Synthesis and Kinetic Analysis of Two Conformationally Restricted

Article pubs.acs.org/biochemistry

Synthesis and Kinetic Analysis of Two Conformationally Restricted Peptide Substrates of Escherichia coli Penicillin-Binding Protein 5 Venkatesh V. Nemmara,† Robert A. Nicholas,‡ and R. F. Pratt*,† †

Department of Chemistry, Wesleyan University, Lawn Avenue, Middletown, Connecticut 06459, United States Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7365, United States

‡

S Supporting Information *

ABSTRACT: Escherichia coli PBP5 (penicillin-binding protein 5) is a DD-carboxypeptidase involved in bacterial cell wall maturation. Beyond the C-terminal D-alanyl-D-alanine moiety, PBP5, like the essential high-molecular mass PBPs, has little specificity for other elements of peptidoglycan structure, at least as elicited in vitro by small peptidoglycan fragments. On the basis of the crystal structure of a stem pentapeptide derivative noncovalently bound to E. coli PBP6 (Protein Data Bank entry 3ITB), closely similar in structure to PBP5, we have modeled a pentapeptide structure at the active site of PBP5. Because the two termini of the pentapeptide are directed into solution in the PBP6 crystal structure, we then modeled a 19-membered cyclic peptide analogue by cross-linking the terminal amines by succinylation. An analogous smaller, 17-membered cyclic peptide, in which the L-lysine of the original was replaced by L-diaminobutyric acid, could also be modeled into the active site. We anticipated that, just as the reactivity of stem peptide fragments of peptidoglycan with PBPs in vivo may be entropically enhanced by immobilization in the polymer, so too would that of our cyclic peptides with respect to their acyclic analogues in vitro. This paper describes the synthesis of the peptides described above that were required to examine this hypothesis and presents an analysis of their structures and reaction kinetics with PBP5. stem peptides3 and act as combined transition state analogue/ mechanism-based inhibitors.3,4 The DD-peptidases have been classified into two main groups, high molecular mass (HMM) and low molecular mass (LMM), where the dividing line, operationally, is ∼50 kDa. With current insight into molecular structure, however, it is clear that the division is actually structurally and functionally based.5,7 The HMM enzymes are essential for bacterial growth and division and have been shown to catalyze the necessary transpeptidase reaction. The HMMA group also has transglycosylase activity that catalyzes glycan polymerization of stem peptide monomers into nascent peptidoglycan, while the HMMB group has only transpeptidase activity. The LMM enzymes are not essential to growth, in the short term at least, and have the DD-carboxypeptidase and DDendopeptidase activity necessary to regulate the degree of

B

acterial DD-peptidases (also known as penicillin-binding proteins or PBPs) are the well-established targets of βlactam antibiotics.1,2 These enzymes catalyze the final steps of peptidoglycan biosynthesis by cross-linking peptides from adjacent strands (Scheme 1). β-Lactams are believed to mimic the C-terminal D-alanyl-D-alanine moiety of cell wall Scheme 1. Roles of Biosynthesis

DD-Peptidases

in Bacterial Cell Wall

Received: June 7, 2016 Revised: July 6, 2016

© XXXX American Chemical Society

A

DOI: 10.1021/acs.biochem.6b00576 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry peptidoglycan cross-linking.8,9 The LMM enzymes are classified into subgroups A−C.5,6 The general reactivity and substrate specificity of DDpeptidases is not well understood. LMM subclass B (LMMB) and C (LMMC) enzymes are specific for stem peptide fragments with free N-termini.10−12 For example, the LMMC Actinomadura R39 DD-peptidase very specifically catalyzes the hydrolysis of peptide 1, which has the free N-terminus of the stem peptide of Actinomadura R39.11 Crystal structures of LMMB and LMMC enzymes clearly reveal the presence of Nterminal binding sites.13,14 No significant specificity of the HMM and LMMA enzymes for small peptidoglycan-mimetic peptides has been demonstrated.15,16 The reasons for this are not clear. One important factor is that these apparently reluctant enzymes have largely been studied in vitro as solubilized constructs. All are cell membrane-bound in vivo, and their reactivity is probably influenced by interactions with the membrane, with peptidoglycan, and/or with other proteins.16−23 Another important unknown is just how much of a peptidoglycan substrate is actually bound to the enzymes during catalysis in vivo. In all cases, however, at least one substrate is a peptidoglycan polymer. The LMMA enzymes are the best-studied of the membranebound DD-peptidases, both structurally and functionally. They are believed to be DD-carboxypeptidases in vivo, responsible for trimming stem pentapeptides to tetrapeptides (Scheme 1).8,9 Among the best known of these are Escherichia coli PBP5 (EcPBP5) and PBP6 (EcPBP6) and Streptococcus pneumoniae PBP3 (SpPBP3). Crystal structures of solubilized constructs are available for all three, including structures with ligands bound at the active site.23−26 Extensive substrate specificity studies have been conducted on EcPBP512,27 and SpPBP3.12 As noted above, no significant small molecule substrate specificity has been revealed. For example, peptide 1 is not measurably turned over by EcPBP5 (kcat/Km < 100 M−1 s−1), and peptide 2 shows only slow turnover (kcat/Km = 150 M−1 s−1); these peptides are clearly closely related structurally to E. coli stem peptide 3. Inclusion of sugar residues, as in 4, gives little improvement (kcat/Km = 300 M−1 s−1 28).

poorly reactive active site.14,29 Indeed, addition of a boronic acid transition state analogue inhibitor leads to contraction of the active site to dimensions more typical of active DDpeptidases.29 Another indication is provided by a crystal structure of 4 bound to the active site of EcPBP6. This is another LMMA PBP, very similar in structure to PBP5 (60.2% identical, 88.6% similar, 387 residues overlap).25 It is apparently somewhat less active than EcPBP5 against small peptides and, in fact, does not catalyze the hydrolysis of L-Ala-D-isoGlu-L-Lys30,31 D-Ala-D-Ala at all. The crystal structure of 4 bound to the active site of EcPBP625 (Figure 1) is informative. The D-Ala-DAla moiety is bound to the active site in what would appear to be a productive conformation. The penultimate D-Ala-carbonyl oxygen is hydrogen-bonded into the oxyanion hole (Ser40 and Thr212 NH groups); the hydroxyl group of Ser40 is hydrogenbounded to the terminal amine of Lys43, and the terminal carboxylate of the peptide is hydrogen-bonded to the Thr210 and Ser106 hydroxyl groups. The nucleophilic hydroxyl group of Ser40 is 2.88 Å from the terminal peptide carbonyl carbon. Despite this apparently productive pose, the terminal peptide is intact in the crystal structure, no hydrolysis has occurred, and the active site has not contracted around the substrate.14 Beyond the nominal reaction center, there are few polar interactions between the glycopeptide and the enzyme. The Llysine α-NH group is hydrogen-bonded to the carbonyl of Gly81 and the lysine carbonyl oxygen to the side chain NH group of Asn108. The extended hydrophobic lysine tetramethylene moiety and the polar N-terminal ammonium ion appear as if they would be free in solution. Similarly, the Llactoyl-L-Ala-isoGlu segment of the peptide, which is connected to the NAM sugar, also does not appear to strongly interact with the enzyme molecule in the crystal. The sugar residue interacts with an adjacent enzyme molecule in the crystal; what it interacts with in solution or in vivo is not known, beyond with water itself, although available kinetic results are not suggestive of a productive interaction with another part of the protein.28 It seems possible therefore that in vivo the stem peptide N-termini and glycosyl termini might interact with macromolecular entities beyond the enzyme itself. It is likely that the same situation applies to PBP5, where activity in vivo does seem to require aggregation of protein components.32 The low activity of EcPBP5 and EcPBP6 in solution against pentapeptide substrates may be limited to some extent by the mobility of the weakly bound and unbound N-termini and glycoside termini of the stem peptide. This proposition is perhaps supported by the observation that bulky substituents on the peptide termini increase rates of turnover.27 We therefore wondered whether a decrease in the mobility (entropy) of these termini would lead to enhanced reactivity. Thus, as described below in more detail, we designed and synthesized cyclic peptides 5 and 6 and studied their structure and their reactivity with EcPBP5. For comparison, uncyclized peptides 7 and 8 were also examined. There is a quite long history in biochemistry and medicinal chemistry of application of the cyclization of linear ligands, especially peptides, to both reduce their mobility in solution and to achieve a bioactive conformation.33−36 Some of these attempts succeeded; others, apparently at least, did not. Below we describe our experience in this arena.

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MATERIALS AND METHODS Commercially available solvents and reagents were purchased from Sigma-Aldrich and Acros Organics, unless otherwise

One factor contributing to the lack of substantial peptidase activity by EcPBP5 is perhaps indicated by the crystal structure of the apoenzyme, which shows an expanded and possibly B


Article

Biochemistry

Figure 1. Detail from the crystal structure of the noncovalent complex of a peptidoglycan stem peptide fragment 4 with EcPBP6 (PDB entry 3ITB) showing the N-termini and glycosyl termini of the peptide, both directed away from the protein. The nitrogen atoms cross-linked to form 5 and 6 are indicated by arrowheads.

water were recorded on an Agilent VNMRS 800 MHz spectrometer equipped with a 5 mm HCN salt-tolerant inverse cold probe (University of Connecticut Health Center, Farmington, CT). M. W. Maciejewski of the University of Connecticut helped with the acquisition and processing of 2D NOESY and COSY spectra using NMRpipe software.38 Molecular Modeling. Cyclic Peptide Design for EcPBP6. Discovery Studio version 2.5 (Accelrys) was used to perform computational modeling of EcPBP6. The CHARMm force field was employed for all computations. A crystal structure of EcPBP6 complexed with glycopeptide 4 (PDB entry 3ITB)25 was used as the starting point for model building. A pH of 7.0 was set to fix the dissociation states of the protein and ligand functional groups. The NAM moiety of 4 was removed to form branched diamine 9. To form 5, diamine 9, while still bound to the enzyme, was cyclized through the N-terminus of L-Ala and the side chain amine of L-Lys by means of a succinate linker. Potential steric clashes with the enzyme active site were assessed and manually minimized. The noncovalent enzyme− substrate complex was hydrated with a 20 Å sphere of water molecules centered at the active site serine. The hydrated structures were energy minimized using a steepest descent algorithm for 250 steps, followed by a conjugate gradient method for 750 steps. The energy-minimized structure was heated to 300 K in 10000 steps (10 ps) with coordinates saved every 100 steps. The heated structure was then subjected to an equilibration run of 10000 steps (10 ps) and then to a production run (10 ns) at 300 K. A typical low-energy snapshot was further energy minimized as described above. It was noted that a smaller ring could also fit into the active site so two carbon atoms were removed from the ring of 5. This was achieved by replacement of the L-Lys of 9 with Ldiaminobutyric acid, forming acyclic peptide 10, and cyclization by a succinate linker as described above, to form 6. A structure of the complex of 6 with EcPBP6 was obtained as described above for 5. Cyclic Peptide Design for EcPBP5. The crystal structure of EcPBP5 as an apoenzyme (PDB entry 1NZO)37 was used as the starting point for model building. The structure of EcPBP6, modeled with cyclic peptide 5, was superimposed on the EcPBP5 crystal structure using the C-α pairs of 11 residues

noted, and were used directly without purification. D-Glutamic acid-α-benzyl ester, N-Boc-D-alanine, N-Boc-L-alanine, Nα-BocNε-Fmoc-L-lysine, Nα-Boc-Nε-Fmoc-L-diaminobutyric acid, Dalanine benzyl ester, and D-glutamine were purchased from Chem Impex. E. coli PBP5 (EcPBP5) was prepared as described previously.37 S. pneumoniae PBP3 (SpPBP3) was a generous gift from A. Dessen of Institut de Biologie Structurale (Grenoble, France). Routine one-dimensional (1D) and two-dimensional (2D) NMR spectra were recorded on Varian Mercury 300 and 400 MHz NMR spectrometers. Absorption spectra of peptides and the spectrophotometric measurement of enzyme activity were obtained by means of Hewlett-Packard 8452A and 8452E spectrophotometers. High-resolution mass spectra were recorded at the Mass Spectrometry Laboratory, School of Chemical Sciences, University of Illinois at Urbana-Champaign (Urbana, IL). Routine ESI mass spectra of peptides were recorded with an in-house ThermoFisher LCQ Advantage ESI Spectrometer. HPLC analysis was conducted using a Varian ProStar unit equipped with a Varian ProStar model 340 UV/vis detector and a Nucleosil 5 C-18 reverse phase column. Circular dichroism (CD) spectra were recorded on a Jasco model J-810 spectropolarimeter. 1D and 2D NMR spectra in C


Article

Biochemistry rij = A + B(Iij)−1/6

[EcPBP5: Ser44, Lys47, Ser110, Ser87, Asn112, Arg198, Lys213, Thr214, Gly215, His216 (Thr in EcPBP6), and Thr217] in the active site (root-mean-square deviation of 1.57 Å). The EcPBP6 structure was deleted, and the alignment of cyclic peptide 5 in the EcPBP5 active site was visualized. Molecular dynamics and energy minimizations of this structure were performed as described above for EcPBP6. A similar procedure was adopted to model the smaller cyclic peptide 6 in the active site of EcPBP5. The energies in vacuo of cyclic peptides 5 and 6 from the active site (after removal of the protein) were calculated for comparison with those of the free peptides (see below). Solution Structure Models of the Cyclic Peptides. Molecular models of the two cyclic peptides were constructed computationally in a sphere of water molecules (10 Å, 1044 water molecules). The hydrated peptides were then subjected to a Standard Dynamics Cascade protocol to replicate a simulated annealing procedure on both cyclic peptides. The protocol involved minimization for 250 steps using the steepest descent algorithm followed by 750 steps by the conjugate gradient method. The minimized structures were heated to 2000 K for 10 ps and then cooled to 300 K. The cooled structures at 300 K were then equilibrated for 10 ps and then subjected to 10 ns production runs. In each case, a typical lowenergy structure was selected for further analysis. A series of eight structures for each peptide were then constructed, where the ϕ angle for each peptide bond was set to the value indicated by the NMR spectra (see below). Each of these structures in a sphere of water, with ϕ angles constrained, was then energy minimized (1000 steps of steepest descent). The in vacuo energy of each conformation (unconstrained) was then calculated. Synthesis of Peptide Substrates. Peptides 5−8 were prepared by standard solution peptide methodology. Details are given in the Supporting Information. NMR Spectroscopy. Samples for the NMR structural analysis of cyclic peptides 5 and 6 were prepared by making a 5.0 mM stock solution of each peptide in 700 μL of a H2O/ D2O (9:1) mixture. 1D and 2D NMR spectra were recorded at 25 °C on an Agilent VNMRS-800 MHz spectrometer equipped with a 5 mm HCN salt-tolerant inverse cold probe (University of Connecticut Health Center). DQF COSY experiments with 5 and 6 were performed using the Watergate pulse sequence for water suppression.40 A 2D TOCSY experiment for 6 was performed on a Varian Mercury 400 MHz NMR spectrometer with a mixing time of 100 ms. 2D NOESY experiments were conducted on this instrument for 600 t1 increments with 32 transients for each t1 increment. The spectral width in both dimensions was 6400 Hz. NOESY spectra were recorded with a mixing time of 150 ms for both samples. All spectra were processed with NMRpipe software38 and analyzed. Complete proton resonance assignments were made from the data provided by 1D 1H NMR, DQF COSY, and 2D TOCSY experiments. From the spectra, 3J coupling constants were obtained for the ϕ dihedral angles of each peptide bond. In the case of 6, for which sufficient data were available from the NOESY spectrum, interproton NOE cross-peaks were converted into distances from the volume integrals of the crosspeaks by incorporating two reference distances, 1.8 Å for the geminal proton interactions of the β-CH2 group in D-isoGlu and 2.4 Å for the interaction between the L-Ala α-CH and CH3 protons.41 The distances rij were calculated using the equation given below:

A=

r1(I1)6 − r2(I2)6 6

6

(I1) − (I2)

B=

r1 −1/6

(I1)

− r2 − (I2)−1/6

where I1 and I2 are corresponding reference intensities, Iij is the intensity of the observed cross-peak, and r1 and r2 are the reference distances. Circular Dichroism Spectroscopy. Circular dichroism (CD) experiments were performed on a Jasco model J-810 spectropolarimeter. Spectra were recorded in a 0.1 cm Jasco cell between 250 and 185 nm at a scanning speed of 100 nm/min with a bandwidth of 2.0 nm, a response time of 2 s, and a sensitivity of 100 mdeg. Stock solutions of peptides 5−8 (5.0 mM) were prepared in deionized water. The final concentrations of peptides in the cell were 350 μM in a volume of 300 μL. CD spectra of the peptides in water were recorded at 25 °C. Molar ellipticity [θ] was calculated from the equation given below: [θ ] = θ /(10cl)

where θ is the measured ellipticity in millidegrees, c is the molar concentration of the sample in the cell, and l is the path length of the cell in centimeters. Enzyme Kinetics. Steady state kinetic parameters for hydrolysis of peptides 5 and 6 by EcPBP5 were determined spectrophotometrically, where the loss of a peptide bond was monitored at wavelengths between 215 and 235 nm. The reactions catalyzed by these enzymes were studied in 0.1 M sodium pyrophosphate buffer containing 10% glycerol at pH 8.5 (this buffer was also used at pH 9.5) and 37 °C.9,16 Peptide concentrations employed were 0−1.0 mM, and the enzyme concentration was 0.60 μM. Because it was found that substrate concentrations approaching KM were not achieved, values of kcat/KM were obtained by least-squares exponential fits to the progress curves. Steady state kinetic parameters for hydrolysis of peptides 5− 8 by S. pneumoniae (Sp) PBP3 were determined spectrophotometrically as described above. The buffer employed was 20 mM sodium phosphate at pH 7.5 and 25 °C. Peptide concentrations employed were 0−1.0 mM, and the enzyme concentration was 0.10 μM. D-Alanine Assay. A well-validated spectrophotometric coupled enzyme assay for D-alanine was employed to confirm substrate turnover.39 The standard assay mixture consisted of 1.0 mL of Tris buffer (0.1 M at pH 8.2), 400 μL of flavin adenine dinucleotide (FAD, 0.3 mg/mL in Tris buffer), 300 μL of horseradish peroxidase (10 μg/mL in H2O), and 100 μL of D-amino acid oxidase (5 mg/mL in Tris buffer). An aliquot (100 μL) of the assay mixture was mixed with 5 μL of odianisidine hydrochoride (10 mg/mL in H2O) along with 30 μL of the reaction mixture (hydrolyzed peptide sample) and the resulting reaction mixture incubated at 37 °C for 30 min. Methanol (0.7 mL) was then added, and after incubation of the reaction mixture for a further 2 min, the absorbance at 460 nm was measured. Calibration was achieved by means of standard D-alanine solutions.

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RESULTS AND DISCUSSION Cyclic peptides 5 and 6 were designed to be conformationally constrained substrates of EcPBP5, as described in the introductory section. Because the active site of EcPBP5 is D


Article

Biochemistry Scheme 2. Synthesis of 5

Structural Studies. Computational Models. Enzyme/ Cyclic Peptide Complexes. Structural models of peptides 5 and 6 noncovalently bound (potential Michaelis complexes) to the active sites of, first, EcPBP6 and then EcPBP5 were constructed as described in Materials and Methods. Figure 2 shows the active site region of these models for both cyclic peptides bound to EcPBP5; those for EcPBP6 are shown in the Supporting Information (Figure S1). Also shown in the Supporting Information (Figure S2) are the EcPBP5 and EcPBP6 complexes of 5 superimposed. This diagram shows that the conformations of the bound peptide are very similar for the two enzymes, which is not unexpected in view of the close structural similarity between the enzymes and how the models were constructed. These models also show that the interactions between the cyclic peptides and the EcPBP5 active site are very similar to those in the reference EcPBP6 crystal structure with its acyclic ligand (Figure S1).25 The reactive C-terminal D-alanyl-D-alanine interacts strongly with the active site in a manner typical of that found in β-lactam-recognizing enzymes. The reactive D-alanyl carbonyl group tightly interacts with the oxyanion hole (NH groups of Ser44 and His216); the terminal carboxylate appears to strongly hydrogen bond to the hydroxyl groups of Ser110 and Thr214, and the Lys47/Ser44 pair appears to be poised to

very similar to that of EcPBP6, the crystal structure of acyclic peptide 4 in a complex with PBP6 was used to design 5, a cyclized form of 7, as described in Materials and Methods. Compounds 6 and 8, which incorporated the two-carbon shorter L-diaminobutyryl (vs L-lysyl) side chain, were also prepared. The L-lysyl side chain of 4 in the EcPBP6 crystal structure is apparently free at the surface of the enzyme, so we envisioned that a shorter chain might also allow a productive complex to form and further show the effects of conformational constraint in comparison with 5 and 8. Synthesis of cyclic peptides 5 and 6 was achieved by means of the reaction sequences of Schemes 2 and 3, respectively. Protected acyclic precursors F11 and G6 were obtained by standard solution peptide chemistry. A succinate group was used to link the two N-termini in each case in a way that appeared in the models to avoid serious steric contact with the protein, either EcPBP5 or EcPBP6. Cyclization was efficiently achieved by treating the precursor with HATU/DIPEA in a dilute DMF solution. The required cyclic peptides were then directly obtained by hydrogenolysis to remove the benzyl protecting groups. Acyclic analogues 7 and 8 were obtained by hydrogenolysis of intermediates F9 and G4, respectively. Experimental details of these syntheses are provided in the Supporting Information. E


Article

Biochemistry Scheme 3. Synthesis of 6

using NMR spectroscopy to narrow down the possibilities as much as possible (see below). Circular Dichroism. CD spectra of cyclic peptides 5 and 6 and acyclic analogue 7 are shown in Figure 3. The shorter acyclic peptide 8 had a spectrum (not shown) similar to that of 7. The spectra of 7 and 8 are very similar to that reported for LAla-D-isoGlu-L-Lys-D-Ala-D-Ala by Naumann et al.48 These are certainly not the spectra of random coils but reflect specific structure. They were interpreted by Naumann et al. to represent an “open turn”, not stabilized by intramolecular hydrogen bonding. More recently, 2D NMR experiments have shown that analogous pentapeptides adopt a relatively narrow population of conformers in aqueous solution.49,50 Presumably, these structures lead to the observed CD spectra of the acyclic peptides. The CD spectra of cyclic peptides 5 and 6 are quite different from those of 7 and 8 and must reflect the presence of different structural elements. The spectra are of classical A/B form,51 suggesting the presence of β-turn-like structures.52,53 Such elements of structure are often found upon cyclization of linear peptides.52−57 Interpretation of the present spectra in terms of classical β-turns is complicated by the presence of nonclassical amino acids and D-amino acids in 5 and 6, such that there is not a classical tripeptide moiety present, as required for β-turn formation, in these cyclic compounds. A β-turn-like element could arise, however, from the ring structure itself by interaction with the pendent D-Ala-D-Ala terminus. Contributions from conformers containing γ-turns are also possible.52,58,59 Open (non-hydrogen-bonded) turns may also contribute to these spectra (see above). The greater intensities of the extrema in 6 versus those of the extrema in 5 indicate a contribution of these secondary structural elements to the conformations adopted by the former peptide greater than that of the latter. A more constrained and less mobile structure might be expected for the smaller ring of 6.

attack the D-alanyl carbonyl group. The L-Lys/D-alanine peptide bond is loosely placed between the side chain NH group of Asn112 and the backbone carbonyl oxygen of His216; these interactions are loose probably because of the unusual expansion of the active site found in EcPBP5 and EcPBP6 crystal structures.24,25 The crystal structure of the complex between EcPBP6 and 4 is striking in that there is little interaction between the Nterminal arms of 4 and the protein (Figure 1). Only a likely hydrogen bond between the carbonyl oxygen of Gly81 and the α-NH group of the ligand L-lysine residue and a loose interaction via a water molecule between the peptide isoglutamyl carboxylate and Arg194 are evident. These interactions are also possible in the EcPBP5/cyclic peptide model structures of Figure 2 (where Gly85 and Arg198 are the corresponding residues). This relative lack of specific interactions may explain why 4 is not perceptibly a substrate of EcPBP625 and one reason why small peptidoglycan-mimetic peptides in general are poor substrates of both EcPBP5 and EcPBP6.12,42 Although the positioning of the C-terminal Dalanyl-D-alanine moiety is very similar in the complexes of 5 and 6, the peptides do diverge at the L-lysyl(L-Dab) carbonyl to accommodate the longer lysine side chain in 5. The bulk of the rings still largely occupy the same space, however, as seen in the superimposed structures (Figure S2). The structures of Figure 2 are analyzed further below with respect to catalysis and specificity. Solution Structures. Attempts were made to determine solution structures of the cyclic peptides by computational means. A variety of simulated annealing procedures were applied, but global minima were not obtained, probably because of the considerable conformational mobility of these structures. This is well-known to be a difficult problem with cyclic peptides, especially when extended amino acid side chains are present in the ring.43−47 We chose therefore to proceed by F


Article

Biochemistry

Figure 2. Top figures show models of cyclic peptides 5 and 6 noncovalently bound at the active site of EcPBP5 (both cyclic peptides are colored aqua). Bottom figures represent a stereodiagram of the superimposition of 5 (thin bonds) and 6 (thick bonds) from the top figures and were achieved by superimposition of the α-C of the conserved active site residues (S44, K47, S110, N112, K213, T214, and G215).

observed 3J values could be obtained. Because of the cosine form of the Karplus relationship, there are two possible Ramachandran-allowed values for each of the intracyclic angles, ϕ1 (L-Ala), ϕ2 (D-isoGlu), and ϕ3 (L-Lys/Dab). Thus, there are eight possible permutations of values for the three dihedral angles (Table S3). For each of these conformations, where the dihedral angles mentioned above were constrained in each case, an energy-minimized structure in water was obtained by molecular mechanics computation (see Materials and Methods) and molecular mechanics energies were computed for each of the minimized structures in vacuo. These values for both cyclic peptides are also listed in Table S3. Averages of the eight conformations of 5 were 84.4 ± 3.5 kcal/mol and those of 6 66.5 ± 2.8 kcal/mol. Despite the conformational differences caused by the different combinations of angles, the calculated energies are quite similar for each peptide, suggesting a rather flat energy landscape at the bottom of the conformational energy well of these mobile peptides. As a caveat to these

NMR Experiments. 1D proton NMR spectra of 5 and 6 are shown in Figures 4 and 5. Assignment of all protons in 5 was accomplished by combination 2D COSY and 2D NOESY experiments (Figure S3); the NH assignments are given in Figure 4. Similarly, assignment of all protons in 6 was achieved by 2D TOCSY and 2D NOESY experiments and confirmed by a 2D COSY spectrum (Figure S4). The NH assignments for 6 are given in Figure 5, and complete assignments for both compounds are listed in Tables S1 and S2. 3 J coupling constants of the amide hydrogens with the adjacent α-CH hydrogens in both 5 and 6 were obtained from the 1D spectra (Tables S1 and S2). Notable among them is the fact that the 3J coupling constants of the L-Ala and D-isoGlu residues in both peptides fall well outside the range observed for random coil peptides,60 suggesting specific conformational restriction of these residues. Those of the pendent D-Ala-D-Ala residues suggest no such restriction. By application of the Karplus equation,52 dihedral angles (ϕ) corresponding to the G


Article

Biochemistry

Figure 5. 1H NMR spectrum (800 MHz) of cyclic peptide 6 in water at 25 °C. Inset are the downfield resonances.

peptides.60,61 In the case of 6, intensities of NOE cross-peaks were converted into distances as described in Materials and Methods, and these are listed in Table S4 along with the comparable distances from the structures computed from the dihedral angles described above. The best match between the two sources of information is probably structure 1, which is shown, as an example, in Figure 6. This structure is notable for

Figure 3. CD spectra of peptides 5 (red), 6 (blue), and 7 (green), recorded in aqueous solution at 25 °C.

Figure 6. Stereodiagram of a possible conformer of 6 in aqueous solution (see the text).

the possibility of an intracyclic hydrogen bond, shown in Figure 6, between the D-isoGlu γ-carbonyl and the L-Dab γ-NH group. Although this hydrogen bond forms an eight-atom ring arising from an amino acid (L-Dab) side chain and is thus not a formal β- or γ-turn, it may be partly responsible for the CD signal observed in solutions of 6 (Figure 3). Kinetic Studies. Spectrophotometric steady state kinetic measurements of turnover of peptides 5−8 by EcPBP5 were taken at pH 8.5, where it is often assayed,12 and at pH 9.5, where it has higher peptidase activity;27 its activity at pH 7 is quite low. An example of typical kinetic data is shown in Figure S5. Similar measurements were obtained at pH 7.5 for turnover of the peptides by SpPBP3, another LMMA DD-peptidase with a structure similar to that of EcPBP5.26 In all cases, the nature of the reaction catalyzed was demonstrated by D-alanine analysis at a time point beyond half-completion. The evidence of a reproducible spectrophotometric reaction was confirmed by substantially quantitative release of D-alanine. The enzymes were therefore acting as DD-carboxypeptidases (Scheme 1). A superficial view of the kinetic results (Table 1) shows that both the cyclic and acyclic L-lysine peptides 5 and 7 were comparably active as substrates of EcPBP5, with greater

Figure 4. 1H NMR spectrum (800 MHz) of cyclic peptide 5 in water at 25 °C. Inset are the downfield NH resonances.

statements, the numbers quoted above, of course, do not represent free energies, which were not estimated as part of this work. The average calculated energies mentioned above, Efree, can, however, be compared with those of the bound peptides, Ebound, 94.7 and 84.4 kcal/mol for 5 and 6, respectively. The comparison of the difference (Ebound − Efree) values for the two peptides shows that, in the absence of other factors, 5 should bind more strongly to EcPBP5 than 6 does by 12.7 kcal/ mol. This result may be indicative of an enthalpic advantage for the binding of 5 to PBP5 (see further discussion below). The NOESY spectra of 5 and 6 are quite different in that the number of cross-peaks observed in the spectrum of 6 is much greater than in that of 5. These observations are, like the CD data, in accord with a less mobile structure of 6 than of 5, again not surprising in view of the smaller ring of the former. In general, few intraresidue NOE signals are exhibited by cyclic H


Article

Biochemistry Table 1. Steady State Kinetic Parameters (kcat/Kma) for Peptide Hydrolysis peptide

EcPBP5 (pH 8.5)b

EcPBP5 (pH 9.5)

SpPBP3

5 7 6 8 Ac2Kaad

100 ± 2 150 ± 2 NRc NRc 300 ± 10

440 ± 10 320 ± 8 NRc NRc 370 ± 25

480 ± 10 970 ± 30 510 ± 10 500 ± 30 (6.2 ± 0.1) × 103e

More striking than the results for 5 and 7 are those for 6 and 8, neither of which is a substrate of EcPBP5. The result for 8 is particularly unexpected because, as noted above, the L-lysine side chain of 4 in the crystal structure with EcPBP6 (Figure 1) is directed out from the protein and presumably solvated by water when the protein is in solution. To rationalize the absence of reactivity of 8, it seems necessary to propose a disruption of substrate binding by this particular side chain. This could arise, for example, from hydrogen bonding between the terminal ammonium ion of 8 and the backbone carbonyl groups of Lys84 and Gly85, which may disrupt the positioning of the backbone loop between Phe83 and Leu88 (EcPBP5 numbering) (Figure 2). The homologue of the Gly85 carbonyl is seen in the EcPBP6 structure to hydrogen bond to the L-Lys NH group of the substrate, and the Ser87 NH and or/side chain OH may assist Asn112 in positioning the substrate L-lysyl carbonyl. Disruption of this loop might well interfere with productive placement of the substrate. Indeed, Nicholas et al.23 have shown that mutation of Ser86 and Ser87 or deletion of the entire 74−90 loop drastically reduced the carboxypeptidase activity of EcPBP5 but had little effect on the turnover of benzylpenicillin. The longer L-lysyl chain of 7 would be less likely to interfere in this way. The lack of reactivity of the smaller cyclic peptide 6 with EcPBP5 can also be rationalized on the basis of the points raised above. First, it seems that the enthalpic barrier of reaching the bound structure (Figure 2) from the substrate in solution may be larger for 6 than for 5 (see above). Second, the energy-minimized bound structure (Figure 5) involves close interaction with the Phe83−Leu88 loop described above. It is also possible that such unfavorable interactions may be enhanced in the transition state conformation. Conformations closer to the likely solution structures involve unfavorable steric interaction with the loop. These are avoided in 5 because of the flexibility allowed by the larger cycle. With respect to the rationale offered in the preceding paragraph, it is interesting to consider the kinetic results obtained for reactions between the new peptides and SpPBP3 (Table 1). SpPBP3, like EcPBP5, is a LMMA DD-peptidase that is considerably homologous in structure and sequence to EcPBP5.26 No crystal structure of a peptide bound to SpPBP3, however, is available. The kinetic results of Table 1 show that all four peptides, 5−8, are of comparable reactivity with SpPBP3. The major difference between these results and those of EcPBP5 is that the peptides with smaller side chains, 6 and 8, are substrates of SpPBP3 but not of EcPBP5. Examination of the crystal structures of the SpPBP3 and EcPBP5 apoenzymes (PDB entries 1XP4 and 1NZO, respectively) shows that the loops on the “left side” of the entrance to the active site, including Phe83−Leu88 of EcPBP5 (corresponding to Asn93− Leu88 of SpPBP3), have rather different conformations in the two enzymes, with that of SpPBP3 extending further into solvent. It may therefore be more flexible than the corresponding loop of EcPBP5 (see above) and thus more able to accommodate 6 without the enthalpic cost of peptide− protein interactions. As Dessen and co-workers have pointed out,26 SpPBP3 also has an omega loop much larger than that of EcPBP5. They argue that this correlates with L-Lys versus DLdiaminopimelic acid in the respective stem peptides. If so, this suggests that the interactions of SpPBP3 (from a Gram-positive organism) with its environment (peptidoglycan and protein) could be very different from those of EcPBP5 (from a Gramnegative organism).

All Km values are >1 mM, and thus, kcat values are >10−3 (kcat/Km) s−1. bEc = E. coli; Sp = S. pneumoniae. cNo reaction observed under the conditions employed. dN,N′-Diacetyl-L-lysyl-D-alanyl-D-alanine. eFrom ref 12; measurements taken at 37 °C. a

reactivity, as anticipated,27 at pH 9.5 than at pH 8.5. In clear contrast, L-diaminobutyric acid peptides 6 and 8 were not substrates of the enzyme at all. All four peptides were comparably active as substrates of SpPBP3. In no cases were Km values less than 1 mM, thus giving no indication of strong noncovalent recognition of these substrates by the enzymes. This latter result is typical of those obtained from studies of LMMA and HMM DD-peptidases with small peptide substrates.12,16 The results in Table 1 are discussed in detail below in terms of substrate and enzyme structures. The rationale for the preparation of cyclic peptides 5 and 7, as described above, was that the anticipated smaller loss of conformational entropy of these compounds upon binding to the enzyme active site, in comparison with their acyclic analogues, 6 and 8, respectively, would lead to more rapid turnover of the cyclic compounds by a DD-peptidase than by the acyclic peptides. Such an increase in the rate of turnover, however, was not directly seen in the results with EcPBP5 and 5 versus 7. There are two ways in which these results can be interpreted. The first possibility is that EcPBP5 exhibits no specificity for a peptide substrate beyond the D-alanyl-D-alanine C-terminus where peptide hydrolysis occurs. This proposition is not true on the basis of previous observations,12,27 which are directly reaffirmed here by the results with 6 and 8, discussed below. The second interpretation is more nuanced. Elements of peptide structure certainly do affect reactivity with EcPBP5, although the absolute overall reactivity is modest, in accord with precedent, even with peptides such as 7. It is likely that the absence of a significant difference in reactivity between 5 and 7 arises from a cancellation of the anticipated favorable conformational entropy effect by an unfavorable enthalpic effect. As discussed above, there may well be such an enthalpic effect arising from the conformational difference between 5 in solution and at the enzyme’s active site; a barrier of several kilocalories is likely. Beyond this major effect, there are also, of course, likely to be differences in solvation entropies that might be significant, although, if present, these may, as often occurs, be damped by entropy−enthalpy compensation.62−64 A survey of past experience with peptide cyclization to promote protein association equilibria finds positive, negative, and neutral outcomes.35,36,65−70 In essentially all cases, the issues of unfavorable enthalpy contributions and entropy−enthalpy compensation are discussed. On the basis of much precedent, therefore, we tend to interpret our results with 5 and 7 in terms of a significant entropic advantage for the binding of 5 to EcPBP5 derived from conformational restriction, although this is offset by an enthalpic disadvantage also derived from conformational restriction. I


Article

Biochemistry

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CONCLUDING ASSESSMENT Cyclic peptides 5 and 6 were designed to reduce the entropic penalty of binding as substrates to the EcPBP5 DD-peptidase, in comparison with their acyclic analogues 7 and 8, respectively. Our initial expectation was that 5 and 6 would be better substrates of the enzyme (as revealed by larger kcat/Km values) than 7 and 8, respectively. The larger cyclic peptide 5, however, was found to be only as active as its acyclic analogue 7. We argue that in this case, the entropic advantage of the cyclic peptide is offset by the enthalpic penalty of achieving the bound conformation from that in solution. The entropic advantage of the cyclic peptide in this case might indeed therefore be a few kilocalories. The smaller peptide 6 is not a substrate of EcPB5, perhaps because in this case the enthalpic cost of conformational adjustment together with an unfavorable steric interaction with the enzyme is sufficient to overcome any entropic advantage. The smaller acyclic peptide may have unfavorable polar interactions with the protein. Both cyclic peptides are substrates of the homologous enzyme SpPBP3, probably because of a more flexible binding site in this enzyme. Hence, with SpPBP3, the entropic advantage of conformational restriction for both cyclic substrates is able to compensate for the enthalpic cost of achieving the bound conformation. The extended peptidoglycan substrates of these enzymes in vivo, therefore, may well achieve considerable reactivity from the stronger immobilization of their extended N-termini upon interaction with the enzyme, particularly if these termini are held in the transition state by interactions with other macromolecules. The results with 5 and 6 reported here suggest that a larger, more rigid cross-link, designed to prevent (or exploit) interaction with the enzyme, would be required to more directly see the entropic advantage of a constrained peptide substrate of EcPBP5.

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mass; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 3morpholinopropanesulfonic acid; NMR, nuclear magnetic resonance; PBP, penicillin-binding protein; PDB, Protein Data Bank; Sp, S. pneumoniae; TAPS, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid; TFA, trifluoroacetic acid; THF, tetrahydofuran; TLC, thin-layer chromatography.

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(1) Blumberg, P. M., and Strominger, J. L. (1974) Interaction of penicillin with the bacterial cell: Penicillin-binding proteins and penicillin-sensitive enzymes. Bacteriol. Rev. 38, 291−335. (2) Waxman, D. J., and Strominger, J. L. (1983) Penicillin-binding proteins and the mechanism of action of β-lactam antibiotics. Annu. Rev. Biochem. 52, 825−869. (3) Tipper, D. J., and Strominger, J. L. (1965) Mechanism of action of penicillin: a proposal based on their structural similarity to acyl-Dalanyl-D-alanine. Proc. Natl. Acad. Sci. U. S. A. 54, 1133−1141. (4) Pratt, R. F. (2002) Functional evolution of the serine β-lactamase active site. J. Chem. Soc., Perkin Trans. 2, 851−861. (5) Ghuysen, J.-M. (1991) Serine β-lactamases and penicillin-binding proteins. Annu. Rev. Microbiol. 45, 37−67. (6) Goffin, C., and Ghuysen, J.-M. (1998) Multimodular penicillinbinding proteins: an enigmatic family of orthologs and paralogs. Microbiol. Mol. Biol. Rev. 62, 1079−1093. (7) Sauvage, E., Kerff, F., Terrak, M., Ayala, J. A., and Charlier, P. (2008) The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis. FEMS Microbiol. Rev. 32, 234−258. (8) Popham, D. L., and Young, K. D. (2003) Role of penicillinbinding proteins in bacterial cell morphogenesis. Curr. Opin. Microbiol. 6, 594−599. (9) Ghosh, A. S., Chowdhury, C., and Nelson, D. E. (2008) Physiological functions of D-alanine carboxypeptidases in Escherichia coli. Trends Microbiol. 16, 309−317. (10) Anderson, J. W., and Pratt, R. F. (2000) Dipeptide binding to the extended active site of the Streptomyces R61 D-alanyl-D-alanine peptidase: The path to a specific substrate. Biochemistry 39, 12200− 12209. (11) Adediran, S. A., Kumar, I., Nagarajan, R., Sauvage, E., and Pratt, R. F. (2011) Kinetics of reactions of the Actinomadura R39 DDpeptidase with specific substrates. Biochemistry 50, 376−387. (12) Nemmara, V. V., Dzhekieva, L., Subarno Sarkar, K. S., Adediran, S. A., Duez, C., Nicholas, R. A., and Pratt, R. F. (2011) Substrate specificity of low-molecular mass bacterial DD-peptidases. Biochemistry 50, 10091−10101. (13) McDonough, M. A., Anderson, J. W., Silvaggi, N. R., Pratt, R. F., Knox, J. R., and Kelly, J. A. (2002) Structures of two kinetic intermediates reveal species specificity of penicillin-binding proteins. J. Mol. Biol. 322, 111−122. (14) Sauvage, E., Powell, A. J., Heilemann, J., Josephine, H. R., Charlier, P., Davies, C., and Pratt, R. F. (2008) Crystal structures of complexes of bacterial DD-peptidases with peptidoglycan-mimetic ligands; the substrate specificity puzzle. J. Mol. Biol. 381, 383−393. (15) Josephine, H. R., Charlier, P., Davies, C., Nicholas, R. A., and Pratt, R. F. (2006) Reactivity of penicillin-binding proteins with peptidoglycan-mimetic β-lactams: What’s wrong with these enzymes? Biochemistry 45, 15873−15883. (16) Pratt, R. F. (2008) Substrate specificity of bacterial DDpeptidases (penicillin-binding proteins). Cell. Mol. Life Sci. 65, 2138− 2155. (17) Popham, D. L., and Young, K. D. (2003) Role of penicillinbinding proteins in bacterial cell morphogenesis. Curr. Opin. Microbiol. 6, 594−599. (18) Matteï, P.-J., Neves, D., and Dessen, A. (2010) Bridging cell wall biosynthesis and bacterial morphogenesis. Curr. Opin. Struct. Biol. 20, 749−755. (19) Typas, A., Banzhaf, M., van den Berg van Saparoea, B., Verheul, J., Biboy, J., Nichols, R. J., Zietek, M., Beilharz, K., Kannenberg, K., von Rechenberg, M., Breukink, E., den Blaauwen, T., Gross, C. A., and

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.6b00576. Synthetic details for the preparation of 5 and 6, models of 5 and 6 bound to PBP6, 2D 1H NMR spectral data for 5 and 6, proton assignments and intramolecular distance estimates, and an example of the kinetic data (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Telephone: 860-685-2629. E-mail: [email protected]. Funding

This research was supported by National Institutes of Health Grants AI-17986 (R.F.P.), AI-113170 (R.A.N.), and GM066861 (R.A.N.). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS DCM, dichloromethane; DIPEA, diisopropylethylamine; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; Ec, E. coli; EDC, N-(dimethylaminopropyl)-N′-ethylcarbodiimide; ESI/ MS, electrospray ionization mass spectrometry; HATU, O-(7azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate; HMM, high molecular mass; LMM, low molecular J


Article

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Synthesis and Kinetic Analysis of Two Conformationally Restricted

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