cases, and strongly so for the p25a/p29e double mutant. furthermore, mutating the single proline residue in the slpa linker peptide to threonine did not appreciably alter the affinity of this peptide for ttslydfl. we therefore conclude that binding of substrates to ttslydfl does not strictly require the presence of proline residues. next, we tested binding of an s mutant peptide where the sole tryptophan residue was replaced by alanine. this peptide retained the ability to bind to both domains of ttslydfl, albeit with moderately reduced affinities. the results using point-mutated peptide variants thus further underline the lax sequence specificity of ttslyd. the thermodynamic parameters of binding to the if domain are similar for the wild-type and point-mutated peptides, but the enthalpies of binding to the fkbp domain are significantly reduced for the p29e, p25a/p29e, p25n/p29n, and w23a variants, which is again partly compensated for by gains in entropy. finally, we also analyzed binding of the s s and mutated variants of the s peptide to a chimeric ttslyd construct, in which the if domain is replaced by the flap loop from human fkbp. as expected, only one binding event was observed in this case. furthermore, the affinities for the fkbp domain were found to be – -times lower in the absence of the if domain, which is primarily due to a more unfavorable binding entropy. overall structure of ttslyd in complex with peptides and fk506 three structures of ttslydfl were known prior to this study: two apo structures and one with the modified tetrapeptide suc-alpf-pna bound to the fkbp domain. however, no structures had been determined for ttslyd, or any other member of the slyd family, in complex with longer, unmodified peptides or with inhibitors. we therefore co-crystallized the full-length and Δif constructs of ttslyd with several of the peptides identified as substrates in the binding studies, namely the s s t s2-plus and s2-w23a peptides. in addition, we carried out co-crystallization with the fkbp inhibitor fk for phasing, we used both molecular replacement and single-wavelength anomalous diffraction, as detailed in the “methods.” in total, we obtained five structures of ttslydfl and three of ttslydΔif at a maximum resolution of up to  Å. furthermore, there is more than one molecule of ttslyd in the asymmetric unit in several cases, which for ttslydfl:s and ttslydfl:s2-w23a display substantial differences in substrate binding. in most of the ttslydfl complexes, a peptide is bound to each of the if and fkbp domains. notably, the positions of the binding sites in the crystal structures are the same as in solution, as confirmed by mapping of the chemical shift perturbations.fig. overview of structures. the structures of all five full-length ttslyd and three ttslyd constructs with the insert-in-flap domain replaced by the flap loop from human fkbp are shown in ribbon representation with the fk506-binding protein domain in white, if domain in blue, inter-domain loops in orange, and flap loop in black. spheres designate bound anions, and pink sticks represent the bound peptides and fk all structures are shown in the same orientation and are labeled according to which substrate is bound. note that the ttslydfl:s and ttslydfl:s2w23a structures display different peptide binding modes for the different ttslydfl copies in their asymmetric units. ttslydfl:s thus presents two different binding modes at the if domains of ttslydfl molecules a and b, while ttslydfl:s2w23a displays two different binding modes at the fkbp domain of molecules a and c contra molecules b and d. additional file shows the binding site for the chloride ion in detail, and additional file shows a metal binding site that was omitted from the main figure for clarity    90 90 90 90   r sym   i/σi   r work / r free   all three ttslyd∆if data sets were processed anomalously, while the full-length ttslyd data sets were not. numbers in parentheses refer to statistics for the outer shell. the ramachandran statistics and clash scores were determined using the molprobity validation tool apart from the differences in substrate binding, the if and fkbp domains show only small structural variations except in the β8–β hairpin of the if domain and the c-terminal tail of the fkbp domain, which is in line with previous results on ttslyd and other members of the slyd family. the if and fkbp domains are connected via loop65− and loop118− which adopt the same conformations as previously described for other members of the slyd family. however, in spite of the loops being structurally well defined, some degree of bending must take place in these regions, because the relative orientation of the two domains is quite variable. a computational analysis suggests that there is a key pivot point in the hinge region comprising residues 62–, which can be considered part of both the fkbp domain and the inter-domain connectors, and which is also conserved in many other fkbps where it forms the n-terminal base of the flap loop. a high mobility of the y side chain is furthermore confirmed by aromatic 1h–13c nmr spectra of apo ttslydfl, where y is the only aromatic residue completely broadened beyond detection due to conformational exchange dynamics affecting both the δ and ε positions. as discussed further below, this variability is intimately connected with interactions between the side chain of y and the peptides bound to the fkbp domain. the fkbp flap loop inserted into ttslydΔif in place of the if domain adopts essentially the same conformation as in full-length fkbp in the case of ttslydΔif:fk while it is partially disordered in ttslydΔif:s2-w23a and ttslydΔif:s.fig. structural flexibility. a superimposition of the insert-in-flap domain for all molecules of the five full-length ttslyd structures. colored as in fig.  except that the structurally most variable region is red. b superimposition of the fk506-binding protein domain for all eight ttslydfl structures and ttslyd constructs with the insert-in-flap domain replaced by the flap loop from human fkbp. colored as in fig.  except that residues 62– which can be considered as part of both the fkbp domain and the connector loops, are violet, and that the structurally most variable region is red. c all ttslydfl molecules of the five ttslydfl structures were superimposed on the inter-domain loops. d a dyndom computational analysis was carried out to identify putative hinge regions between the domains based on pair-wise superimpositions of all possible combinations of ttslydfl molecules. the results are displayed in a “putty” representation: the more commonly a given residue was found to be part of a hinge region, the thicker the putty trace. for loop65– the thick part clearly converges around residues 62– at the n-terminal junction, while it is more diffusely distributed for loop118– additional file shows 1h–13c transverse relaxation optimized spectroscopy hetero single quantum coherence spectra of the fδ,ε/yδ and yε region data that corroborate the results from the dyndom analysis a previously unrecognized chloride anion is bound near the binding site of the fkbp domain in all of the new structures, except ttslydfl:s2-plus where it is replaced by sulfate, and a metal ion is bound in a previously identified binding site near the c-terminus in the ttslydΔif structures, but not the ttslydfl structures. the role of the chloride ion is unknown, whereas the metal binding site could be important for facilitating metal insertion and folding of metalloproteins. peptide binding to the if domain a peptide is bound to the if domain in four structures: ttslydfl:s ttslydfl:s2-w23a, ttslydfl:s2-plus and ttslydfl:t. indeed, the only structure with a substrate-free if domain is ttslydfl:fk, which was crystallized in the absence of a peptide. the binding site consists of a highly hydrophobic groove comprising v f f l v and f as well as the edge of the β8–β hairpin. the binding mode is highly variable among the structures. moreover, in the case of ttslydfl:s there are two ttslydfl molecules in the asymmetric unit, which bind the peptides in very different ways. the resolution of this structure is, however, rather low, and although the electron density map clearly shows that the peptide binds differently to the if domains of molecules a and b, the side chains are poorly defined for the peptide bound to molecule b. the s peptide inserts w23s and i31s into the binding groove of molecule a, whereas it appears to insert p29s and f32s in molecule b. we therefore expected that a peptide where w23s is mutated to alanine would bind similarly to the s peptide bound to molecule b in the ttslydfl:s structure. surprisingly, the structure of ttslydfl:s2-w23a revealed instead a third binding mode where f30s and i31s are inserted into the binding groove. furthermore, although the s2-plus peptide encompasses the same residues that form most of the intermolecular contacts in both the ttslydfl:s and ttslydfl:s2-w23a structures, including w23s p29s i31s and f32s, it was found to bind in yet a fourth mode with v38s and i40s inserted into the binding groove. interestingly, although the binding mode is variable, three aspects are shared for all structures: the peptides bind to the β8–β hairpin through β-strand augmentation with two to four hydrogen bonds, the peptides generally insert two hydrophobic side chains into the binding groove as detailed above, and peptide binding does not perturb the hydrophobic groove. the only exception to rule is the t peptide, which inserts only one hydrophobic residue, but this peptide also binds with much lower affinity than the s s2-w23a, and s2-plus peptides. in addition to the shared core interactions, a number of highly variable peripheral interactions are also formed. these mainly include van der waal interactions, but also some hydrogen bonds, as well as a single salt bridge in the case of ttslydfl:s2-plus. the fact that rather extensive interactions are formed between the if domain and the peptides is also reflected in the favorable binding enthalpy measured by itc. interestingly, the proline residues of the peptides bound to the if domains are all in the trans conformer—with the possible exception of the s peptide bound to molecule b in the ttslydfl:s structure, where p29s could be in cis configuration —and in most cases do not form direct interactions with the if domain. this is in good agreement with the binding studies, which showed that the if domain is capable of binding peptides both with and without proline residues with high affinity.fig. binding of peptides to the insert-in-flap domain. a the substrate-free if domain of ttslydfl:fk is depicted as in fig.  except that the backbone of β is shown in sticks, and the side chains of residues forming a hydrophobic binding groove are shown in green sticks and semi-transparent spheres. b binding of the s peptide to the if domain of molecule a in the ttslydfl:s structure. the backbone of the peptide is shown in pink sticks, and the hydrophobic side chains that are sequestered in the hydrophobic binding groove are shown in sticks and semi-transparent spheres. dashes indicate main chain hydrogen bonds between the peptide and β c peptide binding in ttslydfl:s. d peptide binding in ttslydfl:s2-w23a. e peptide binding in ttslydfl:s2-plus f peptide binding in ttslydfl:t additional file shows the electron density map for the s peptides bound to the if domain, additional file shows an analysis of the structural changes in the if domain induced by substrate binding, and additional file shows the peripheral substrate:if domain interactions the binding site of the fkbp domain we determined several crystal structures of ttslydfl and ttslydΔif with either a long peptide or fk bound to the fkbp domain, namely ttslydfl:s ttslydfl:s2-w23a, ttslydfl:t ttslydfl:fk ttslydΔif:s2-w23a, ttslydΔif:s and ttslydΔif:fk whereas the ttslydfl:s2-plus structure displayed a substrate-free fkbp domain. to the best of our knowledge, these represent the first structures of an fkbp domain in complex with long, unmodified peptides, and the first structures of a member of the slyd family in complex with a macrolide inhibitor. both fk and the peptides bind in the hydrophobic pocket, which is composed of numerous hydrophobic and aromatic residues, including y l l y l i l l and f and is flanked by d y and h as well as the bound anion. binding of fk or peptides strongly affect the position of y as further described below, and the loop encompassing l and i also moves slightly, but otherwise the binding site remains relatively unperturbed.fig. binding of peptides and fk to the fk506-binding protein domain. a the substrate-free fkbp domain of ttslydfl:s2-plus the color scheme is the same as used in fig.  except that residues 62– are violet, and the residues forming the hydrophobic binding pocket are green. residues involved in binding are shown in sticks. b binding of fk to full-length ttslyd. dashes indicate distances up to  Å between conventional hydrogen bond donors and acceptors. the pipecolinyl ring and selected atoms of fk are labeled. c binding of the s2-w23a peptide to molecule a in ttslydfl:s2-w23a. a similar binding mode is also seen for molecule c and ttslydfl:s for clarity, side chains are only shown for residues 27– of the peptide. the residues of the peptide are labeled. d binding of the s2-w23a peptide to molecule d in ttslydfl:s2-w23a. a similar binding mode is also seen for molecule b and ttslydΔif:s2-w23a. e ramachandran plots for residues k28s and p29s of ttslydfl:s as well as ttslydfl:s2-w23a molecules a and c compared to the standard values for i +  and i +  residues of a type via β-turn. there is a clear match. f similar ramachandran plots for residues k28s and p29s of ttslydfl:s2-w23a molecules b and d as well as ttslydΔif:s2w23-a compared to the standard values of a type vib β-turn. there is a partial match, but the phi angle of k28s is off by 40–60°. additional file shows an analysis of the structural changes in the fkbp domain induced by substrate binding, additional file illustrates the non-canonical binding modes of the t and s peptides, and additional file shows the peripheral substrate:fkbp domain interactions binding of fk to the fkbp domain fk binds in a similar way to all three copies of ttslyd in the asymmetric unit of ttslydfl:fk and in both copies in ttslydΔif:fk, but exhibits some structural variation in parts of the molecule that are more distal to the binding pocket. the binding mode is similar to that observed in the fk506–fkbp complex. specifically, the pipecolinyl ring, which mimics a proline side chain, is inserted into the center of the hydrophobic pocket, and four hydrogen bonds are formed: two to the backbone of n and i, and one each to the side chains of d and y. apart from these conventional hydrogen bonds, a number of weaker ch–o hydrogen bonds are also present. most noteworthy are three potential interactions between the c carbonyl oxygen of fk and ch groups of y l and f which are reminiscent of three putative ch–o interactions observed between fk and fkbp residues y f and f. indeed, y and f are equivalent to ttslyd residues y and f respectively. peptide binding to the fkbp domain the s and s2-w23a peptides both bind by inserting the side chain of p29s in cis form into the center of the binding pocket. the binding mode is the same for both ttslydfl molecules in the ttslydfl:s structure, whereas the s2-w23a peptide binds in two different ways to the four molecules in the ttslydfl:s2-w23a structure: one similar to the s peptide, and one in a different but partially overlapping fashion. this latter binding mode was also observed in the ttslydΔif:s2-w23a structure. the ttslydfl:t and ttslydΔif:s structures both show alternative binding modes. the t peptide adopts a reverse orientation as compared to the s and s2-w23a peptides, and forms only few interactions with the fkbp domain. the s peptide binds to ttslydΔif by inserting a valine instead of a proline residue into the binding pocket, which supports the conclusion that in vitro binding to the fkbp domain does not strictly require the presence of proline residues. the non-canonical binding modes agree well with the weaker affinities of these peptides, but it is unclear if they mimic any physiologically relevant interactions. the two different binding modes observed for the s and s2-w23a peptides are characterized by different β-turn conformations. β-turns consist by definition of four residues with a distance between the cα atoms of residues i and i +  of 7 Å or less, and are divided into nine types according to the phi and psi torsion angles of residues i +  and i +  with the additional requirement for types via via and vib that i +  must be a cis-proline. we found that the two binding modes observed for the peptides bound to both ttslydfl molecules in the ttslydfl:s crystal, and for the peptide bound to ttslyd molecules a or c in the ttslydfl:s2-w23a crystal, conform to a type via β-turn with cis-p29s in position i + , whereas the conformation of the peptide bound to ttslyd molecules b or d in the ttslydfl:s2-w23a crystal, as well as in ttslydΔif:s2-w23a, conforms to a type vib-like β-turn with a distorted i +  phi angle. a number of interactions are shared between the via and vib-like binding modes: two β-strand type interactions are formed between k28s and n and i of ttslyd, the backbone nitrogen of p29s is within potential hydrogen-bonding distance of the hydroxyl group of y from ttslyd, the side chains of m27s k28s and p29s interact in a similar way with the binding pocket via van der waal and hydrophobic interactions, and k28s interacts electrostatically with the bound chloride ion. note that although k28s is well accommodated, the binding pocket clearly has room for larger side chains. indeed, when the activity of slyd from e. coli was screened with an ala-x-pro-phe tetrapeptide with each of the proteinogenic residues in the “x” position, aromatic residues were found to yield the highest kcat/km values, while lysine was in the middle range. notwithstanding the listed similarities, there are, however, a number of differences between the two binding modes. most notably, y forms different interactions in the two forms apart from the shared potential hydrogen bond with the backbone nitrogen of p29s2: in the via form, the hydroxyl group of the y side chain is within hydrogen-bonding distance of the backbone nitrogen of f30s and the carbonyls of m27s and i31s, while in the vib-like form, it is instead within hydrogen-bonding distance of the backbone carbonyl of p29s. in addition, a hydrogen bond is formed between f30s and h for the vib-like form, but not the via form, and several differences are also observed in peripheral interactions. notably, k26s m27s and k28s adopt almost the same conformations in both binding modes. the differences in binding mode of the via and vib-like forms thus lie mainly in the residues that are found in the c-terminal direction from p29s. comparison of the binding modes of fk and the peptides a comparison of the fk and peptide binding modes reveals that the interactions between fk and the backbone atoms of n and i which are mediated by the o carbonyl oxygen atom at c and the o hydroxyl group at c respectively, overlap with the two β-strand type interactions formed by k28s in the s and s2-w23a peptides. it furthermore shows that the pipecolinyl ring partially overlaps with the side chain of cis-p29s in both peptide binding modes, but is in a roughly orthogonal orientation relative to these, which enables it to reach considerably deeper into the pocket. moreover, there is a partial overlap between the large appendage at the c atom of fk and the side chain of m27s. the pipecolinyl ring is flanked by a dicarbonyl moiety encompassing both an amide carbonyl at c and an α-keto carbonyl at c, which are both candidates for mimicking the carbonyl group of a bound proline residue. the carbonyl at c is in the trans form, as was also observed in the fkbp12:fk complex. yet, it still hydrogen bonds with y similarly to the cis-p29s residue of the type vib-like peptide, though at a different angle. this is made possible through the rotation of the pipecolinyl ring described above combined with a difference in the proline phi and psi angles relative to the equivalent angles in fk and a slight change in the position of y. the carbonyl group at c is almost orthogonal to the carbonyl at c, which has been suggested to enable fk to mimic the twisted transition state. it does not form conventional hydrogen bonds, but points directly into a sub-pocket of the binding site formed by y l and f with which it forms ch–o hydrogen bonds, as described above. as hinted above, the position of y differs depending on which substrate is bound. indeed, it does not only differ between fk and peptides, but also between peptides adopting different binding modes. this may suggest that the flexibility of the hinge region encompassing y is important for enabling the fkbp domain to adapt to different substrates. furthermore, the different positions of y also translate into different positions of the if and fkbp domains relative to each other, which may be relevant in relation to the reported cross talk between them.fig. comparison of binding modes for the fk506-binding protein domain. a superimposition of the fkbp domains of ttslydfl:s2-w23a molecule b, ttslydfl:s2-w23a molecules d, and ttslydfl:fk, showing the variation in interactions with the backbone of n35–i selected amino acid residues and fk atoms are labeled. b same overlay but showing instead the interaction with y c superimposition of the fkbp domains of all full-length ttslyd molecules in the ttslydfl:s2-w23a, ttslydfl:s ttslydfl:fk and ttslydfl:s2-plus structures, showing how the orientation of the insert-in-flap domain varies with the position of y and thus with which substrate is bound. ttslydfl molecules binding the peptides in the type via or vib-like modes are purple and light blue, respectively, ttslydfl:fk is yellow, and ttslydfl:s2-plus is gray enzymatic activity in order to accurately measure the catalytic activity of ttslydfl on peptidyl-prolyl cis/trans isomerization, we carried out michaelis-menten studies under equilibrium conditions using nmr lineshape analysis, which is capable of monitoring the rate of exchange between cis and trans conformations of the peptide substrate. initially, we benchmarked the method against literature data using the standard suc-alpf-pna tetrapeptide. we measured a kcat/km value of  ±   μm−1s− for the wild-type ttslydfl, which is in good agreement with previously published data obtained using other methods. for the ttslydΔif construct, we obtained kcat/km =   ±   μm−1s− showing a minor influence of the if domain. interestingly, no such effect of the if domain was detected for e. coli slyd when isomerization of suc-alpf-pna was monitored using uv/vis spectroscopy rather than nmr.fig. michaelis-menten analysis of ttslyd at 25 °c. the data for full-length ttslyd domain replaced by the flap loop from human fkbp are shown as gray open circles. a determination of kcat/km using the suc-alpf-pna tetrapeptide as substrate. the enzyme concentrations were varied, while 500 μm suc-alpf-pna was used in all experiments. we determined kcat/km to be  ±  • 106 m−1s− for ttslydfl and  ±  • 106 m−1s− for ttslydΔif, using linear regression analysis. b full michaelis-menten analysis using the s2-p25a peptide as substrate. a quantity of  μm of enzyme was used in all experiments, while the peptide concentration was varied. by using the previously determined cis content of  %, these concentrations were transformed into concentrations of s2-p25a cis. michaelis-menten fitting of ttslydfl resulted in a kcat/km of  ±  • 108 m−1s− with kcat =   ±   s− and km = 2000 ± 410 μm. because less curvature and precision was obtained for ttslydΔif, only the kcat/km of  ±  • 108 m−1s− is reported having validated the method, we next turned to the longer unmodified peptides of interest here. we opted to use the s2-p25a peptide for these studies because it has the advantage over the s peptide that it contains only one proline residue, making data interpretation more straightforward, while it still binds well to both domains. the 13c shifts of proline in the s2-p25a peptide show the same characteristics as an isolated proline amino acid. by comparing intensities between the cis and trans forms, the relative population of the cis form was found to be  ± 1 %. we determined apparent rate constants for ttslydfl and ttslydΔif as a function of peptide concentration. the resulting kcat/km value was 368 ± 4 μm–1s– for ttslydfl, which is a factor of 100– higher compared to results obtained previously for various members of the slyd family using tetrapeptides or unfolded proteins, and even exceeds those generally reported for fkbps and other ppiases. for ttslydΔif the obtained kcat/km was 248 ± 1 μm–1s– clearly indicating that the if domain affects the activity of the fkbp domain, similarly to what was observed using tetrapeptide substrates. however, the difference in kcat/km between ttslydfl and ttslydΔif is much smaller than what has been observed using protein substrates, and may simply relate to the lower binding affinity of ttslydΔif relative to ttslydfl. in the case of ttslydfl we could separate the two parameters, yielding kcat =  106 s– and km =   ±   mm. neither value is very precisely determined, but it is obvious from the modest curvature of the michaelis-menten plot and the substrate concentrations used here that kcat > >   s− and km > > 100 μm, where the latter value is significantly higher than kd. taken together, these results underline the high catalytic efficiency of ttslyd when acting on unfolded substrates. mutational probing of catalytically important residues to better understand which residues play an important role for binding and catalysis, we generated a number of mutations in ttslydfl and tested their ability to bind the wild-type s peptide and catalyze cis/trans isomerization. specifically, we mutated y and h of the inter-domain loops; y d n i and f of the fkbp domain; and a y q and m of the if domain. the affinities for binding of the s peptide to these mutants were measured by itc and the activity determined by nmr using the standard suc-alpf-pna tetrapeptide, in order to generate results comparable to previously published activity data. to separate contributions to binding from contributions to catalysis, the nmr-derived kcat/km values were plotted against the fkbp domain-specific kd values of the s peptide determined by itc, and compared to the results expected when a mutation affects only binding or only the turnover rate. although the kcat/km and kd values refer to different substrates, this analysis should provide valuable insights into the relative impact of a given mutation on binding and turnover.fig. mutational studies. a mapping of mutated residues on the structure. the side chains of mutated residues are shown in sticks and semi-transparent spheres for ttslydfl:s molecule a and colored according to activity relative to that of the wild type: dark red, 0–30 %; pink, 31–40 %; pale pink, 41–50 %; pale cyan, 50–60 %; bright blue, 61–80 %; dark blue, 81–100 %. note: y was also mutated to phe, which caused a reduction to 76 % of that of the wild type. b sequence conservation. same as in panel a, except that the structure is colored by increasing level of conservation, ramped from teal over cyan, white, and pink to purple. c effect of mutations on activity and binding. kcat/km values from the suc-alpf-pna tetrapeptide assay plotted against fk506-binding protein domain-specific kd values of the s peptide. full-length ttslyd is shown in black, ttslyd constructs with the insert-in-flap domain replaced by the flap loop from human fkbp are shown in gray, if domain mutants are shown in red, fkbp-domain mutants in blue, and linker mutants in green. the solid gray curve crossing the ttslydfl wild-type data point shows the result of varying km only. the dashed gray curve was generated with kcat =  · kcat. the effect of all mutations close to the solid curve can be explained primarily by binding, assuming that the effects on kd and km are the same. mutations with data points falling on the dashed curve can be seen to reduce kcat by a factor of two compared to the wild type, while showing variable binding strength. f128a and y63a have apparently greater effects on kcat, highlighting the importance of these residues ttslydfl ttslydΔif the affinities and thermodynamic parameters were determined at 20 °c for the wild type s peptide using isothermal titration calorimetry, while the activities were determined for the suc-alpf-pna tetrapeptide using nuclear magnetic resonance spectroscopy. ttslyd Δif ttslyd constructs with the insert-in-flap domain replaced by the flap loop from human fkbp ttslyd fl full-length ttslyd the changes in affinities are about twofold or less in all cases, except for mutations of y where a fivefold increase in the affinity of the if domain was observed, thus supporting the notion of inter-domain cross talk. the mutation with the strongest effect on ppiase activity was y63a, which retained only 21 % activity. it is therefore likely that this residue plays an important role in the catalytic mechanism, in keeping with its ideal position for forming hydrogen bonds with the substrate. this interpretation is further supported by its high degree of sequence conservation as well as the kcat/km versus kd plot. however, the effect on activity is clearly not exclusively due to hydrogen bonds formed by the hydroxyl group, because the y63f mutant retained as much as 76 % activity. interestingly, the affinity of the fkbp domain was only negligibly decreased for y63a, but was -times higher for y63f with a significantly greater enthalpy and largely unperturbed entropy of binding as compared to the wild type. this is similar to what has been observed for binding of fk or rapamycin to the equivalent y82f mutant of fkbp where detailed analysis of the binding thermodynamics indicate that the effect of the mutation is due to altered solvation. the second-most detrimental mutations were y92a, m96a, and f128a, which each retained 30–40 % activity. f forms part of the hydrophobic binding site and is highly conserved, whereas y and m are found in the dynamic β8–β hairpin of the if domain and are rather poorly conserved. considering the effect of the binding affinity on kcat/km, f seems to be as important for catalysis as y interestingly, while the absence of the if domain has a positive effect on kcat, the y92a and m96a mutations seem instead to have a negative effect. we hypothesize that the effect of these two mutations on the catalytic activity is due to interference with inter-domain cross talk. discussion substrate selectivity of the if domain itc experiments revealed that the if domain binds long peptides with up to nanomolar affinity, and that substantial sequence variation of the substrate can be accommodated through enthalpy-entropy compensation. notably, we also found that peptides without proline residues can bind to the if domain, which is in line with a previous study showing that proline-free substrates can inhibit binding of proline-containing substrates to e. coli slyd. four structures were obtained with 15-residue-long peptides bound to the if domain. the peptides bind in the large hydrophobic groove delineated by the 310-helix and β8–β hairpin through β-strand augmentation, as was also previously observed for the linker of the uncleaved purification tag in the structure of e. coli slpa, and originally predicted based on structural analysis of apo ttslydfl. the binding modes are highly variable, but seem to nonetheless be governed by common recognition principles. it thus appears that the flexible β8–β hairpin and the likewise flexible unfolded polypeptide stretch of the substrate are able to structurally adapt to each other, such that one or more hydrophobic side chains of the substrate can be sequestered in the quite rigid hydrophobic groove. this highly adaptable binding strategy explains how the if domain is able to bind extended/flexible polypeptide stretches containing hydrophobic residues in various different sequence contexts. notably, such stretches are a hallmark of unfolded proteins, and the if domain therefore seems ideally suited for its function as a folding chaperone. substrate selectivity of the fkbp domain most of the peptides tested in the itc binding studies were found to bind to the fkbp domains of both ttslydfl and ttslydΔif, though the affinities were almost invariably highest for the former. interestingly, as was also found to be the case for the if domain, the presence of proline residues in the substrate is not a requirement for binding to the fkbp domain in vitro. it is questionable whether such proline-independent binding is physiologically relevant, but this could potentially be the case if the fkbp domain can serve as an auxiliary binding site for the chaperone domain, such as has been suggested for trigger factor, or if it can engage non-proline dimerization motifs of cognate interaction partners, such as has been described for human fkbp. five structures were obtained of ttslydfl or ttslydΔif with a 15-residue-long peptide bound to the fkbp domain. the peptide inserts a cis-proline residue into the hydrophobic binding pocket in all cases, except in the ttslydΔif:s structure, where a trans-valine residue is inserted instead, thus further supporting the conclusion that proline residues are not essential for binding to the fkbp domain. two peptides, s and s2-w23a, adopt β-turn structures with a cis-proline in the i +  positions. a major role of cis-prolines is to enable the formation of type via via and vib β-turns, which all strictly require the presence of a cis-proline in the i +  position. based on early computational studies, it was suggested that fkbp may be specific for type via β-turns, whereas the crystal structure of a tetrapeptide–cyclophilin a complex showed the substrates bound as a type vib β-turn. taken together, this indicated that there might be a “division of labor” in the cell, with fkbps and cyclophilins acting on different types of cis-proline β-turns. however, our results speak against such a scenario, because both type via and distorted vib β-turns are observed in our ttslydfl:s ttslydfl:s2-w23a, and ttslydΔif:s2-w23a structures, which strongly suggests that fkbps can catalyze cis/trans isomerization of prolines present in both these two types of β-turns. a putative mechanism for transition state stabilization at the fkbp domain the catalytic mechanism of ppiases does not involve any bond formation or breakage, but hinges instead on rotation around the peptidyl-prolyl bond, which is at least partially mediated by preferential stabilization of the twisted transition state. in the case of the cyclophilins, there has been some debate as to whether it is the part n- or c-terminal to the peptidyl-prolyl bond that rotates. the fkbp field has not seen a similar debate, which is probably mainly due to a dearth of informative substrate complexes. however, on the basis of the structures presented here, we find it most likely that fkbps operate with c-terminal rotation, though we concede that a definitive conclusion regarding this question will require that structures representing the trans form are also obtained. the basis for our assertion is that the two residues n-terminal to the peptidyl-prolyl bond are anchored in the same way in both the type via and vib-like binding modes through two β-strand type hydrogen bonds supplemented by side chain interactions with the hydrophobic binding pocket, whereas the residues found c-terminal to the peptidyl-prolyl bond adopt very different positions in the via and vib-like β-turns, suggesting that this part would have more freedom to rotate during catalysis. in line with this, it has been shown that fkbp exhibits higher sequence specificity towards the residue that immediately precedes the proline than the one that immediately follows it. in addition, we also found that fk forms two hydrogen bonds that mimic the β-strand type hydrogen bonds formed by the peptides. moreover, this is not unique to ttslyd:fk but is also commonly observed in structures of fkbp12:inhibitor complexes. indeed, based on such structures, it was already predicted that peptides would bind via two β-strand type hydrogen bonds in the same manner, as we have now observed for ttslyd. furthermore, the large appendage at the c position of fk overlaps with the side chain of m27s that is, the residue that precedes the proline by two positions. we therefore conclude that binding of fk involves several interactions that mimic those of the n-terminal part of a bound polypeptide, which supports the notion that this is the part that is kept anchored during catalysis.fig. model for fk506-binding protein -mediated rotation around the peptidyl-prolyl bond. a s2-w23a peptide bound to full-length ttslyd molecule d. the color scheme is the same as for fig.  except that the colors are more subdued. the hypothesized rotations of the p29s proline residue are indicated. we suggest that the residue found immediately n-terminal to the peptidyl-prolyl bond are kept anchored during catalysis through β-strand type hydrogen bonds and side chain interactions with the hydrophobic pocket. the c-terminal part, including the side chain of the proline, then rotates from the trans to the cis form via the twisted transition state or vice versa, as indicated. notably, the model predicts that the proline side chain penetrates deepest into the pocket in the twisted transition state, which may confer preferential stabilization of this form over the ground states. b binding of fk to ttslydfl. the orientation and color scheme are the same as in panel a. the pipecolinyl ring is in the same position as expected for the proline side chain when in the syn form, which supports the notion that fk can be viewed as a mimic of the twisted transition state interestingly, a consequence of c-terminal rotation is that the proline side chain would reach considerably deeper into the binding pocket and interact more extensively with it in the syn form relative to the cis and trans forms, which could be a key factor in conferring preferential stabilization of the twisted transition state. in keeping with this notion, it is well established that the hydrophobic environment of the binding pocket is critically important for the catalytic mechanism, as is also supported by our finding that the f128a mutation in the bottom of the binding pocket reduced the catalytic activity to 35 % of that of the wild type. if, on the other hand, ttslyd mediates n-terminal rotation, then the proline side chain would remain throughout catalysis in the shallow position observed in the present structures. fk as a potential mimic of the twisted transition state fk has been proposed to mimic the twisted transition state with the pipecolinyl ring being a surrogate of the proline side chain, and the roughly orthogonal α-keto carbonyl group at c being a surrogate of its twisted backbone carbonyl group. the situation is, however, complicated by the fact that a trans amide carbonyl is found at the c position between the ring and the α-keto carbonyl, which could also serve as a mimic of the proline carbonyl group. interestingly, we found that y hydrogen bonds to both the c carbonyl oxygen atom of fk and the cis-proline carbonyl atoms of peptides bound as a type vib-like β-turn. it may therefore be argued that fk mimics aspects of the type vib-like cis-state, though it should be pointed out that the angles of the y63-carbonyl hydrogen bonds are markedly different in the two cases, as a consequence of the different positions of the two carbonyl groups. a probably more significant observation is that the pipecolinyl ring is roughly orthogonal to the side chains of the cis-prolines of the bound peptides regardless of their adopted binding mode, and penetrates considerably deeper into the binding pocket. indeed, under the premise that catalysis proceeds through c-terminal rotation around the peptidyl-prolyl bond, this finding strongly supports the notion that fk mimics the twisted transition state of the substrate. the role of tyrosine- in the catalytic mechanism in our mutational analysis of the binding site, the y63a mutation was found to be the most severe, with a residual activity of only 21 %. the corresponding y residue in fkbp has been proposed to aid in catalysis by forming a hydrogen bond to the proline imide nitrogen, thereby lowering the rotational barrier of the peptidyl-prolyl bond. in line with this, we found that the hydroxyl group of y is indeed within hydrogen-bonding distance of the imide nitrogen of cis-p29s in both the type via and vib-like peptide binding modes, if applying a generous cut-off. the hydroxyl group of y is furthermore within hydrogen-bonding distance of the backbone nitrogen of f30s and the carbonyls of m27s and i31s in the case of the type via binding mode, and of the p29s carbonyl group in the case of the type vib-like binding mode, which might implicate it in appropriately orienting the substrate in the binding site. the functional role of y is, however, not entirely dependent on the hydroxyl group, given that the y63f mutant retained as much as 76 % activity. in line with this, a mutational study on human fkbp where the equivalent y residue was replaced by each of the other proteinogenic amino acids showed that aromatic residues, arginine, and proline retained or even improved activity, whereas most other residues caused a substantial reduction. furthermore, similar results have also been obtained for the equivalent y residue in plasmodium vivax fkbp except that arginine and, in particular, proline replacements were less well tolerated. finally, it may be noted that although y is highly conserved, a phenylalanine is found in its place in some catalytically active fkbps, for example, e. coli slpa. it is therefore clear that other interactions must be able to partially substitute for any functionally important hydrogen bonds formed by the hydroxyl group, for example, ch–π, ch–o, or ch–n hydrogen bonds and/or van der waal interactions. in relation to this point, it may be noted that several putative ch–o hydrogen bonds between fkbps and bound inhibitors have been identified in fkbp:inhibitor structures, and that an nmr analysis of aromatic ring flips has identified a hydrogen bond between the ζ hydrogen of f and rapamycin in the fkbp12:rapamycin complex. comparisons of the crystal structures obtained for ttslydfl indicate that y is found in a highly mobile hinge region, which we further confirmed by aromatic 1h–13c nmr studies on apo ttslyd. furthermore, the flap loop of fkbp which encompasses the equivalent y residue, has also been shown to be flexible or mobile. this mobility of y can be expected to enable a certain level of dynamic remodeling of the binding site, which could be important for allowing it to optimally interact with structurally different substrates and/or for adapting to their motions during catalysis. in conclusion, the role of y is not yet fully clarified, but likely hinges on a combination of its ideal position for interacting with the substrate and its high level of mobility. evidence for inter-domain cross talk it has been reported that there is cross talk between the if and fkbp domains in the sense that binding to one domain affects dynamics, substrate affinity, and stability of the other. here we show that 15-residue-long peptides can be bound to each of the two domains at the same time, and that deletion of the if domain results in reduced affinity and activity of the fkbp domain of ttslyd. interestingly, the reduction in affinity stems mainly from less favorable entropy, suggesting that it may be a consequence of the enhanced dynamics of the fkbp domain that reportedly results from substrate binding at the if domain. we furthermore show that mutating y or h which are part of both the inter-domain connectors and the active site of the fkbp domain, increases the affinity of the if domain up to fivefold, and that y adopts different orientations depending on the substrate, which interestingly correlates with different positions of the if and fkbp domains relative to each other. this may suggest that y and the connector loops may be important for inter-domain cross talk, but more studies will be needed to confirm this. finally, we show that mutating y or m in the if domain to alanine reduces the ppiase activity to 36–39 % relative to the wild type without substantially affecting affinities, indicating that these mutations may affect the dynamics of catalytically important fkbp residues. in conclusion, our results clearly support the notion of inter-domain cross talk in slyd, and highlight the need for further experiments to pinpoint the molecular mechanisms underlying this phenomenon. catalytic activity our nmr lineshape analysis of ttslydfl activity on the commonly used tetrapeptide yielded kcat/km =  106 m−1s− which is in line with previously published values obtained for e. coli slyd using a fluorescence-based assay. the presence of the if domain was found to have a small effect on the activity towards tetrapeptides, which contrasts with previous studies, where no effect was observed. this effect probably relates to the effect of the if domain on the affinity of the fkbp domain. for partially folded proteins, similar kcat/km values but much higher affinities have been reported. however, in these cases the absence of the if domain decreased the activity by a factor of 100–. our studies using 15-residue-long peptides paint a different picture. these peptides bind with affinities that are similar to those of partially folded proteins, and thus much higher than those of tetrapeptides. however, the nmr-derived activity for the s2-p25a peptide is 108 m−1s− and thus a factor of 100-times higher than for both tetrapeptides and partially folded proteins. indeed, the activity is of the same order as that of super-efficient enzymes, with kcat/km in the range 108–1010 m−1s− whose activity is considered diffusion-limited. this might appear surprising given that ttslyd does not have a defined optimal substrate, but rather catalyzes the isomerization of a broad range of proline-containing peptides that form variable contacts with ttslyd. however, it can be explained on the grounds that the catalytic efficiency of ttslyd is not hindered by substrate specificity. the if domain was found to have a definite but small effect on the activity, similar to what we observed for suc-alpf-pna. our michaelis-menten analysis of ttslydfl with the s2-p25a peptide revealed kcat =   s– and km = 2000 μm, a result that highlights the high efficiency of ttslyd. for comparison, the homologous human fkbp yields a kcat above  s− with suc-alpf-pna as a substrate, again indicating high catalytic efficiency. this contrasts with kcat values determined for partially folded protein substrates, which have been estimated to be around 1 s–. our findings can be summarized as follows: a high catalytic rate constant of kcat >   s− is obtained with both s2-p25a and the suc-alpf-pna tetrapeptide as substrates. however, because the longer s2-p25a peptide can make more contacts with the protein, or retain more flexibility in the bound state, it has lower kd and km values, resulting in higher activity. by contrast, with partially folded proteins as substrates, one observes a very low kcat, a strong impact of the if domain, and a lower overall activity than what is found using peptide substrates. taken together, the available data suggest that while slyd can isomerize various kinds of substrates, including partially folded or unfolded proteins, it is most efficient when acting on unfolded polypeptide chains.  we have shown that 15-residue-long unmodified peptides bind to 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 fkbps. we therefore conclude that long unmodified peptides are better mimics of unfolded protein substrates than the classical tetrapeptides. we have furthermore 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. in addition, we have presented several structures of ttslyd in complex with 15-residue-long peptides, which represent the first structures of an fkbp protein in complex with long unmodified peptides. these structures show that unfolded polypeptides bind to the if domain in a highly adaptable fashion involving β-strand augmentation and hydrophobic interactions, which agrees well with the low sequence specificity of this domain. furthermore, they also show that substrates can bind to the fkbp domain as both types via and vib-like β-turns, indicating that fkbps are functionally more versatile than previously appreciated. based on a comparison of our structures, we have proposed 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 partially rotated transition state in the hydrophobic binding site. our results furthermore establish that y is important for the catalytic activity of ttslyd. why this is the case is still not entirely clear, but it likely relates to its ideal position for interacting with substrates bound to the fkbp domain and its high mobility. finally, we have found further evidence for the previously reported phenomenon of inter-domain cross talk between the if and fkbp domains, and propose that y may also be important in this context. to sum up, we show in great detail how the if and fkbp domains recognize unfolded protein mimics, and provide novel insights into the general catalytic mechanism of fkbps. 
in addition, we present a series of crystal structures of ttslyd with the inhibitor fk 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 via and vib-like cis-proline β-turns. we have characterized the binding of 15-residue-long unmodified peptides to slyd from thermus thermophilus in terms of binding thermodynamics and enzyme kinetics through the use of isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, and site-directed mutagenesis. our results furthermore provide important clues to the catalytic mechanism and support the notion of inter-domain cross talk. 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. peptidyl-prolyl isomerases catalyze cis/trans isomerization of peptidyl-prolyl bonds, which is often rate-limiting for protein folding. 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 found that 15-residue-long unmodified peptides can serve as better substrate mimics for the if and fkbp domains than chemically modified tetrapeptides. 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.
 peptidyl-prolyl isomerases 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 domain and an insert-in-flap 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.  we have characterized the binding of 15-residue-long unmodified peptides to slyd from thermus thermophilus 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 fk 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 via 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.  we 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 material the online version of this article contains supplementary material, which is available to authorized users. keywords peptidyl-prolyl isomerase fk506-binding protein chaperoneprotein foldingprolinebeta-turnfk506slydnmrx-ray crystal structurehttp://dx.doi.org/ /501100004963seventh framework programmefp/2007- biostruct x nr 783nordlund pär http://dx.doi.org/ /501100004359vetenskapsrådet2014-5815621-2013-5905akke mikael göran gustafsson foundation for research in natural sciences and medicine http://dx.doi.org/ /501100004063knut och alice wallenbergs stiftelseissue-copyright-statement© the author 2016 