Differential Modes of Peptide Binding onto Replicative Sliding Clamps

Article pubs.acs.org/jmc

Differential Modes of Peptide Binding onto Replicative Sliding Clamps from Various Bacterial Origins Philippe Wolff,§,+ Ismail Amal,¶,+ Vincent Oliéric,‡ Olivier Chaloin,# Gudrun Gygli,¶,▼ Eric Ennifar,§ Bernard Lorber,§ Gilles Guichard,○ Jérôme Wagner,∥ Annick Dejaegere,¶ and Dominique Y. Burnouf*,§ §

Université de Strasbourg, UPR9002, Architecture et Réactivité de l’ARN, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15, rue René Descartes, 67084 Strasbourg, France ¶ Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Département de Biologie Structurale et Génomique, 1 rue Laurent Fries, BP10142, 67404 Illkirch, France ‡ Swiss Light Source (SLS), Paul-Scherrer-Institute (PSI), 5232 Villigen, Switzerland # Le Centre National de la Recherche Scientifique (CNRS), Institut de Biologie Moléculaire et Cellulaire, Laboratoire d'Immunopathologie et Chimie Thérapeutiques, 15 rue René Descartes, 67084 Strasbourg cedex, France ○ Université de Bordeaux, CNRS, IPB, UMR 5248, CBMN, Institut Européen de Chimie et de Biologie, 2 rue Robert Escarpit, 33607 Pessac, France ∥ CNRS UMR7242, ESBS, Université de Strasbourg, BP 10413, 67412 Strasbourg Cedex, France S Supporting Information *

ABSTRACT: Bacterial sliding clamps are molecular hubs that interact with many proteins involved in DNA metabolism through their binding, via a conserved peptidic sequence, into a universally conserved pocket. This interacting pocket is acknowledged as a potential molecular target for the development of new antibiotics. We previously designed short peptides with an improved affinity for the Escherichia coli binding pocket. Here we show that these peptides differentially interact with other bacterial clamps, despite the fact that all pockets are structurally similar. Thermodynamic and modeling analyses of the interactions differentiate between two categories of clamps: group I clamps interact efficiently with our designed peptides and assemble the Escherichia coli and related orthologs clamps, whereas group II clamps poorly interact with the same peptides and include Bacillus subtilis and other Gram-positive clamps. These studies also suggest that the peptide binding process could occur via different mechanisms, which depend on the type of clamp.

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INTRODUCTION The faithful replication of chromosomes is a major challenge for all organisms. For that purpose, they have evolved highly sophisticated mechanisms not only to copy the genetic material in an error-free manner but also to regulate the whole replicative process.1 In Escherichia coli (Ec), the multisubunit DNA polymerase III completes the full chromosomal replication within 40 min under optimal growth conditions, at a rate of about 750 nucleotides/second.2,3 Central to the efficiency of this process is the replicative processivity factor, also referred to as β ring or sliding clamp (SC), that anchors the polymerase onto DNA, thus conferring high processivity to © 2014 American Chemical Society

the replicative enzyme. This homodimeric factor is loaded on DNA by the so-called γ complex in an ATP-dependent manner, slides rapidly along the double stranded helix, and interacts with the various polymerases (Pol I, II, III, IV, and V),4 as well as with other proteins involved in DNA metabolism, such as MutS, Hda, or DNA ligase.5−7 The interaction motif of all these proteins with SC was first identified using a bioinformatics approach.5 This work identified a short interacting peptide in many eubacterial SC Received: February 28, 2014 Published: August 29, 2014 7565

dx.doi.org/10.1021/jm500467a | J. Med. Chem. 2014, 57, 7565−7576

Journal of Medicinal Chemistry

Article

Figure 1. Comparison of the different sliding clamps (SC) used in this study. (A) SC are compared in terms of total residues number and residues identities and similarities. Alignments were performed with LALIGN program available on the EXPASY Bioinformatics Resource Portal. EcSC is considered as the reference protein. (Spy: S. pyogenes, Spn: S. pneumoniae) Spy and Spn have been published elsewhere18 and DOI:10.2210/ pdb1awa/pdb and were included in this analysis. (B) Phylogeny of the different sliding clamps used in this study. The graph was drawn using the freely available program Phylogeny.fr (http://phylogeny.lirmm.fr/phylo_cgi/index.cgi), in the automatic “one-click” mode, with carefully selected default options and parameter values suitable for most analyses. Despite the high similarity between the different SC, the phylogenic tree clearly differentiates between SC from Gram− and Gram+ bacteria. Although it belongs to the Gram+ class, MtSC maps at an intermediate position between these two classes. (C) Sequence alignments obtained from the structural superposition of 3D structures of SC from Gram− (1OK7: E. coli; pseudo: P. aeruginosa, this work, 4TR8; 1VPK: Thermotoga maritima) and Gram+ (myco: M. tuberculosis, this work, 4TR7; 2AVT: S. pyogenes; 2AWA: S. pneumoniae, 3T0P: Enterobacter rectale; subt: B. subtilis, this work, 4TR6). Only domains 2 and 3 of the SC monomer, which contact the peptides, are presented. The numbering refers to Ec (1OK7). Residues that form the peptide binding pocket (cf. Table 1) are highlighted with ◇ underneath the sequences.

further structurally defined.8,9 On one hand, the pentapeptide is anchored at the surface of SC by a bidentate contact where its C-terminal hydrophobic part is nested in a deep leucine rich

partners and allowed the authors to define the consensus sequence QL[SD]LF. The interaction of the Ec DNA PolIV binding peptide (QLDLGL) with its cognate SC (EcSC) was 7566



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(SI.1). Amino acids 351−352 of EcSC are however at about 20 Å from the peptide binding site. A phylogenic tree drawn between the various SC (Figure 1B) underlines the high similarity of all sequences but also differentiates between the most related ones. There is a clear partition between proteins from Gram+ and Gram− organisms, with two subgroups (BsSC and SaSC versus SC from Streptococcus pyogenes (Spy) and Streptococcus pneumoniae (Spn)) emerging in this last group. Although Mt belongs to the Gram+ class because of a lack of external membrane, MtSC stands apart of these two subgroups and is closer to the Gram− group, in agreement with our ligand interaction data (see below). The EcSC binding pocket is shaped by 19 residues (Table 1)9 located in the second and third domains of the SC monomers. From the sequence alignment obtained by superimposing the structures of SC from different Gram+ and Gram− bacteria (Figure 1C), the amino acids contacting the peptide are rather conserved among the different SC, although this conservation is not strict. In particular, the EcSC binding pocket has a sequence identity of 93% with the pocket of PaSC but only 56% with that of SaSC and MtSC and 50% with that of BsSC. For all Gram+ bacteria, SC residues homologous to EcS346 are changed into a P residue. Other additional non conservative changes are observed in MtSC at positions corresponding to EcG174 (G to R), EcH175 (H to F) and EcR152 (R to L) (Table 1). In BsSC, Mt SC and SaSC, a G to S substitution occurs at position corresponding to EcG174, while for both streptococcus strains, a M to T substitution occurs at position corresponding to EcM362. Otherwise, several conservative changes of hydrophobic residues are observed in Gram+ bacteria (Table 1). For Pa SC, a single non-conservative change (V to N) is observed at position corresponding to residue EcV344. Ec High Affinity Peptides Differentially Interact with the Various Rings. We used surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) to study the interactions between the various SC and several peptides that were previously designed for targeting the EcSC with improved affinities. 1 0 The binding constants of peptide P 1 (R1Q2L3V4L5G6L7), the natural interacting peptide of the Ec DNA polymerase IV, were determined by SPR (Table 2 and SI.2). The analysis yields quite similar equilibrium constant values for all SC, ranging from 1.7 to 4.6 × 106 M−1 for the association constants (KA) and from 0.23 to 0.58 μM for KD, viz., a 3-fold variation. These results are in agreement with a previous work5 showing that all bacterial rings recognize peptide sequences related to the QL(S/D)LF consensus sequence. We then used a competition assay to analyze the interaction of peptide P14 (AcQ1ChaD3L4diClF), previously identified as a high affinity ligand for EcSC,10 with the other SC. Only EcSC, PaSC, and MtSC interact with P14, with IC50 of 80, 140, and 850 nM, respectively (Figure 2A, Table 3 and SI.3), whereas BsSC and SaSC failed to interact efficiently with the very same peptide (Table 3 and SI.3). These results were confirmed by ITC analyses (Figure 2B, Tables 3 and 4). As found previously,10 KD values determined by ITC for the interaction of P14 with EcSC, PaSC, and MtSC were not much different from the respective IC50 determined by SPR (Table 3), with less than a 2-fold variation. As for SPR experiments, no interaction was detected for BsSC and SaSC with this peptide (SI.4). In order to elucidate this lack of interaction between P14 and BsSC and SaSC, we used another peptide, P7 (AcQ1ChaD3L4F5) with no modification on the C-

pocket (subsite 1, SS1), and on the other hand, the conserved Q1 residue forms a complex net of interactions with SC and peptide residues in subsite 2 (SS2).10 Finally, the overall binding area is flexible, and rearrangements are observed upon peptide binding that lead to the formation of a groove between the two binding subsites and to the extension of a hydrophobic platform on which the second peptide residue stacks.10 Recently, it was suggested that peptide binding onto EcSC could occur via a two steps process, with an initial interaction in SS1, followed by the binding in SS2.11 Because SC is a central element for the physiological functions of enzymes involved in DNA metabolism4 and their trafficking,12,13 the binding pocket has been considered as a target for the development of new antibacterial drugs.5,9,14 Several studies have explored this potentiality either by screening a chemical bank14 or by a structured-based design approach, opening the way for structure−activity studies.10,15 These studies led to the design of peptides which bind to the Ec SC in the nM range with a 4-fold higher affinity as compared to the natural ligand DNA PolIV polymerase.10 Another aspect of the drug development strategy is to assess the compound’s specificity in terms of molecular targets but also bacterial species, which will define, ultimately, the activity spectrum of the drugs. In the present work, we have studied the interaction of replication processivity factors from various bacteria, namely, P. aeruginosa (Pa), B. subtilis (Bs), S. aureus (Sa) and M. tuberculosis (Mt), with selected peptides previously shown to bind to EcSC with high affinity. We show that although the binding pockets of the different SC are structurally similar, the peptides display very different affinities to them. The thermodynamic signature of peptide binding to the different SC is also significantly different, with large negative entropies of binding for Gram-negative (Gram−) (Ec, Pa) SC and positive (or near zero) entropies of binding for Gram-positive (Gram+) (Bs, Mt, Sa) bacteria. Enthalpic contributions to binding on the other hand are significantly more favorable for binding to Gram− (Ec, Pa) SC than to Gram+ (Bs, Mt, Sa). Our combined structural and thermodynamics studies thus reveal that the molecular determinants of peptide binding differs between Gram− (Ec, Pa) or Gram+ (Bs, Mt, Sa) bacteria and relies on subtle differences in the structural dynamics of the binding pocket residues. This finding adds to our understanding of SC interacting protein trafficking processes and should improve the design of specific drugs targeting the SC binding pockets.

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RESULTS SC Amino Acid Sequence Alignments. Amino acid sequences of the various SC considered in this study were aligned using T-Coffee package16 (Supporting Information (SI), SI.1). The sequence identity between the PaSC and EcSC is 55% but only 26 to 28% for the three other clamps BsSC, MtSC, and SaSC (Figure 1A). Sequence similarities with EcSC range around 50% for all Gram+ bacteria clamps and more than 70% for PaSC (Figure 1A). The alignments display few gaps of one or two residues between the different sequences. There are very few larger insertions, and these are generally located far from the peptide binding site. The insertion located closest to the peptide binding site is found in MtSC, where the sequence (VSGDDRPVAGLNGNGPFP) is inserted between amino acids aligned with the region 351−352 in the EcSC sequence 7567


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Residue symbol letters are followed by position numbers in the peptidic sequence. Non conservative changes relative to the EcSC sequence are highlighted in bold, and conservative changes are in italic. Row 1 specifies the location of each residue in SS1 or SS2 according to the EcSC binding pocket description.9

terminal residue. This peptide interacts with EcSC with a KD of about 200 nM.10 We measured a 10-fold lower interaction of this peptide with BsSC and SaSC by SPR (Table 3), although ITC experiments yielded KD values ranging from 10 to 30 μM (Figure 2 and Tables 3 and 4), that is, 50- to 150-fold lower than those measured for EcSC. The differential interaction of the clamps with P7 and P14 peptides is also illustrated by the results of thermodynamic analysis (Table 4). The binding process is markedly exothermic for both EcSC/P14 and PaSC/P14 complexes at 25 °C, and ΔH is about 2-fold smaller for the MtSC/P14 interaction. Similarly, the enthalpy variation for P7 binding to BsSC and SaSC is 5- to 10fold smaller as compared to the EcSC/P7 value (Figure 2B). A strong enthalpy−entropy correlation is observed (SI.5), which reveals a ΔH/ΔS compensatory effect that accounts for the overall slight variation in ΔG of the peptide−protein interactions, independently of the SC origin. The ITC experiments were performed at 25 and 30 °C (SI.6). A significant difference in ΔH values at both temperatures is observed for complex formation involving EcSC and PaSC and to a lesser extent MtSC (ΔΔH = −4.00, −5.65, −7.69, and −1.35 kcal/mol for EcSC/P7, EcSC /P14, PaSC/P14 and MtSC/ P14, respectively). Such a difference is not observed for BsSC/P7 and SaSC/P7 interaction (ΔΔH = 0.01 and 0.05 kcal/mol, respectively). Resulting ΔCp for that temperature window are −1.13, −1.53, −0.27, −0.8, 0.002, and 0.01 kcal/mol/deg for Ec SC/P14, PaSC/P14, MtSC/P14, EcSC/P7, BsSC/P7, and SaSC/P7, respectively (Table 4). Similarity in Crystal Structures of the Various Bacterial Rings. To investigate the difference in peptide interaction with the various SC, we determined the structure of Pa SC, BsSC, and MtSC at 1.8, 1.5, and 2.3 Å resolution, respectively (SI.7 and SI.8). Previous studies also reported the structure of MtSC at 2.9 Å17 and 3.08 Å resolution, and from two other Gram+ bacteria, S. pyogenes (2.6 Å) (SpySC)18 and S. pneumoniae (2.5 Å) (SpnSC) (DOI: 10.2210/pdb1awa/pdb). To our knowledge, no structures have been reported for PaSC and Bs SC yet. The three structures have the same organization as the Ec SC, forming head-to-tail homodimers that assemble in a closed ring (SI.8). Moreover, each monomer is organized in three modules sharing a homologous structural organization. The sequence insertions are situated in loops and do not affect the structural organization of the different SC. We also solved the structure of the PaSC/P14 complex. Each ring of the unit cell is bound with two P14 peptides, located in the interacting pocket defined previously.8−10 All eight monomers are structurally similar, and their Cα chains superimpose with rmsd values ranging from 0.05 to 0.27 Å for more than 333 aligned residues. Similarly, the eight peptides adopt the same conformation in their binding pocket as indicated by their exact superimposition (data not shown). When compared to the same peptide bound into the EcSC binding pocket (PDB 3Q4L),10 the N-terminal part of P14 establishes with PaSC the same interactions as with EcSC (SI. 9).9,10 Its C-terminal part, which interacts in SS1, is however displaced upward by 0.97 Å for the Cε of the phenylalanine ring and 1.25 Å for its Cθ (Figure 3). The rings of the two F residues form an angle of 11.78°. As a consequence, the distances with interacting pocket residues (EcT172, EcL177 and EcV247)10 are increased by 0.25, 0.56, and 0.78 Å, respectively. As for EcSC/ P14, the meta chloro atom of PaSC/P14 establishes an halogen bond with the Oγ atom of residue PaT172, although both

a

R365 R366 R376 R376 R375 R376 R399 M364 M365 V375 V375 I374 V375 V398 P363 P364 P374 P374 P373 P374 P397 M362 M363 T373 T373 L372 L373 M396 V361 V362 I372 I372 I371 I372 L395 V360 V361 L371 L371 L370 L371 L394 S346 S347 P356 P356 P356 P357 P362 V344 N345 V354 V354 M354 M355 G360 Y323 Y324 Y333 Y333 Y332 Y340 Y339 N320 N321 N330 N330 N329 S337 N336 V247 V247 L255 L255 L255 L255 L264 P242 P242 P250 P250 P250 P250 P259 L177 L177 L185 M184 L185 L184 L186 R176 R176 R184 R183 R184 R183 R185 H175 H175 H183 H182 H183 H182 F184 G174 G174 S182 S182 S182 S181 R183 T172 T172 T180 T180 T180 T179 T181 L155 L155 L162 L162 L163 L162 L164 R152 R152 R159 R159 R160 R159 L161 SC SC Spn SC Spy SC Sa SC Bs SC Mt SC Pa

SS2 SS2 SS2 SS1 SS1 SS1 SS2 SS2 SS2 SS1 SS1 SS1 SS1 SS1 SS1 SS1 SS1 SS1

Table 1. Comparison of Residues Shaping the Peptide Binding Pocket in the Different SCa

Ec

SS2


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Table 2. Binding Rates and Equilibrium Constants of Peptide P1 (RQLVLGL) Interacting with the Various SC as Measured by SPR at 25°Ca Ec

SC kon (103 M−1 s−1) koff (10−3 s−1) KA (106 M−1) KD (10−7 M) a

8.0 1.9 4.2 2.3

± ± ± ±

Pa 1.2 0.3 1.2 1.3

7.1 3.6 1.7 5.8

± ± ± ±

Mt 1.5 0.3 0.2 0.8

8.7 2.1 4.6 2.4

± ± ± ±

Bs 0.3 0.1 1.8 1.2

4.8 1.5 3.5 2.9

± ± ± ±

Sa 1.2 0.7 0.5 0.4

9.2 1.9 4.1 2.5

± ± ± ±

0.5 0.1 0.9 0.5

Sensograms are presented in SI.2. Data are obtained from at least three independent experiments.

distance and angle are slightly increased as compared to EcSC/ P14 (d = 3.38 vs 3.17 Å, Θ = 157.13 vs 148.71°).10 Attempts to cocrystallize the other rings with the different peptides have not been successful so far. Comparison of Binding Pockets. We have compared the structure of each binding pocket of PaSC, BsSC, and MtSC with the EcSC pocket (Table 5 and SI.10), using the lsqman program (Uppsala Software Factory). Global rms values were determined by superimposition of full monomers. Local rms values were obtained by considering, for each SC monomers, the residues of the binding pocket and measuring the deviation of their Cα’s from the position of the cognate atom in the EcSC, taken as a reference structure. Superimposition of monomers A and B of the same structure give a measure of the intrinsic deviation in the six different structures (two EcSC structures 10K7 and 3Q4L, PaSC, BsSC, MtSC, and PaSC/P14) (Table 5A).9,10 For the superimposition of monomers of the same crystals, the global rms values vary between 0.3 and 0.7 Å, and the local rms values between 0.05 and 0.77 Å. (Table 5A). Interestingly, the local rms measured with 1OK7 (0.25 Å), which has a free pocket on monomer A and a P1 bound pocket on monomer B, suggests that P1 binding does not strongly perturb Cα’s positions of residues that shape the pocket. A similar conclusion can be drawn from the comparison between Pa SC and PaSC/P14 structures, which yield a local rms of 0.22 Å. The structure of the monomer A of the EcSC/P1 complex was used as a reference in the comparison with each monomer of the other rings (Table 5B). The global rms range from 1.1 to1.5 Å, whereas the local rms vary from 0.29 to 0.82 Å for MtSC (Table 5B and Figure 4). We define Δ as the difference between local rms measured from the superposition of two monomers from different structures (1OK7A/PaSC (or BsSC or Mt SC)) and local rms measured from the superposition of the two monomers of the control structure (1OK7A/B) (Table 5B). These values (ΔPa/Ec, ΔBs/Ec, ΔMt/Ec) are in the same range as the local rms measured between monomers of the same structure (PaSC (0.16 Å), BsSC (0.22 Å), or MtSC (0.77 Å)) (Table 5A). In other words, there is no more difference between two monomers of the same structure (PaSC, BsSC or Mt SC) than between monomers of two different structures (1OK7A/PaSC, 1OK7A/BsSC, 1OK7A/MtSC), suggesting that the binding pockets of the various rings are conformationally similar. This statement is further supported by the discrete analysis of Cα’s deviations of pocket residues, as measured by superimposing monomers A and B of PaSC, BsSC, and MtSC rings onto monomer A of the EcSC 1OK7 structure (Figure 4, SI.10 and SI.11). In all three cases, the Cα’s deviations are in the range of those measured by comparing monomers of identical sequence, except for residues located in loops, essentially P242, V247, V344, and S346, and to some extend G174 and V360 (EcSC numbering). EcM364 shows larger deviations because of its Cterminal position in the protein sequence. Thus, the structure

of the binding pockets of PaSC and BsSC rings are not much different from that of EcSC, although larger deviations are observed (Figure 4C) in both subsites of the MtSC ring pocket. Representations of pockets superimposition are presented in Supporting Information, SI.10. In order to further analyze the molecular interactions of the SC pocket residues with the different peptides, we performed molecular dynamics of the different complexes, followed by an analysis of the intermolecular interactions using free energy decomposition (FED). Details of the simulation protocols are available as Supporting Information (SI.12). We first considered the SC/P 1 complexes. In all cases, FED (SI.12A,C) reveal that all P1 residues, except G6, make substantial interactions with SC pocket residues. However, the balance of interactions appears somewhat different between the different complexes: the N-terminal of P1 forms slightly stronger interaction with MtSC than with EcSC, while for the Bs SC and SaSC complexes, the C-terminal side of P1 forms slightly more favorable interactions. Notably, L7 residue interactions are stronger with BsSC and SaSC than with EcSC (SI.12A). This additional stabilization appears to be correlated, at least in part, to interactions between the charged C-terminal of P1 and a conserved BsR159 (Table 1). MD trajectories reveal that the orientation of this R residue differs between EcSC and Bs SC, related to a strain specific change of a nearby clamp residue (EcY154 to BsI161), so that the average distance between the charge of this R residue and the C-terminal of the peptide is 4.8 ± 1 Å in BsSC and 8.1 ± 1.4 Å in EcSC (Figure 5A). Such differential interaction is also observed in SaSC (data not shown). Consequently, although the overall affinity of native P1 peptide is similar for all complexes, MD analyses shows that the balance of discrete interactions vary in subtle ways between the different complexes. We then analyzed the SC interactions with non natural peptides P14 and P7 (SI.12B,D−F). All P7 and P14 residues make favorable interactions with the different SC, and the energetic contributions of these peptide residues are largely similar, as revealed by the energetic analysis (SI.12B,D−F). However, when analyzing which amino acids are most involved in stabilizing the complexes on the SC side, differences are apparent between the different SC, that can be related to their differences in sequence. Residues in the C-terminal of MtSC that are involved in stabilizing the N-terminal R of the native P1 peptide are not involved in stabilizing P14 (SI.12C,D). For Bs SC/P14 and SaSC/P14, the long-range interaction between the C-terminal end of the native P1 peptide and BsR159 (or SaR160) is not observed in the simulations with P14 (Figure 5B, SI.12A,B). Indeed the distance between the C-terminal charges of this peptide residue and BsR159 (9.3 Å) or SaR160 (9.2 Å) are significantly increased with respect to the distances observed in complexes with P1 (4.8 Å in BsSC/P1 and 3.9 Å in Sa SC/P1) (Figure 5B). This differential effect may be related to both the peptide sequence (X-ray structural data have shown that the C-terminal residue of different peptides occupies 7569



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Figure 2. Interaction of peptides P7 (AcQChaDLF) and P14 (AcQChaDLdiClF) with the different sliding clamps. (A) SPR binding isotherms: IC50 values were determined by competition experiments where SC were preincubated with various concentrations of challenger peptide (P7 or P14) before being injected on the chip loaded with P1 peptide. All experiments were performed at least twice at 25 °C. A: EcSC/P14; B: PaSC/P14; C: Mt SC/P14; D: BsSC/P7; E: SaSC/P7. (B) ITC titration curves. SC concentrations ranged between 20 to 60 μM and peptide/SC ratio was 10. Complete thermodynamic data from these experiments are compiled in Table 4. A: EcSC/P14 (25 °C); B: PaSC/P14 (25 °C); C: MtSC/P14 (25 °C); D: BsSC/P7 (25 °C) ; E: BsSC/P7 (30 °C) ; F: SaSC/P7 (30 °C); G: EcSC/P7 (30 °C).

slightly different positions in the hydrophobic SS19,10) and to the pocket conformation, modulated by the local residues sequence. Such subtle changes may account for drastic differences in binding, such as observed for P7 and P14

differential interaction between SC from Gram− and Gram+

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Table 3. Comparison of IC50 and KD Values Determined, Respectively, by SPR and ITC Experiments, for the Interaction of P7 and P14 Peptides with the Various SCa Ec

Pa

SC

Mt

SC

Bs

SC

Sa

SC

SC

SPR

ITC

SPR

ITC

SPR

ITC

SPR

ITC

SPR

ITC

IC50 (nM)

KD(nM)

IC50 (nM)

KD(nM)

IC50 (nM)

KD(nM)

IC50 (nM)

KD(nM)

IC50 (nM)

KD(nM)

P7

170 ± 46

230 ± 52

nd

nd

nd

2095 ± 1200

80 ± 15

50 ± 1.6

140 ± 41

850 ± 165

750 ± 150

lb

8200 ± 500 (30 °C) nd

1535 ± 380

P14

640 ± 10 (30 °C) 99 ± 18

29 000 ± 3000 (30 °C) nd

lb

a

Experiments were performed at 25°C unless otherwise specified. SPR sensograms and ITC titration curves are presented in Figures 2 and SI.3. IC50 were determined by competition assays where pre-incubation with different concentrations of peptide P7 or P14 challenged SC binding to P1 loaded on the chip. Data are obtained from at least three (SPR) and two (ITC) independent experiments. lb: low binding. nd: not determined.

Table 4. Thermodynamics Data of the Titration Experiments of the Different SC by Peptides P7 and P14 As Measured by ITC Experimentsa SC

Ec

Pa

Mt

Bs

Ec

Bs

Sa

peptides

P14

P14

P14

P7

P7

P7

P7

25 0.95 ± 0.2 0.05 ± 0.001 −15.9 ± 1.2 −20 ± 0.7 −9.94 −1.13

25 0.77 ± 0.3 0.09 ± 0.02 −12.8 ± 2.4 −13 ± 0.3 −8.92 −1.53

25 0.45 ± 0.01 0.75 ± 0.15 −7.7 ± 1.0 −2 ± 0.5 −7.15 −0.27

25 0.88 ± 0.02 12.3 ± 0.5 −4.01 ± 1 9 ± 0.4 −6.69

30 0.52 ± 0.01 1 ± 0.12 −25 ± 1.3 −55 ± 4.5 −8.2 −0.8

30 0.82 ± 0.02 8.2 ± 0.5 −4 ± 0.2 10 ± 1.8 −6.96 0.002

30 0.54 ± 0.2 29 ± 3 −2.25 ± 0.7 13 ± 2.4 −6.27 0.01

temperature (°C) N KD (μM) ΔH (kcal/mol) ΔS (cal/mol/deg) ΔG (kcal/mol) ΔCp (25−30 °C) (kcal/mol/deg)

Raw ITC data have been treated with Origin software. N: number of sites per monomers. The experiments have been performed twice at least. ΔG = ΔH − TΔS. ΔCp = ΔΔH/ΔT. See also SI.6 for the ΔH variation with temperature. a

pathogens, and in particular, their multidrug resistant strains are responsible for nosocomial infections. M. tuberculosis (Mt) remains the most threatening infectious organism with more than 10 million new infections per year, and its prevalence is increasing worldwide.19 Finally, although B. subtilis (Bs) is not considered as a pathogenic agent, it is the most studied bacteria of the Gram+ group, and extensive studies have shown that its replication differs from that of Ec.20 Peptide Binding Pocket Structural Similarity among Bacterial Rings. Structures analysis of the different SC studied here shows that they all share the same general organization, viz., dimeric rings with a head-to-tail interaction of the two monomers (SI.8). Variations in overall shapes and sizes are observed, in line with previous observations.18,17 Major differences with EcSC structure result from the introduction of few amino acid stretches after the β4 sheet for Gram+ strains and, for the Mt ring, in the β′7 sheet and at the C-terminal extremity of β′′7 (SI.1). Comparison of peptide binding pockets did not reveal any major differences in Cα’s positions (Figure 4, Table 5, SI.10 and SI.11), except for residues located in the loops. In line with their almost identical sequence (Table 1C and SI.1), pocket residues of EcSC and PaSC are nicely superimposed, including side-chain orientations (local rms = 0.37 Å) (Table 5 and SI.10A). This close fit is also observed when both pockets are bound to P14 peptide (Figure 3). Comparison of EcSC and BsSC pockets (rms = 0.37 Å) (SI.10B) leads to the same conclusion, as most of the different residues keep the hydrophobic character, particularly in SS2 (Table 1). Only EcG174 and EcS346 are changed into S181 and P357 in BsSC, respectively. Considering the close homology between BsSC and SaSC (54% of identity and 73% of similarity) and the almost strict identity of pocket residues, with only two conservative substitutions (Table 1), one can suggest that the Sa SC pocket is structurally similar to that of BsSC. Finally,

Figure 3. Superimposition of EcSC/P14 and PaSC/P14 structures. The figure displays the detail of the interacting pockets bound to P14. Cognate pockets residues of both SC place well on top of each other (see also SI.10A and Table 5). Although the three N-terminal residues (AcQChaD) of P14 peptide are nicely superimposed for both complexes, the two C-terminal peptide residues in the PaSC/P14 complex are slightly displaced upward. EcSC/P14 complex: light green and orange, PaSC/P14 complex: dark green and yellow.

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DISCUSSION The fight against antibiotic-resistant strains relies notably on the development of new drugs, and bacterial SC are new potential targets for developing such compounds. Preliminary studies have been undertaken along these lines, using the EcSC as a model target.10,11,15 Here, we analyzed the interaction of SC from different Gram− and Gram+ bacteria with ligands previously designed to target the EcSC binding pocket.10 The bacteria were chosen due to their pathogenic impact on human health: P. aeruginosa (Pa) and S. aureus (Sa) are major human 7571



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Table 5. Structural Comparison of the Various SC and Their Binding Pocketsa A global rmsd local rmsd B global rmsd local rmsd

Ec

SC A/B 1OK7

0.56 (365) 0.25 1OK7A/ PaSC A

Ec

Pa

SC A/B 3Q4L

0.45 (361) 0.13 1OK7A/ PaSC B

1.17 (355) 1.05 (343) 0.37 0.29 Δ1 = 0.12 ; 0.04 mean ΔPa/Ec = 0.08

SC A/B

0.72 (365) 0.16 1OK7A/ BsSC A

Bs

SC A/B

0.73 (311) 0.22 1OK7A/ BsSC B

1.42 (343) 1.30 (338) 0.37 0.63 Δ2 = 0.12 ; 0.38 mean Δ Bs/Ec = 0.26

Mt

SC A/B

0.71 (344) 0.77 1OK7A/ MtSC A

Pa

SC/P14 A/B

0.29 (365) 0.05 1OKA7/ MtSC B

1.58 (322) 1.52 (311) 0.82 0.46 Δ3 = 0.21 ; 0.57 mean Δ Mt/Ec = 0.36

Root mean square deviations of Cα’s (Å) are obtained by superimposing full monomers (global rmsd) or only the 19 residues shaping the binding pocket (local rmsd). Numbers in brackets indicate the number of aligned residues. (A) Intrinsic rms deviations (Å) measured by superimpositions of monomer A onto monomer B of a given structure. (B) rms deviations (Å) between EcSC monomer A (1OK7 structure) and monomers A or B of Pa SC, BsSC and MtSC structures. Δn is the difference between local rmsd measured from the superposition of two monomers from different structures (1OK7A/PaSC (or BsSC or MtSC)) and local rmsd measured from the superposition of the two monomers of the control structure (1OK7A/B). a

superposition of EcSC and MtSC pockets reveals a larger difference between the two sites (local rms = 0.82 Å) (SI.10C), but the overall pocket conformation is maintained. These conclusions based on structures analyses are supported by SPR data showing that the native peptide P1 has a similar affinity for all SC, as indicated by KA values that vary only by a factor of 3 (Table 2). This is in agreement with the concept proposed by Dalrymple5 that all SC-binding eubacterial proteins contain a universal interaction motif, which in turn suggests that peptides with the consensus sequence should interact with a different but structurally related pocket. This has been supported by the fact that the PolC replicative subunit of several Gram+ strains can productively interact with Ec SC.21,22 Pocket Residues Modulate Peptide Interaction. In striking contrast with the P1 interaction data, we show here that peptides specifically designed as tight EcSC binders10 indeed bind to the closely related PaSC but fail to bind productively with BsSC and SaSC and poorly interact with MtSC. Namely, P14 shows a 10- to 15-fold lower affinity for MtSC as compared to Ec SC or PaSC and poorly interacts with BsSC and SaSC (Figure 2, Table 3, SI.4, SI.5). P7 also inefficiently interacts with these SC, with a 8- to 12-fold decrease as compared to EcSC (Figure 2, Table 3). The native peptide P1 is stabilized in the EcSC binding pocket9 by a combination of direct and water-mediated hydrogen bonds (mostly for the highly conserved Q2 residue, mediated by main chain atoms of the SC, see SI.9C) and hydrophobic interactions formed by the three leucine residues (L3, L5, and L7). Although the C-terminal L5 and L7 residues are found in a hydrophobic pocket (SS1) that is lined with conserved residues such as EcL177 and EcP242 (Table 1), L3 is more solvent exposed and is stabilized by interactions with EcM362 and EcP363. Interestingly, the orientation of EcM362 differs between the apo and peptide bound structures of EcSC10 and has been shown to be in dynamic equilibrium between these two conformations.11 These essential interactions are maintained in all SC/ peptides complexes studied, whether the structural information is available experimentally or by docking of the native peptide on the apo SC structures (see SI.12A,B,E). However, the changes in sequence between the different SC may affect the detailed energetic balance between the different interaction sites. In particular, we observed that the C-terminal L7 amino acid of P1 makes particularly favorable interactions with the SS1 of BsSC and SaSC (SI.12A), which results from electrostatic interactions between the C-terminal of the peptide and a conserved arginine (equivalent to EcR152, Table 1) of the SC

(Figure 5A). This interactions appears specific to BsSC and SC, owing to nearby sequence changes. (EcY154 to BsI161, indeed EcY154 affects the orientation of EcR152 and does not allow interactions with the C-terminal of the peptide, although BsI161 does not restrict the flexibility of BsR159 to the same extent.) The modified P14 and P7 peptides differ from the native P1 peptide in different aspects: they are significantly shorter (five residues versus seven for P1), and they contain unnatural amino acids, cyclohexyl-alanine, and 3,4-dichloro phenylalanine at positions 2 and 5, respectively. The increase in hydrophobic contacts with respect to P1, and formation of a halogen bond with residue EcT172 of the modified P14 are observed in the docked structures of the peptides with MtSC, BsSC, and SaSC, yet the affinity of the P7 and P14 peptides is significantly lower than what is observed for EcSC and PaSC (Table 4). The underlying molecular origin of this different behavior remains somewhat elusive, considering the similarities in structure between the different SC. For BsSC and SaSC, the observed electrostatic interaction between the C-terminal of P1 and BsR159 and SaR160 (equivalent to EcR152) is lost in the shorter P7 and P14 peptides, which may explain the decreased binding. Peptide Ring Interaction Occurs via Two Different Binding Modes. We have previously proposed a peptide/ Ec SC binding mechanism,10 stating that the pocket can adopt two conformations: in absence of a ligand, a closed configuration is preferentially observed where the EcM362 side chain blocks the path between SS1 and SS2. A more recent study highlights a dynamic equilibrium between open and closed conformations of this residue.11 Upon peptide interaction, EcM362 side chain shifts by 180°, along with that of EcS346, to allow the carving of a groove that joins the two subsites and in which the extended peptide lies. Concomitantly, a platform opens where the L3 of P1 interacts9 and which has been exploited to increase affinity by extending hydrophobic contacts in the modified peptides by the insertion of a cyclohexylalanyl residue.10 Besides these hydrophobic interactions, EcM362 main chain carbonyl oxygen is central to the network of hydrogen bonds that stabilize the side chain of the essential glutamine Q 2 in P1 (Q 1 in P 7/P14) (SI.9). Interestingly, in Gram+ bacteria, EcM362 is not generally found and is replaced by shorter, less flexible amino acids such as BsL373 (Table 1). The mechanism of binding of the central amino acids of the peptides may be affected by these changes, resulting in a different binding mechanism characterized by a different balance of enthalpic and entropic contributions to binding, as observed when comparing the binding of P7 to BsSC Sa

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or SaSC, relative to EcSC. Indeed, in this particular example, binding of P7 to EcSC is enthalpy driven and entropy opposed, although binding to BsSC and SaSC, while weaker, is favored by entropy and enthalpy (Table 4). Experience with designed synthetic ligands for medicinal chemistry has often underlined the importance of the underlying thermodynamic mechanism for the success of affinity improvement and selectivity in drug design.23 Our experience with the synthetic peptides presented here further emphasizes this point in the context of peptidomimetics binding to highly related SC targets. Finally, we observed that ΔCp ≠ 0 for EcSC, PaSC and to a lesser extend Mt SC complexes (group I), while for the BsSC and SaSC complexes (group II), ΔCp is almost null (Table 4 and SI.6). A previous in depth analysis of ΔCp in light of protein remodeling upon ligand binding24 has shown that different ΔCp magnitudes were associated with different modes of binding. We propose that peptide binding may occur via different processes depending on which group the SC belongs to. It is noteworthy that group I SC are essentially found in Gram− bacteria, while those of group II are present in Gram+ bacteria (findings of D.Y.B., unpublished). This difference in peptide interaction process will impact the design strategies of specific antibiotic compounds. In conclusion, our study shows that ligands specifically designed to bind with high affinity to the EcSC retain their full activity toward evolutionary-related targets such as PaSC but are poor ligands for more distantly related proteins such as BsSC and SaSC. Further developments of these compounds may open the path to the synthesis of strain specific drugs. Alternatively, they may also serve as starting molecules for the design of improved ligands targeting weaker binders such as MtSC. Moreover this study reveals that, despite the high structural conservation of both partners of the interaction, subtle modifications in residues sequence may dramatically modulate the binding efficiency. Finally, it suggests that different modes of interaction have been selected to adapt to the pocket sequence variability while maintaining binding efficiency.

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MATERIAL AND METHODS Cloning, Expression, and Purification of Processivity Factors (SC). DnaN genes were PCR amplified with Pf u turbo DNA polymerase (Agilent) according to usual procedures. Oligonucleotides for PCR amplification were from Sigma. PCR products were purified on agarose gel and ligated in pET-15b plasmids (Novagen) in the NdeI-BamHI restriction sites. Selected recombinant plasmids were sequenced (GATC, Kontanz, Ge) and transformed in BL21 (DE3) pLys E. coli strains. For expression of dnaN proteins (SC), cells were grown in LB at 37 °C to OD 0.5, then induced by IPTG (0.1 mM) at 28 °C overnight. SC fractions were first enriched on a Ni-NTA column, eluted with an imidazole step (300 mM) and further purified on a MonoQ column in buffer containing 20 mM Tris HCl pH 7.5, 0.5 mM EDTA and 10% glycerol, using a gradient from 0 to 0,5 M NaCl. After a final ultracentrifugation (100000 g, 1 h, 20 °C), soluble SC were concentrated on a Centricon 30K (Millipore) in the same buffer and stored at 4 °C. Protein quality was assessed by mass spectrometry in denaturing conditions and by DLS analysis (SI.13 and SI.14). Peptides Design and Synthesis. Procedures for the synthesis of the different peptides used in this study, namely P1, P7, and P14 have been described elsewhere.10 Their chemical formulas are shown in Supporting Information (SI.15).

Figure 4. C′α deviations of pocket shaping residues between EcSC and Pa SC (A), BsSC (B), and MtSC (C). C′α deviations (Δ) are expressed as rmsd (in Å) for each residue shaping the pocket. Global rms refers to the superimposition of the whole monomers, whereas local rms refers to the superimposition of the 19 pocket shaping residues only (see Table 5). (A) Comparison between EcSC (1OK7 monomer A) and Pa SC (monomer A and B): global rms 1.17 Å (monomer A) and 1.04 Å (monomer B); local rms: 0.37 Å (A) and 0.29 Å (B). (B) Comparison between EcSC (1OK7 monomer A) and BsSC (monomer A and B). Global rms 1.42 Å (A) and 1.30 Å (B) ; local rms: 0.37 Å (A) and 0.63 Å (B). (C) Comparison between EcSC (1OK7 monomer A) and MtSC (monomer A and B). Global rms 1.58 Å (A) and 1.52 Å (B); local rms: 0.82 Å (A) and 0.46 Å (B). For each graph, controls are presented, which measure the C′α deviation between the two monomers of the same structure (1OK7 A/B; PaSC A/B; BsSC A/B; Mt SC A/B). 7573



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Figure 5. Comparison of interactions networks in the SS1 pockets of EcSC and BsSC for the P1 and P14 peptides. Superposition of the average structure from the MD simulation of Ec (green) and Bs (blue) complexed with P1 (A) and P14 (B) peptides. (A) Differential positions of the conserved EcR152 and BsR159 residues result in a different electrostatic interaction network in the two complexes. The interaction between the Cterminal L7 residue of P1 and BsR159 can be related to the more favorable free energy contribution of L7 in the BsSC/P1 complex (SI.12A). Amino acids EcY154 and BsI161 affect the orientation of EcR152 and BsR159. (B) No interaction is observed between the C-terminal of P14 and either EcR152 or BsR159. In the shorter modified peptide, the orientation of the C-terminal of diClF5 differs from the native peptide. This modified network can be related to the reduced free energy contribution of diClF5 in the BsSC/P14 as compared to L7 in the BsSC/P1 complex (see SI.12B). The structures displayed are average structures from the MD simulations of the different complexes. The distances shown on Figure 5A are averages calculated over the entire MD trajectory. 1OK7 and 3Q4L were used as initial structures for the MD of EcSC/P1 and EcSC/P14 complexes, respectively. As no experimental complex structure was available for Bs, the 4TR6 Apo structure was used to dock the P1 and P14 peptides. See SI.12 for details of the protocol.

Crystallogenesis, Data Collection, and Processing. Crystallization experiments were conducted using the diffusion vapor technique. Condition screening and optimization were performed using Index screen and a Mosquito robot for dispatching droplets (200 nL). Once crystallization conditions have been optimized, crystals were grown in hanging drops. All SC were used at 10 μg/μL final concentration. For EcSC, crystallogenesis conditions were as described.9,10 Crystal of Pa SC and cocrystals of PaSC/P14 were obtained in 0,1 M sodium acetate pH 4,5 and PEG 3350, at 20 °C. Crystal of BsSC grew in 0,1 M bis tris pH 6,5 and 28% PEG MME 2000 at 20 °C. Finally, crystal of MtSC were obtained at 4 °C in 0,2 M sodium acetate trihydrate pH 7,0 and 20% PEG 3350. Cryoprotection was achieved by soaking crystals in the same buffer supplemented with 20% glycerol. Cryoprotected crystals were frozen in liquid ethane and X-ray diffraction data were collected at 100 K at beamline X06SA, X10SA and X06DA at the Swiss Light Source (Villigen PSI, Switzerland). Diffraction images were processed with XDS, XSCALE, and XDSCONV.25 Structures were solved by molecular replacement with MOLREP2926 or PHASER,29 using the known EcSC structure (PDB ID 1OK7) as a search model.9 Proteins crystallized in space group P212121 (PaSC) and P1 (BsSC and MtSC), with 1 dimer per asymmetric unit in each case. Alternate rounds of rebuilding and refinement, including noncrystallographic symmetry restraints, were carried out with BUSTER27 and COOT3128 and CNS32. Model statistics were obtained with Molprobity.30 Molecular visualizations and structures illustrations were performed using PyMOL.31 Data processing and refinement statistics are summarized in SI.7. Detailed analysis of the PaSC structure reveals a long tail of 16 residues in monomer A, while only two residues (SH) are noted in monomer B. These additional residues are encoded by

the His-tag of pET-15b vector in which the dnaN gene has been cloned. In both monomers, residues PaR23 and PaR24 are missing in the loop connecting α1 and β2 (according to the nomenclature of Kong et al.32), but the corresponding loops are complete in modules 2 and 3 of each monomer. For the BsSC structure, in both monomers, 4 residues are missing in the loop connecting α1 and β2, and the additional sequence EEGDKEI (as compared to EcSC) (SI.1) is forming a longer loop connecting β4 and β4b. Finally, in the MtSC structure, the additional sequence (SI.1) KDGLLGI forms a longer loop between β6′ and β7′ in the second module, whereas in the third one, the sequence VAGLNGNGPF forms an extended loop which joins β7″ and β8″, but its Nt part (SGDDR) is not seen in the structure, probably because of an intrinsic high mobility. The PaSC/P14 complex was formed during the course of an ITC experiment, and was concentrated to 47 mg/mL. Cocrystals are obtained in conditions similar to those leading to the crystallization of the apo form. The structure was solved at 2.0 Å resolution by molecular replacement using our previously determined structure of PaSC (this work, 4TR8). The complex crystallized in space group P1 and height rings were observed per unit cell. Surface Plasmon Resonance Assays. SPR experiments were performed on a Biacore 3000 at 25 °C in buffer containing 10 mM Hepes pH 7.4, 0.15 M NaCl and 3 mM EDTA. The association constant (KA) of SC with the natural C-terminal heptamer (P1, Table 2) of the DNA polymerase IV of E. coli were determined as follow: SC (0.125−2 μM) was injected on the immobilized P1 peptide at a flow rate of 50 μL.min−1. After subtraction of the background response, the data were fit to the 1:1 Langmuir model using BIAevaluation (BiacoreTm). The inhibition of SC/P1 interaction by peptides P7 and P14 (Table 3) was used to measure their affinity to SC and was assessed 7574



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Accession Codes

according to the following procedure: complexes of SC (0.25 μM) with various concentrations of challenging peptides (1.5 nM to 100 μM) were formed and injected on a chip loaded with the P1 peptide. IC50 values for each challenging peptide were determined by plotting peptide concentration against the percentage of binding inhibition. In Table 2, which presents the binding rates and equilibrium constants of peptide P 1 interacting with the various SC, we noted a discrepancy between our present data for EcSC values and previously obtained data.10 However, in the present study, binding of EcSC to P1 peptide anchored onto the chip was used systematically to validate the chip. Consequently, we are confident in the present values, but have at present no explanation for the aforementioned difference. Isothermal Titration Calorimetry. ITC was performed using a iTC200 (MicroCal LLC). Peptides (200 to 600 μM) were titrated at 25 or 30 °C in sequential injections (2 μL each) into SC solution (300 μL, 20 to 60 μM). Data were corrected from control experiments in which peptides were injected in buffer solution (Hepes 10 mM pH 7.4, NaCl 0.15 M, EDTA 3 mM). Data analysis was performed with Origin 7.0 software. Molecular Modeling. The initial structures used for the molecular dynamics and energetic analysis were crystallographic structures of the EcSC/P1 complex (PDB codes 1OK7, chains B and C) and new structures solved during this work (see below). For SC for which only an apo structure was available (PaSC, Mt SC and BsSC), the peptides were docked by superimposing the structure of the EcSC/P1 complex to the various apo structures, and transferring the coordinates of the peptide into the PDB file of the apoprotein, ensuring that no steric clash occurs between the peptide and SC. For SaSC clamp, homology models of the complex were built using the program Modeler,33 using standard modeling procedures. Explicit solvent molecular dynamics simulations of the different complexes were done using the NAMD program and the all atom force field CHARMM2734 with CMAP corrections.35 To obtain a semiquantitative estimate of the contributions of all amino acids to the binding free energy for the formation of the SC/ peptide complexes, a molecular free energy decomposition scheme based on the Molecular Mechanics/Poisson−Boltzmann Surface Area (MM/PBSA) analysis was performed, using the MD trajectories as a starting point, following the approach presented in refs 36 and 10. Details of the procedure are given in SI.12. With this approach, the total binding free energy can be decomposed into individual energetic contributions per residue. Decomposition of the binding free energy to individual amino acid contributions leads to the identification of amino acids that play a dominant role in binding and can contribute to reliable predictions of the role of particular amino acids in stabilizing complexes.

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Coordinate and structure factors have been deposited with the PDB under codes (BSSC: 4TR6), (PaSC: 4TR8), (PaSC/P14: 4TSZ) and (MtSC: 4TR7).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: 33 3 88 602 218. Tel.: 33 3 88 417 002. Present Address ▼

WU Agrotechnology & Food Sciences, Laboratory of Biochemistry, P.O. Box 8128, 6700ET Wageningen, Dreijenlaan 3, 6703HA Wageningen (G.Gyli). Author Contributions +

P.W. and I.A. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS For the gift of bacterial genomic DNA or strains, we acknowledge the contributions of Dr. Alain Baulard (Institut Pasteur, Lille, Mt H37Rv gDNA), Dr. Pierre Fechter (IBMC, Strasbourg, Sa strain RN6390), and Prof. Jacky de Montigny (UMR7156, Université de Strasbourg, Pa PAO1 strain). C. da Vega is gratefully acknowledged for her help in ITC experiments. We are indebted to Dr. C. Sauter for his help with USF programs.

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ABBREVIATIONS ITC, isothermal titration calorimetry; SPR, surface plasmon resonance; Ac, acetyl group; Cha, beta-cyclohexyl-L-alanyl; (3,4di-Cl)Phe, 3,4-dichloro-L-phenyl-alanyl

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REFERENCES

(1) O’Donnell, M.; Langston, L.; Stillman, B. Principles and concepts of DNA replication in bacteria, archaea, and eukarya. Cold Spring Harbor Perspect. Biol. 2013, 5 (7), 1−13. (2) Fay, P. J.; Johanson, K. O.; McHenry, C. S.; Bambara, R. A. Size classes of products synthesized processively by DNA polymerase III and DNA polymerase III holoenzyme of Escherichia coli. J. Biol. Chem. 1981, 256 (2), 976−983. (3) Hingorani, M. M.; O’Donnell, M. Sliding clamps: a (tail)ored fit. Curr. Biol. 2000, 10 (1), R25−R29. (4) Becherel, O. J.; Fuchs, R. P.; Wagner, J. Pivotal role of the betaclamp in translesion DNA synthesis and mutagenesis in E. coli cells. DNA Repair 2002, 1 (9), 703−708. (5) Dalrymple, B. P.; Kongsuwan, K.; Wijffels, G.; Dixon, N. E.; Jennings, P. A. A universal protein-protein interaction motif in the eubacterial DNA replication and repair systems. Proc. Natl. Acad. Sci. U.S.A. 2001, 98 (20), 11627−11632. (6) Lopez de Saro, F. J.; O’Donnell, M. Interaction of the beta sliding clamp with MutS, ligase, and DNA polymerase I. Proc. Natl. Acad. Sci. U.S.A. 2001, 98 (15), 8376−8380. (7) Wijffels, G.; Dalrymple, B. P.; Prosselkov, P.; Kongsuwan, K.; Epa, V. C.; Lilley, P. E.; Jergic, S.; Buchardt, J.; Brown, S. E.; Alewood, P. F.; Jennings, P. A.; Dixon, N. E. Inhibition of protein interactions with the beta 2 sliding clamp of Escherichia coli DNA polymerase III by peptides from beta 2-binding proteins. Biochemistry 2004, 43 (19), 5661−5671. (8) Bunting, K. A.; Roe, S. M.; Pearl, L. H. Structural basis for recruitment of translesion DNA polymerase Pol IV/DinB to the betaclamp. EMBO J. 2003, 22 (21), 5883−5892. (9) Burnouf, D. Y.; Olieric, V.; Wagner, J.; Fujii, S.; Reinbolt, J.; Fuchs, R. P.; Dumas, P. Structural and biochemical analysis of sliding clamp/ligand interactions suggest a competition between replicative

ASSOCIATED CONTENT

S Supporting Information *

SC sequences alignments, SPR sensograms, ITC titration curves, enthalpy/entropy compensatory effect, ΔH variation with temperature, statistics and crystallographic information, structures of the different bacterial SC, binding pockets structures, Cα’s distances variations, peptide Q residue interactions, free energy contributions procedures and graphs, MS data, DLS data and peptides chemical structures. This material is available free of charge via the Internet at http:// pubs.acs.org 7575



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Differential Modes of Peptide Binding onto Replicative Sliding Clamps

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