Stereoelectronic effects on polyproline conformation

The polyproline type II (PPII) helix is a prevalent conformation in both folded and unfolded proteins, and is known to play important roles in a wide variety of biological processes. Polyproline itself can also form a type I (PPI) helix, which has a disparate conformation. Here, we use derivatives of polyproline, (Pro)10, (Hyp)10, (Flp)10, and (flp)10, where Hyp is (2S,4R)-4-hydroxyproline, Flp is (2S,4R)-4-fluoroproline, and flp is (2S,4S)-4-fluoroproline, to probe for a stereoelectronic effect on the conformation of polyproline. Circular dichroism spectral analyses show that 4R electron-with-drawing substituents stabilize a PPII helix relative to a PPI helix, even in a solvent that favors the PPI conformation, such as n-propanol. The stereochemistry at C4 ordains the relative stability of PPI and PPII helices, as (flp)10 forms a mixture of PPI and PPII helices in water and a PPI helix in n-propanol. The conformational preferences of (Pro)10 are intermediate between those of (Hyp)10/(Flp)10 and (flp)10. Interestingly, PPI helices of (flp)10 exhibit cold denaturation in n-propanol with a value of T(s) near 70 degrees C. Together, these data show that stereoelectronic effects can have a substantial impact on polyproline conformation and provide a rational means to stabilize a PPI or PPII helix.     

Direct and continuous assay for prolyl 4-hydroxylase

Prolyl 4-hydroxylase (P4H) is a nonheme iron dioxygenase that catalyzes the posttranslational hydroxylation of (2S)-proline (Pro) residues in protocollagen strands. The resulting (2S,4R)-4-hydroxyproline (Hyp) residues are essential for the folding, secretion, and stability of the collagen triple helix. P4H uses α-ketoglutarate and O₂ as cosubstrates, and forms succinate and CO₂ as well as Hyp. Described herein is the first assay for P4H that continuously and directly detects turnover of the proline-containing substrate. This assay is based on (2S,4S)-4-fluoroproline (flp), a proline analogue that is transformed into (2S)-4-ketoproline (Kep) and inorganic fluoride by P4H. The fluoride ion, and thus turnover by P4H, is detected by a fluoride ion-selective electrode. Using this assay, steady-state kinetic parameters for the human P4H-catalyzed turnover of a flp-containing peptide were determined and found to be comparable to those obtained with a discontinuous HPLC-based assay. In addition, this assay can be used to characterize P4H variants, as demonstrated by a comparison of catalysis by D414A P4H and the wild-type enzyme. Finally, the use of the assay to identify small-molecule inhibitors of P4H was verified by an analysis of catalysis in the presence of 2,4-pyridine dicarboxylate, an analogue of α-ketoglutarate. Thus, the assay described herein could facilitate biochemical analyses of this essential enzyme.     

Modulating the folding stability and ligand binding affinity of Pin1 WW domain by proline ring puckering

The pyrrolidine side chain makes proline play a unique role in protein structure and function. The C^γ ring pucker preference and the cis– trans peptidyl bond ratio can be mediated via stereoelectronic effects. Here we used a compact triple‐stranded antiparallel β‐sheet protein, the human Pin1 WW domain, to study the consequences of implanting a preorganized C^γ ring pucker on protein structure and function. The conserved Pro37 is a key residue involved in one hydrophobic core, plays an important role in the WW domain, and adopts a C^γ‐endo ring pucker in the native structure. Pro37 was replaced with C^γ‐exo biased pucker derivatives: (2S,4R)‐4‐hydroxyproline (4R‐Hyp), (2S,4R)‐4‐fluoroproline (4R‐Flp), (2S,4R)‐4‐methoxyproline (4R‐Mop), and C^γ‐endo biased pucker derivatives: (2S,4S)‐4‐hydroxyproline (4S‐hyp), (2S,4S)‐4‐fluoroproline (4S‐flp), (2S,4S)‐4‐methoxyproline (4S‐mop) to examine how a preorganized pucker affects the folding stability and ligand‐binding affinity. Circular dichroism measurements indicate that among the variants, only the one with 4S‐flp substitution (P37flp) is more stable than the wild type, suggesting that the stabilization effects originated from preorganization of the backbone conformation and the hydrophobicity of C? F group. Analysis of ligand‐binding affinity using isothermal titration calorimetry revealed that only P37flp has a stronger ligand affinity than the wild type, showing that 4S‐flp can stabilize the WW domain and increase its ligand affinity. Together we have used 4‐substituted proline derivatives and the WW domain to demonstrate that proline ring puckering can be a key factor in determining the folding stability of a protein but the choice of the derivative groups is also critical. Proteins 2014; 82:67–76. © 2013 Wiley Periodicals, Inc.     

Peptidyl prolyl cis/trans-isomerases: comparative reactivities of cyclophilins, FK506-binding proteins, and parvulins with fluorinated oligopeptide and protein substrates

Peptidyl prolyl cis/trans-isomerases catalyze the cis-trans isomerization of prolyl bonds in oligopeptides and various folding states of proteins. The proline residue in PPIase substrates at the P1' subsite, which follows the isomerizing peptide bond, appears to be the common recognition element for all subfamilies of this enzyme class. The molecular principles that govern substrate specificity at the P1' subsite were analyzed using 4-fluoroproline-containing tetrapeptide 4-nitroanilides and barstar Cys40Ala/Cys82Ala/Pro27Ala/Pro48-->4-fluoroproline quadruple variants. Generally, PPIase catalysis demonstrated stereospecificity for monofluoro substitutions at the 4-position of the pyrrolidine ring. However, the replacement of hydrogens with fluoro atoms did not impair productive interactions for the majority of PPIase-substrate complexes. Comparison of specificity constants for oligopeptide and protein substrates revealed striking differences in the 4-fluoroproline substituent effects between members of the PPIase families. Introduction of 4(R)-fluoroproline resulted in an oligopeptide substrate completely resistant to catalytic effects of FKBP-like PPIases. By contrast, the 4(R)-fluoroproline barstar variant demonstrated only slightly reduced or even better catalytic susceptibility when compared to the parent barstar Cys40Ala/Cys82Ala/Pro27Ala/Pro48 substrate. On the other hand, Suc-Ala-Ser-4(S)-FPro-Phe-pNA exhibits a discriminating specificity toward the prototypic parvulin, the Escherichia coli Par10. The E. coli trigger factor, in the extreme, catalyzes Cys40Ala/Cys82Ala/Pro27Ala/4-F(2)Pro48 with a more than 20-fold higher efficiency when compared to the proline-containing congener. These findings support the combined subsite concept for PPIase catalysis in which the positioning of a substrate in the active cleft must activate a still unknown number of remote subsites in the transition state of the reaction. The number of critical subsites was shown to vary between the PPIase families.     

Origin of the stability conferred upon collagen by fluorination

According to a prevailing theory, (2S,4R)-4-hydroxyproline (Hyp) residues stabilize the collagen triple helix via a stereoelectronic effect that preorganizes appropriate backbone torsion angles for triple-helix formation. This theory is consistent with the marked stability that results from replacing the hydroxyl group with the more electron-withdrawing fluoro group, as in (2S,4R)-4-fluoroproline (Flp). Nonetheless, the hyperstability of triple helices containing Flp has been attributed by others to the hydrophobic effect rather than a stereoelectronic effect. We tested this hypothesis by replacing Hyp with 4,4-difluoroproline (Dfp) in collagen-related peptides. Dfp retains the hydrophobicity of Flp, but lacks the ability of Flp to preorganize backbone torsion angles. Unlike Flp, Dfp does not endow triple helices with elevated stability, indicating that the hyperstability conferred by Flp is not due to the hydrophobic effect.     

Interstrand dipole-dipole interactions can stabilize the collagen triple helix

The amino acid sequence of collagen is composed of GlyXaaYaa repeats. A prevailing paradigm maintains that stable collagen triple helices form when (2S)-proline (Pro) or Pro derivatives that prefer the C(γ)-endo ring pucker are in the Xaa position and Pro derivatives that prefer the C(γ)-exo ring pucker are in the Yaa position. Anomalously, an amino acid sequence in an invertebrate collagen has (2S,4R)-4-hydroxyproline (Hyp), a C(γ)-exo-puckered Pro derivative, in the Xaa position. In certain contexts, triple helices with Hyp in the Xaa position are now known to be hyperstable. Most intriguingly, the sequence (GlyHypHyp)(n) forms a more stable triple helix than does the sequence (GlyProHyp)(n). Competing theories exist for the physicochemical basis of the hyperstability of (GlyHypHyp)(n) triple helices. By synthesizing and analyzing triple helices with different C(γ)-exo-puckered proline derivatives in the Xaa and Yaa positions, we conclude that interstrand dipole-dipole interactions are the primary determinant of their additional stability. These findings provide a new framework for understanding collagen stability.     

The crystal structure of a collagen-like polypeptide with 3(S)-hydroxyproline residues in the Xaa position forms a standard 7/2 collagen triple helix

Collagen has a triple helical structure comprising strands with a repeating Xaa-Yaa-Gly sequence. L-Proline (Pro) and 4(R)-hydroxyl-L-proline (4(R)Hyp) residues are found most frequently in the Xaa and Yaa positions. However, in natural collagen, 3(S)-hydroxyl-L-proline (3(S)Hyp) occurs in the Xaa positions to varying extents and is most common in collagen types IV and V. Although 4(R)Hyp residues in the Yaa positions have been shown to be critical for the formation of a stable triple helix, the role of 3(S)Hyp residues in the Xaa position is not well understood. Indeed, recent studies have demonstrated that the presence of 3(S)Hyp in the Xaa positions of collagen-like peptides actually has a destabilizing effect relative to peptides with Pro in these locations. Whether this destabilization is reflected in a local unfolding or in other structural alterations of the collagen triple helix is unknown. Thus, to determine what effect the presence of 3(S)Hyp residues in the Xaa positions has on the overall conformation of the collagen triple helix, we determined the crystal structure of the polypeptide H-(Gly-Pro-4(R)Hyp)3-(Gly-3(S)Hyp-4(R)Hyp)2-(Gly-Pro-4(R)Hyp)4-OH to 1.80 A resolution. The structure shows that, despite the presence of the 3(S)Hyp residues, the peptide still adopts a typical 7/2 superhelical symmetry similar to that observed in other collagen structures. The puckering of the Xaa position 3(S)Hyp residues, which are all down (Cgamma-endo), and the varphi/psi dihedral angles of the Xaa 3(S)Hyp residues are also similar to those of typical collagen Pro Xaa residues. Thus, the presence of 3(S)Hyp in the Xaa positions does not lead to large structural alterations in the collagen triple helix.     

Collagen-like triple helix formation of synthetic (Pro-Pro-Gly)10 analogues: (4(S)-hydroxyprolyl-4(R)-hydroxyprolyl-Gly)10, (4(R)-hydroxyprolyl-4(R)-hydroxyprolyl-Gly)10 and (4(S)-fluoroprolyl-4(R)-fluoroprolyl-Gly)10.

For the rational design of a stable collagen triple helix according to the conventional rule that the pyrrolidine puckerings of Pro, 4-hydroxyproline (Hyp) and 4-fluoroproline (fPro) should be down at the X-position and up at the Y-position in the X-Y-Gly repeated sequence for enhancing the triple helix propensities of collagen model peptides, a series of peptides were prepared in which X- and Y-positions were altogether occupied by Hyp(R), Hyp(S), fPro(R) or fPro(S). Contrary to our presumption that inducing the X-Y residues to adopt a down-up conformation would result in an increase in the thermal stability of peptides, the triple helices of (Hyp(S)-Hyp(R)-Gly)(10) and (fPro(S)-fPro(R)-Gly)(10) were less stable than those of (Pro-Hyp(R)-Gly)(10) and (Pro-fPro(R)-Gly)(10), respectively. As reported by B?chinger's and Zagari's groups, (Hyp(R)-Hyp(R)-Gly)(10) which could have an up-up conformation unfavorable for the triple helix, formed a triple helix that has a high thermal stability close to that of (Pro-Hyp(R)-Gly)(10). These results clearly show that the empirical rule based on the conformational preference of pyrrolidine ring at each of X and Y residues should not be regarded as still valid, at least for predicting the stability of collagen models in which both X and Y residues have electronegative groups at the 4-position.     

Impacts of terminal (4R)-fluoroproline and (4S)-fluoroproline residues on polyproline conformation

Many interests have been focused on prolyl cis–trans isomerization which is related to protein folding and isomer-specific biochemical recognition. Since polyproline can adopt either type I (PPI) helices with all cis amide bonds or type II (PPII) helices with all trans amide bonds, it has been a valuable model to study the prolyl isomerization. Recent studies have shown that stereoelectronic effects govern the stability of PPII structure and the rate of PPII → PPI conversion. To further explore the terminal stereoelectronic effects on polyproline conformation, herein we synthesized a series of host–guest peptides in which (2S,4S)-4-fluoroproline (flp) or (2S,4R)-4-fluoroproline (Flp) residues are incorporated into the C- or N-terminal end of a peptide and studied the thermodynamic and kinetic consequences on polyproline conformation. Circular dichroism measurements revealed that inserting 4-fluoroproline residues into the C terminus of a polyproline peptide induces a great stereoelectronic effect on PPII stability and PPII → PPI conversion rates. From the C terminus, a (Flp)₃ triplet stabilizes PPII structure and increases the transition barrier of PPII → PPI conversion by 1.53 kJ mol^?1 while a (flp)₃ triplet destabilizes PPII conformation and reduce the PPII → PPI transition barrier by 4.61 kJ mol^?1. In contrast, the 4-fluoroproline substitutions at the N terminus do not exhibit distinct stereoelectronic effects on PPII stability and PPII → PPI conversion rates. Our data demonstrate that the C-terminal stereoelectronic effects have a more dramatic impact on PPII stability and PPII → PPI conversion kinetics.     

Specificity of prolyl endopeptidase

A series of tetrapeptides, Cbz(Bz)-Gly-X-Leu-Gly, were synthesized and the kinetic parameters, kcat and kcat/Km, determined for their hydrolyses by prolyl endopeptidase from Flavobacterium. The peptides with X = N-Me-Ala, Sar and Ala as well as the standard substrate (X = Pro) were found to be good substrates, while those with X = alpha-aminobutyryl, Hyp, Ser and Gly were poor substrates, and those with X = pipecolyl, alpha-aminoisobutyryl, N-Me-Val, N-Me-Leu, Hyp(O-Bzl) and Ser(O-Bzl) were not cleaved at all. These results suggest that the specificity-determining site or S1 subsite of the enzyme is designed to fit exactly the proline residue of the substrate with allowance for the residues carrying substituents at the N and/or C alpha which must not exceed the size of the pyrrolidine ring of proline.     

Rational design of protein stability: effect of (2S,4R)-4-fluoroproline on the stability and folding pathway of ubiquitin

Background

Many strategies have been employed to increase the conformational stability of proteins. The use of 4-substituted proline analogs capable to induce pre-organization in target proteins is an attractive tool to deliver an additional conformational stability without perturbing the overall protein structure. Both, peptides and proteins containing 4-fluorinated proline derivatives can be stabilized by forcing the pyrrolidine ring in its favored puckering conformation. The fluorinated pyrrolidine rings of proline can preferably stabilize either a C^γ-exo or a C^γ-endo ring pucker in dependence of proline chirality (4R/4S) in a complex protein structure. To examine whether this rational strategy can be generally used for protein stabilization, we have chosen human ubiquitin as a model protein which contains three proline residues displaying C^γ-exo puckering.

Methodology/Principal Findings

While (2S,4R)-4-fluoroproline ((4R)-FPro) containing ubiquitinin can be expressed in related auxotrophic Escherichia coli strain, all attempts to incorporate (2S,4S)-4-fluoroproline ((4S)-FPro) failed. Our results indicate that (4R)-FPro is favoring the C^γ-exo conformation present in the wild type structure and stabilizes the protein structure due to a pre-organization effect. This was confirmed by thermal and guanidinium chloride-induced denaturation profile analyses, where we observed an increase in stability of −4.71 kJ·mol⁻¹ in the case of (4R)-FPro containing ubiquitin ((4R)-FPro-ub) compared to wild type ubiquitin (wt-ub). Expectedly, activity assays revealed that (4R)-FPro-ub retained the full biological activity compared to wt-ub.

Conclusions/Significance

The results fully confirm the general applicability of incorporating fluoroproline derivatives for improving protein stability. In general, a rational design strategy that enforces the natural occurring proline puckering conformation can be used to stabilize the desired target protein.     

Conformational preferences of substituted prolines in the collagen triple helix

Researchers have recently questioned the role hydroxylated prolines play in stabilizing the collagen triple helix. To address these issues, we have developed new molecular mechanics parameters for the simulation of peptides containing 4(R)-fluoroproline (Flp), 4(R)-hydroxyproline (Hyp), and 4(R)-aminoproline (Amp). Simulations of peptides based on these parameters can be used to determine the components that stabilize hydroxyproline over proline in the triple helix. The dihedrals F-C-C-N, O-C-C-N, and N-C-C-N were built using a N-beta-ethyl amide model. One nanosecond simulations were performed on the trimers [(Pro-Pro-Gly)(10)](3), [(Pro-Hyp-Gly)(10)](3), [(Pro-Amp-Gly)(10)](3), [(Pro-Amp(1+)-Gly)(10)](3), and [(Pro-Flp-Gly)(10)](3) in explicit solvent. The results of our simulations suggest that pyrrolidine ring conformation is mediated by the strength of the gauche effect and classical electrostatic interactions.     

The triple helical structure and stability of collagen model peptide with 4(S)-hydroxyprolyl-Pro-Gly units

Extensive studies on the structure of collagen have revealed that the hydroxylation of Pro residues in a variety of model peptides with the typical (X-Y-Gly)(n) repeats (X and Y: Pro and its analogues) represents one of the major factors influencing the stability of triple helices. While(2S,4R)-hydroxyproline (Hyp) at the position Y stabilizes the triple helix, (2S,4S)-hydroxyproline (hyp) at the X-position destabilizes the helix as demonstrated that the triple helix of (hyp-Pro-Gly)(15) is less stable than that of (Pro-Pro-Gly)(15) and that a shorter peptide (hyp-Pro-Gly)(10) does not form the helix. To clarify the role of the hydroxyl group of Pro residues to play in the stabilization mechanism of the collagen triple helix, we synthesized and crystallized a model peptide (Pro-Hyp-Gly)(4) -(hyp-Pro-Gly)(2) -(Pro-Hyp-Gly)(4) and analyzed its structure by X-ray crystallography and CD spectroscopy. In the crystal, the main-chain of this peptide forms a typical collagen like triple helix. The majority of hyp residues take down pucker with exceptionally shallow angles probably to relieve steric hindrance, but the remainders protrude the hydroxyl group toward solvent with the less favorable up pucker to fit in a triple helix. There is no indication of the existence of an intra-molecular hydrogen bond between the hydroxyl moiety and the carbonyl oxygen of hyp supposed to destabilize the triple helix. We also compared the conformational energies of up and down packers of the pyrrolidine ring in Ac-hyp-NMe(2) by quantum mechanical calculations.     

Substrate specificity of beta-collagenase from Clostridium histolyticum

The substrate specificity of beta-collagenase from Clostridium histolyticum has been investigated by measuring the rate of hydrolysis of more than 50 tri-, tetra-, penta-, and hexapeptides covering the P3 to P3' subsites of the substrate. The choice of peptides was patterned after sequences found in the alpha 1 and alpha 2 chains of type I collagen. Each peptide contained either a 2-furanacryloyl (FA) or cinnamoyl (CN) group in subsite P2 or the 4-nitrophenylalanine (Nph) residue in subsite P1. Hydrolysis of the P1-P1' bond produces an absorbance change in these chromophoric peptides that has been used to quantitate the rates of their hydrolysis under first order conditions ([S] much less than KM) from kcat/KM values have been obtained. The identity of the amino acids in all six subsites (P3-P3') markedly influences the hydrolysis rates. In general, the best substrates have Gly in subsites P3 and P1', Pro or Ala in subsite P2', and Hyp, Arg, or Ala in subsite P3'. This corresponds well with the frequency of occurrence of these residues in the Gly-X-Y triplets of collagen. In contrast, the most rapidly hydrolyzed substrates do not have residues from collagen-like sequences in subsites P2 and P1. For example, CN-Nph-Gly-Pro-Ala is the best known substrate for beta-collagenase with a kcat/KM value of 4.4 X 10(7) M-1 min-1, in spite of the fact that there is neither Pro nor Ala in P2 or Hyp nor Ala in P1. These results indicate that the previously established rules for the substrate specificity of the enzyme require modification.     

The crystal structure of the collagen-like polypeptide (glycyl-4(R)-hydroxyprolyl-4(R)-hydroxyprolyl)9 at 1.55 A resolution shows up-puckering of the proline ring in the Xaa position

The collagen triple helix is characterized by the repeating sequence motif Gly-Xaa-Yaa, where Xaa and Yaa are typically proline and (2S,4R)-4-hydroxyproline (4(R)Hyp), respectively. Previous analyses have revealed that H-(Pro-4(R)Hyp-Gly)(10)-OH forms a stable triple helix, whereas H-(4(R)Hyp-Pro-Gly)(10)-OH does not. Several theories have been put forth to explain the importance of proline puckering and conformation in triple helix formation; however, the details of how they affect triple helix stability are unknown. Underscoring this, we recently demonstrated that the polypeptide Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2) forms a triple helix that is more stable than Ac-(Gly-Pro-4(R)Hyp)(10)-NH(2). Here we report crystal the structure of the H-(Gly-4(R)Hyp-4(R)Hyp)(9)-OH peptide at 1.55 A resolution. The puckering of the Yaa position 4(R)Hyp in this structure is up (Cgamma exo), as has been found in other collagen peptide structures. Notably, however, the 4(R)Hyp in the Xaa position also takes the up pucker, which is distinct from all other collagen structures. Regardless of the notable difference in the Xaa proline puckering, our structure still adopts a 7/2 superhelical symmetry similar to that observed in other collagen structures. Thus, the basis for the observed differences in the thermodynamic data of the triple helix<--> coil transition between our peptide and other triple helical peptides likely results from contributions from the unfolded state. Indeed, the unfolded state of the H-(Gly-4(R)Hyp-4(R)Hyp)(9)-OH peptide seems to be stabilized by a preformed polyproline II helix in each strand, which could be explained by the presence of a unique repeating intra-strand water-mediated bridge observed in the H-(Gly-4(R)Hyp-4(R)Hyp)(9)-OH structure, as well as a higher amount of trans peptide bonds.     

The peptides acetyl-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 and acetyl-(Gly-Pro-3(S)Hyp)10-NH2 do not form a collagen triple helix

Hydroxylation of proline residues in the Yaa position of the Gly-Xaa-Yaa repeated sequence to 4(R)-hydroxyproline is essential for the formation of the collagen triple helix. A small number of 3(S)-hydroxyproline residues are present in most collagens in the Xaa position. Neither the structural nor a biological role is known for 3(S)-hydroxyproline. To characterize the structural role of 3(S)-hydroxyproline, the peptide Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 was synthesized and analyzed by circular dichroism spectroscopy, analytical ultracentrifugation, and 1H nuclear magnetic resonance spectroscopy. At 4 degrees C in water the circular dichroism spectrum indicates that this peptide was in a polyproline-II-like secondary structure with a positive peak at 225 nm similar to Ac-(Gly-Pro-4(R)Hyp)10-NH2. The positive peak at 225 nm almost linearly decreases with increasing temperature to 95 degrees C without an obvious transition. Although the peptide Ac-(Gly-Pro-4(R)Hyp)10-NH2 forms a trimer at 10 degrees C, sedimentation equilibrium experiments indicate that Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 is a monomer in water at 7 degrees C. To study the role of 3(S)-hydroxyproline in the Yaa position, we synthesized Ac-(Gly-Pro-3(S)Hyp)10-NH2. This peptide also does not form a triple helix in water. 1H Nuclear magnetic resonance spectroscopy data (including line widths and nuclear Overhauser effects) are entirely consistent, with neither Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 nor Ac-(Gly-Pro-3(S)Hyp)10-NH2 forming a triple helix in water. Therefore 3(S)-hydroxyproline destabilizes the collagen triple helix in either position. In contrast, when 3(S)-hydroxyproline is inserted as a guest in the highly stable -Gly-Pro-4(R)Hyperepeated host sequence, Ac-(Gly-Pro-4(R)Hyp)3-Gly-3(S)Hyp-4(R)Hyp-(Gly-Pro-4(R)Hyp)4-Gly-Gly-NH2 forms as stable a trimer (Tm=49.6 degrees C) as Ac-(Gly-Pro-4(R)Hyp)8-Gly-Gly-NH2 (Tm=48.9 degrees C). Given that Ac-(Gly-Pro-4(R)Hyp)3-Gly-4(R)Hyp-Pro-(Gly-Pro-4(R)Hyp)4-Gly-Gly-NH2 forms a triple helix nearly as stable as the above two peptides (Tm=45.0 degrees C) and the knowledge that Ac-(Gly-4(R)Hyp-Pro)10-NH2 does not form a triple helix, we conclude that the host environment dominates the structure of host-guest peptides and that these peptides are not necessarily accurate predictors of triple helical stability.     

Discovery of potent, selective 4-fluoroproline-based thrombin inhibitors with improved metabolic stability

Previous reports from our laboratories described potent tripeptide thrombin inhibitors which incorporate heterocycle-substituted chlorophenyl groups in the P1 position. Using these as lead compounds for further optimization, we identified sites of metabolism and designed analogs with 4-fluoroproline in P2 and cyclopropane-containing side chains in P3 as an approach to reducing metabolism and improving their oral pharmacokinetic performance. The large (300-fold) difference in potency between analogs containing (4R)- and (4S)-4-fluoroproline was rationalized by analyzing inhibitor-enzyme interactions in crystal structures of related compounds and by molecular modeling which indicated that the more potent (4R)-4-fluoroproline isomer stabilizes a proline ring conformation that is preferred for binding to the enzyme. An optimal compound from this work, 41, exhibits high potency in a coagulation assay in human plasma (2xAPTT=190 nM), excellent selectivity versus the digestive enzyme trypsin (K(i)=3300 nM), and excellent oral bioavailability in dogs with moderate clearance (F=100%, CL=12 mL/min/kg).     

Extended investigation of the substrate specificity of dipeptidyl peptidase IV from pig kidney.

The substrate specificity of dipeptidyl peptidase IV (dipeptidyl peptide hydrolase, EC 3.4.14.5) from pig kidney was investigated, using a series of substrates, in which the amino-acid residue in position P1, a structural derivative of proline, was altered with respect to ring size and substituents. It was demonstrated that dipeptidyl peptidase IV hydrolyses substrates of the type Ala-X-pNA, where X is proline (Pro), (R)-thiazolidine-4-carboxylic acid (Thz), (S)-pipecolic acid (Pip), (S)-oxazolidine-4-carboxylic acid (Oxa), or (S)-azetidine-2-carboxylic acid (Aze). The ring size and ring structure of the residue in the P1 position influence the rate of enzyme-catalysed hydrolysis of the substrate. The highest kcat value (814 s-1) was found for Ala-Aze-pNA. In contrast, the kcat value for Ala-Pro-pNA is nearly 55 s-1. With all substrates of this series, the rate-limiting step of the hydrolysis by dipeptidyl peptidase IV is the deacylation reaction. Compounds of substrate-like structure, in which the P2 residue has an R-configuration, are not hydrolysed by dipeptidyl peptidase IV.     

Identification of conserved prolyl residue important for transport activity and the substrate specificity range of yeast plasma membrane Na+/H+ antiporters

Yeast plasma membrane Na+/H+ antiporters are divided according to their substrate specificity in two distinct subfamilies. To identify amino acid residues responsible for substrate specificity determination (recognition of K+), the Zygosaccharomyces rouxii Sod2-22 antiporter (non-transporting K+) was mutagenized and a collection of ZrSod2-22 mutants that improved the KCl tolerance of a salt-sensitive Saccharomyces cerevisiae strain was isolated. Several independent ZrSod2-22 mutated alleles contained the replacement of a highly conserved proline 145 with a residue containing a hydroxyl group (Ser, Thr). Site-directed mutagenesis of Pro145 proved that an amino acid with a hydroxyl group at this position is enough to enable ZrSod2-22p to transport K+. Simultaneously, the P145(S/T) mutation decreased the antiporter transport activity for both Na+ and Li+. Replacement of Pro145 with glycine resulted in a ZrSod2-22p with extremely low activity only for Na+, and the exchange of a charged residue (Asp, Lys) for Pro145 completely stopped the activity. Mutagenesis of the corresponding proline in the S. cerevisiae Nha1 antiporter (Pro146) confirmed that this proline of the fifth transmembrane domain is a critical residue for antiporter function. This is the first evidence that a non-polar amino acid residue is important for the substrate specificity and activity of yeast Nha antiporters.     

Mechanism of proline-specific proteinases: (I) Substrate specificity of dipeptidyl peptidase IV from pig kidney and proline-specific endopeptidase from Flavobacterium meningosepticum

The substrate specificity of dipeptidyl peptidase IV (dipeptidyl peptide hydrolase, EC 3.4.14.5) from pig kidney and proline-specific endopeptidase from Flavobacterium meningosepticum, was investigated with a series of N-terminal unprotected (dipeptidyl peptidases IV) and succinylated dipeptidyl-p-nitroanilides (proline-specific endopeptidase). Both enzymes are specific for the S configuration of the amino-acid residue in P1 and P2 position if the penultimate residue is proline. In the case of alanine substrates (Ala in P1, dipeptidyl peptidase IV hydrolyzes such compounds where the configuration of the P2 residue is R. The penultimate residue with dipeptidyl peptidase IV can be, beside proline and alanine, dehydroproline, hydroxyproline and pipecolic acid. Proline substrates (Pro in P1) with an R configuration in P2 are inhibitors of the hydrolysis of proline substrates with an S,S configuration in an uncompetitive (dipeptidyl peptide IV) or mixed inhibition type (proline-specific endopeptidase). Derivatives of Gly-Pro-pNA where the N-terminal amino group is methylated are hydrolyzed by dipeptidyl peptidase IV.