Allostery, Recognition of Nascent Peptidoglycan, and Cross-linking of

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Allostery, Recognition of Nascent Peptidoglycan and Crosslinking of the Cell Wall by the Essential PenicillinBinding Protein 2x of Streptococcus pneumoniae Noelia Bernardo-Garcia, Kiran V. Mahasenan, Maria T. Batuecas, Mijoon Lee, Dusan Hesek, Denisa Petrá#ková, Linda Doubravová, Pavel Branny, Shahriar Mobashery, and Juan A. Hermoso ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00817 • Publication Date (Web): 22 Jan 2018 Downloaded from http://pubs.acs.org on January 23, 2018

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ACS Chemical Biology

Allostery, Recognition of Nascent Peptidoglycan and Crosslinking of the Cell Wall by the Essential Penicillin-Binding Protein 2x of Streptococcus pneumoniae Noelia Bernardo-García,†,§ Kiran V. Mahasenan,‡,§ María T. Batuecas,† Mijoon Lee,‡ Dusan Hesek,‡ Denisa Petráčková,# Linda Doubravová,#Pavel Branny,# Shahriar Mobashery‡,* and Juan A. Hermoso†,* Department of Crystallography and Structural Biology, Institute of Physical Chemistry "Rocasolano", CSIC, 28006 Madrid, Spain; ‡Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA; # Institute of Microbiology, v.v.i, Czech Academy of Sciences, Prague 4, 142 20, Czech Republic Supporting Information Placeholder peptidoglycan, as Lipid II is polymerized on the surface ABSTRACT: Transpeptidases, members of the penicillinof plasma membrane (Figure 1A), but it is rapidly binding protein (PBP) families, catalyze crosslinking of the processed to various lengths, including cases that the bacterial cell wall. This transformation is critical for the stem peptide entirely might be removed. The survival of bacteria and it is the target of inhibition by βcrosslinking reaction is catalyzed by transpeptidases, lactam antibiotics. We report herein our structural insights members of the penicillin-binding protein (PBP) family into catalysis by the essential PBP2x of Streptococcus (Figure 1A). β-Lactam antibiotics inhibit the pneumoniae by disclosing a total of four X-ray structures, transpeptidases in manifesting their activity, whereby two computational models based on the crystal structures the cell-wall crosslinking is prevented and bacteria die. and molecular-dynamics simulations. The X-ray structures Transpeptidases undergo acylation in their active site by are for the apo PBP2x, the enzyme modified covalently in the nascent peptidoglycan, with the attendant release of the active site by oxacillin (a penicillin antibiotic), the the terminal D-Ala. It is the acyl-enzyme species that enzyme modified by oxacillin in the presence of a synthetic accepts the incoming nucleophile from the second strand tetrasaccharide surrogate for the cell-wall peptidoglycan of the peptidoglycan that gives the crosslinked product. and a non-covalent complex of cefepime (a cephalosporin The Tipper-Strominger hypothesis stipulates that the antibiotic) bound to the active site. A pre-requisite for backbone of β-lactam antibiotic mimics the terminal catalysis by transpeptidases, including PBP2x, is the acyl-D-Ala-D-Ala in the peptide stem of the bacterial molecular recognition of nascent peptidoglycan strands, peptidoglycan (Figure 1B).1 These antibiotics acylate the which harbor pentapeptide stems. We disclose that the active-site serine, as does the peptidoglycan, at a recognition of nascent peptidoglycan by PBP2x takes place corresponding site supported by the mimicry (Figure by complexation of one pentapeptide stem at an allosteric 1B). The notion of this mimicry is further supported by site located in the PASTA domains of this enzyme. This judiciously conceived designer β-lactam antibiotics, binding predisposes the third pentapeptide stem in the which structurally had been incorporated with same nascent peptidoglycan strand to penetration into the functionalities that demonstrate the approach of two active site for the turnover events. The complexation of the strands of peptidoglycan to one another, once covalently two pentapeptide stems in the same peptidoglycan strand is bound at the active sites of transpeptidases.2, 3 A 1.2-Å a recognition motif for the nascent peptidoglycan, critical resolution structure of one of these designer antibiotics for the cell-wall crosslinking reaction. in complex with a transpeptidase provided a snapshot of the two strands of peptidoglycan en route to crosslinking.3 We have documented that the cell-wall The cell wall is critical for survival of bacteria. The cell mimicry is also seen for binding, albeit non-covalently, wall is comprised primarily of the peptidoglycan, which of the antibiotic at the allosteric site of PBP2a of has a repeating disaccharide, N-acetylglucosamine methicillin-resistant Staphylococcus aureus (MRSA), (GlcNAc)-N-acetyl-muramic acid (MurNAc), for its where the peptidoglycan stem peptide also binds.4, 5 We backbone. A unique pentapeptide (L-Ala-γ-D-Glu- L-Lys realized that one additional likely example for allostery or m-DAP-D-Ala-D-Ala) is appended to the MurNAc might be apparent from the earlier crystallographic unit, which is the site of 4,3-crosslinking with literature, that of PBP2x of Streptococcus pneumoniae.6 neighboring strands of peptidoglycan (Figure 1A), as PBP2x is an essential transpeptidase, critical for septal opposed to the less common 3,3-crosslinking. The peptidoglycan synthesis that leads to cell division7 pentapeptide stem is found in the nascent Mutant variants of PBP2x represent the primary †

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resistance mechanism for β-lactam antibiotics in S. pneumoniae. Gordon et al. solved the structure of PBP2x in the presence of the β-lactam antibiotic cefuroxime.6 The authors observed one molecule of the antibiotic bound covalently to the active site and another noncovalently approximately 27 Å away at the site of the PASTA domains found adjacent to the catalytic domain (Figure 2B). The function of these additional domains in PBP2x is not known, but the PASTA domain is generally considered a site for binding of the peptidoglycan.8 PASTA domains are also present in the S. pneumoniae serine-threonine kinase StkP, which plays an important role in regulating septal cell-wall synthesis.9, 10,11 However the role of PASTA domains in PBPs remains unclear. Whereas the fold is similar between both kinds of PASTA domains, there is poor sequence conservation. 12 We provide evidence in the present report that the PASTA domains in PBP2x serve as an allosteric site in recognition of the nascent peptidoglycan, which would guide the donor strand of the peptidoglycan (strand 1) into the active site for acylation of Ser337, prior to crosslinking to the acceptor strand (strand 2). This binding site in the PASTA domains is hereafter referred to as the allosteric site. We add parenthetically that the allosteric sites in PBP2a (S. aureus) and PBP2x (S. pneumoniae) are not functionally, structurally, or mechanistically related to one another. Figure 1. (A) The scheme for the catalytic reaction of PBP2x. The pentapeptide stems interact with the allosteric and active sites of PBP2x. (B) Chemical structures of antibiotics cefuroxime, cefepime and oxacillin highlighting the mimicry of the antibiotic backbone (blue atoms) to the peptide terminus of the natural substrate, the peptidoglycan stem peptide (boxed). The chemical structure of compound 5 is shown.

RESULTS & DISCUSSION The pbp2x gene (without the segment for the membrane anchor) from S. pneumoniae RX1 was cloned and expressed and the protein was purified to homogeneity. Crystallization was performed by the sitting-drop vapor-diffusion method in 0.1 M sodium acetate, 2.3-3.0 M NaCl, pH 4.5. The apo structure was determined at 2.67 Å resolution (Figure 2A and Table 1). The structure of the transpeptidase and the two PASTA domains (P1 and P2, residues 632–691 and 692–750, respectively) nicely superimposed with the known apo structure, the N-terminal region being more variable due to intrinsic flexibility of this domain. The active site presents the catalytic Ser337 occluded and oriented towards the core of the protein (Figure 3A and Supplementary Figure S1), which requires a requisite conformational change to make it available for catalysis. Consistent with the Tipper-Strominger hypothesis, we propose that the backbone of cefuroxime bound to the allosteric site (Figure 1B and 2B) would spatially mimic that of the terminal D-Ala-D-Ala of the peptidoglycan stem peptide. The cefuroxime carboxylate at the allosteric site is anchored by Arg426 (Figure 2B, left image). Our complex of PBP2x with a different β-lactam antibiotic, cefepime, is

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Figure 2. (A) Stereo view of the ribbon representation of the apo structure of PBP2x showing the PASTA (pink) and transpeptidase (green) domains. The two PASTA domains (P1 and P2) are labelled and the position of the catalytic Ser337 is indicated by an arrow (2 o’clock). (B) The surface representation of the X-ray structures showing the PASTA (pink) and transpeptidase (green) domains. The previously reported X-ray structure on the left shows binding of two molecules of cefuroxime (spheres); one covalently to the active site and another non-covalently to the allosteric site. The PBP2x:cefepime X-ray structure (present work) is shown on the right, where the allosteric site exists in a closed conformation.

unique and of interest in two respects. First, an intact cefepime has entered the active-site groove on its way to Ser337 for acylation. This β-lactam mimics the stem peptide approaching the site of acylation, but it is present in the crystal structure at a distance of 4 Å from the serine hydroxyl (distance of the β-lactam carboxyl carbon to Ser337 hydroxyl oxygen; Figure 3B). Second, the allosteric site is in a “closed” conformation (Figure 2B, right image) by the gatekeeper Arg426 side chain actually blocking the site. For antibiotic binding at this site—also for peptidoglycan binding, as we will explain—Arg426 needs to move its side chain away to make room. We performed a molecular-dynamics simulation on our X-ray structure for the apo enzyme to explore whether both conformations, those of the “closed” and “open” conformations for the allosteric site, could be sampled. The X-ray structure was immersed in a box of TIP3P water molecules, energyminimized, and subjected to molecular-dynamics simulation with PMEMD module of AMBER1613 using ff12SB forcefield (see Methods for details). This was indeed the case, as we saw a smooth transition from one conformation to the other and then back to the first (Supplementary Movie S1) in the course of the simulation. As Arg426 opens up the site, it interacts favorably with Glu651 and Asp648 via salt-bridges. In the presence of the nascent peptidoglycan with its pentapeptide stem—or that of its mimetic, the β-lactam antibiotic—the open conformation will be selected for complexation. As such, the allosteric site is poised for recognition of the full-length stem peptide. Figure 3. The PBP2x active site in the apo, pre-acylated and acylated states. (A) Stereo view showing the catalytic Ser337 oriented towards the core of the protein. (B) Stereo view for the active site of the non-covalent PBP2x:cefepime complex with the antibiotic shown in capped sticks and color coded by atom types (gray for carbon). Polar interactions are represented by dotted lines. (C) Stereo view showing the active site of the covalent PBP2x:oxacillin complex.

As indicated earlier, our non-covalent cefepime complex within the active site shows the path that the stem peptide traverses to reach Ser337, the site of acylation. This X-ray structure assisted molecular docking with the program Glide (v. 5.6, Schrödinger Inc, NY), which brought the pentapeptide stem nicely to the


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pre-acylation complex, as revealed earlier for the PBP1b of S. pneumoniae by the quantum mechanical (QM)/molecular mechanical (MM) method.14 The backbone carbonyl of the penultimate D-Ala, the site of nucleophilic attack by Ser337, occupied the “oxyanion hole” formed by two hydrogen bonds with the backbone of Thr550 and Ser337, while the methyl side chain of DAla was positioned in a small hydrophobic pocket defined by Phe450 (Figure 4). The lysine of the pentapeptide made hydrogen bonds with the highly conserved Asn397 (through its carbonyl oxygen) and with Gln552 (through its side chain), the function of which appears to be anchoring of the peptide terminus for proper contact to the catalytic residues. Our covalent complex of oxacillin (Figure 3C and Supplementary Figure S1) and that of cefuroxime6 reveal the structure of the acylated active site at Ser337, one step beyond that visualized by our non-covalent cefepime complex within the active site. Why would the protein have the allosteric site for binding to the full-length pentapeptide stem? In search of an answer to this question, we interrogated the solution NMR structure of an octasaccharide peptidoglycan.15 This structure is that of a right-handed helix with a periodicity of three GlcNAc-MurNAc repeats per turn of helix.15 A view down the axis of the helix reveals the stem peptides at approximately 120° of each other. In this conformation for the peptidoglycan, when the stem pentapeptide at the first MurNAc is ensconced in the allosteric site with the carboxylate of the ultimate D-Ala interacting with Arg426 (Figure 4), the third MurNAc is predisposed for penetration into the active site (Figure 4 and Figure 5). The stem peptide at the second MurNAc position protrudes into the milieu and is stabilized by the β3-β4 loop. This complex was studied by molecular-dynamics simulations, showing stable occupancies of both the allosteric and active sites (i.e., the preacylation complex), in each case by a full-length pentapeptide (Supplementary Movie S2). Therefore, PASTA domains are not merely playing a structural role, but also recognizing uncross-linked pentapeptide stems and promoting transpeptidation. This allosteric site is built at the interface of the transpeptidase domain on one side, and the complete P1 and part of the P2 (the first 23 amino acids) on the other. It is worth mentioning that among the five independent combinations of mutations providing β-lactam (cefotaxime) resistance in laboratory mutants,16 two of them (Arg426Cys, Gly422Asp) are located in the core of the allosteric site (Supplementary Figure S2); interestingly, two others, Gly601Val and Gly597Asp, are located in the β5-α11 loop (Supplementary Figure S2), a region that undergoes an important structural rearrangements in the pre-acylation state (our PBP2x:cefepime complex) in order to approach the substrate to the catalytic residue (see below). Furthermore, it was reported that removal of both PASTA domains in PBP2x abolishes β−lactam binding at the active site, and among them, the whole P1 and first residues of P2 were essential. 17 All these previous data are nicely interpreted in the light of our results.

Figure 4. Recognition of the pentapeptide stem at the allosteric and active sites of PBP2x. The energy-minimized preacylation complex of PBP2x (gray surface) with peptidoglycan strand (strand 1, the donor strand, depicted in capped sticks with green for carbons) shows the reach of the D-Ala terminus at the allosteric site of the PASTA domain. The insert on the left, a zoomed-in view of the allosteric site, shows interaction of the peptide terminus to the PBP2x residues. The residue Arg426 is in ‘open’ conformation (see Supplementary Movie 1) allowing direct interaction of the D-Ala terminus carboxylate group with a salt-bridge. A network of residues (labelled) interacts with Arg426 via hydrogen bonds. During the MD simulation of the complex, lysine of the peptide stem formed additional interactions with Glu695 and Asp440, thus demonstrating a pocket ideal for the recognition of a peptidoglycan peptide terminus. The insert of the right, a 60° rotated view of the structure in the surface representation displayed in the background, shows interaction of the peptide terminus at the active site. Hydrogen bonds are displayed as yellow broken lines. The peptide stem at the allosteric and active sites are labeled. In addition, MurNAc residues which attaches these two peptide stems are also labeled. The protein structure is based on the PBP2x:cefepime X-ray structure complex, which showed an extended β3-β4 loop (reported in this work, Figure 2B) and the peptidoglycan model (built as an octasaccharide; see Figure 1A for chemical structure) is based on previously reported solution NMR structure.15

Our fourth X-ray structure for PBP2x was that of the crystals soaked with both oxacillin and the synthetic tetrasaccharide peptidoglycan 5 (Figure 1B). Compound 5 was prepared in 33 steps by a known synthetic method for this study.18 We hoped to capture in the structure of the complex both the active site in its acylated form by oxacillin, as well as have non-covalent complexation by the tetrasaccharide along the surfaces where the peptidoglycan would bind. The X-ray structure revealed the active-site Ser337 modified covalently by oxacillin (a surrogate for acylation by the stem peptide) (Supplementary Figure S1D), however, compound 5 was not seen in the electron density. Nonetheless, interactions between PBP2x and compound 5 were registered in conformational changes onto the protein structure. These conformational changes represent binding event by 5 at the site for the donor peptidoglycan strand (strand 1, the stretch Ser361– Thr381) and for the acceptor peptidoglycan strand (strand 2, the stretch Tyr524–Gly533). These conformational changes were consistent with the models for binding between the two strands of peptidoglycan that emerged (Figure 5, top panel and Supplementary Figure S3). Figure 5. (Upper left) Model of peptidoglycan donor strand (strand 1, atoms in sphere representation), built as an



octasaccharide (n = 3 for compound 2 in Figure 1), bound to PBP2x X-ray structure (gray surface) representing the preacylation complex. Two distinct stem peptides in strand 1 reach the active (at 2 o’clock) and the allosteric sites (at 9 o’clock). (Upper right) The structure was rotated 60° along the vertical axis to show both subsites for binding of the peptidoglycan for the product complex (showing occupancy of both subsites for peptidoglycan binding). The crosslinked stem peptides span the active site (yellow broken circle). The steps in the catalytic process (species I–IV) are as computed by our previous QM/MM calculation. 14

It is important to note that the active site in the apo structure is not sufficiently open to allow entry by the pentapeptide stem. This hindrance is caused by the β3β4 loop spanning residues Gln552–Tyr558, in which Tyr561 adopts a conformation that blocks the approach of the peptidoglycan into the active site (Supplementary Figure S4).6 We have previously reported important structural variations around this region of the active sites in other PBPs. Such variations result in an open active site in PBP1b of S. pneumoniae14 and a closed active site in PBP2a of S. aureus.19 Interestingly, our X-ray structure of PBP2x complex with cefepime bound noncovalently to the active site reveals a conformational motion for the β3-β4 loop, showing it essentially extended (Supplementary Figure S4). This significantly opens the previously occluded region of the active site, enabling the approach of the pentapeptide from the peptidoglycan strand 1. This complex also shows a unique conformation for the β5-α11 loop (Supplementary Figure S4), which enabled approach of the substrate towards the catalytic Ser337 in the center of the active site and simultaneously opening the groove that strand 2 would occupy (Supplementary Figure S4). As indicated above, the program Glide docked the pentapeptide stem to the pre-acylation complex that was computed by QM/MM methods (Figure 5-I) in the X-ray structure of PBP1b, inclusive of the requisite interactions within the oxyanion hole. As the seats of the crosslinking reaction between PBP1b and PBP2x (both from S. pneumoniae) are essentially identical, the subsequent steps of active-site acylation (Figure 5-II), nucleophilic addition by the acceptor strand (Figure 5-III) and the deacylation step leading to the formation of the crosslinked peptidoglycan (Figure 5-IV) are expected to be essentially the same. All these steps in the chemical transformation that leads to crosslinking were studied previously by QM/MM computations for PBP1b14and we consider them relevant for PBP2x as well. As crosslinking of the cell wall takes place (Figure 5IV), the catalytic cycle comes to its conclusion with the two strands of peptidoglycan covalently tethered through the stem peptides. We modeled this species to the active site, guided by structural information from our previous work in transpeptidases. 14, 19 The model for crosslinked peptidoglycan in complex with PBP2x was subjected to molecular-dynamics simulation applying the same protocol described for the pre-acylation complex. We observed concurrent stable interactions of

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the stem peptides at the allosteric and active sites (Figure 5, Supplementary Movie S3). In addition, we note that the sugar backbone of the peptidoglycan strand 2 interacts directly with α9-β3 loop (Tyr524–Gly533). Conformational motion of these residues was observed in our X-ray structures in the presence of compound 5 (Supplementary Figure S3), thus providing support for the model that is depicted in Figure 5. Less than 3% of peptapeptide stem remains in the mature cell wall in S. pneumoniae. 20, 21 22 However, the nascent peptidoglycan, the immediate product of polymerization of Lipid II (Figure 1A), is comprised of all pentapeptide stems, located at the septum where Lipid II flippase is located. 23, 24 25 This provides the requisite peptidoglycan with pentapeptide stems, which is the substrate for the 4,3-crosslinking catalyzed by PBP2x. PBP2x interacts with the aforementioned protein kinase StkP at the division site, though it is not a substrate for it.26 Interestingly, this complex formation between PBP2x and StkP is inhibited in the presence of cellwall fragments.26 As we have described in this report, the existence of allosteric binding site in PBP2x allows recognition of the pentapeptide stems at the first and third MurNAc units within the donor (strand 1) peptidoglycan. Pentapeptide recognition at the allosteric site is likely a key event in distinguishing of a nascent peptidoglycan from the ones that have undergone modification en route to the mature cell wall. This has also important implications in localization of PBP2x to the center of septa through an unknown mechanism, 27 which incidentally has been shown to be dependent on the PASTA domains of PBP2x.28 While still subject of debate, 29 we note that localization of PBP2x is exclusive of the distribution of the DD-carboxypeptidase (PBP3).30 PBP3 catalytically removes the terminal D-Ala from the stem peptide, an activity that moderates the degree of crosslinking by depleting the pentapeptide substrate for the transpeptidases. To have sequential pentapeptide stems (or as in this case at MurNAc postions 1 and 3) is statistically improbable outside of the nascent peptidglycan, and this affords a molecular-recognition mechanism of the nascent peptidoglycan for the crosslinking reaction by PBP2x. Yet, one can also envision that recognition of the pentapeptide at the allosteric site would present an entropic advantage for PBP2x in sequestration of the other pentapeptide stem within the active site for the turnover events. Both these factors would work in concert in selection of the appropriate/suitable peptidoglycan for the crosslinking reaction at the biosynthetic edge of the cell wall of the duplicating bacterium. We have described here an allosteric mechanism for PBP2x, which enables catalysis at the site of Lipid II polymerization. We hasten to add that such facilitation of catalysis by allostery is likely more common in bacteria (and in nature) than the dearth of examples implies. 31 METHODS


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Table 1. Crystallographic data collection and refinement statistics* PBP2x

PBP2x:Cefepime Complex

PBP2x:Oxacillin Complex

PBP2x:Oxacillin:5 Complex

0.97947

0.97947

0.97924

0.97947

Data collection Wavelength (Å) Space group

P312

P312

P312

P312

Unit cell a, b, c (Å)

99.55, 99.55, 189.66

100.42, 100.42, 189.82

100.84, 100.84, 189.35

Unit cell α,β,γ (º)

100.26, 100.26, 189.81 90, 90, 120

90, 90, 120

90, 90, 120

90, 90, 120

T (K)

100

100

100

100

X-ray source

Synchrotron

Synchrotron

Synchrotron

Synchrotron

Resolution range (Å)

34.8–(2.8–2.67)

39.2–(2.8–2.66)

86.79–(2.8–2.7)

48.7–(3.0–2.85)

Unique reflections

31872

31872

30875

26783

Completeness (%)

99.3 (99.1)

99.6 (99.6)

99.7 (99.0)

100.0 (100.0)

Redundancy

3.5 (3.3)

4.2 (4.3)

3.8 (3.8)

6.9 (7.0)

Rpim

0.09 (0.48)

0.12 (0.69)

0.078 (0.67)

0.078 (0.70)

Average I/σ(I) CC1/2

7.5 (1.7)

5.7 (1.4)

8.5 (1.2)

9.3 (1.3)

0.99 (0.76)

1.00 (0.66)

0.99 (0.58)

0.99 (0.58)

Refinement Resolution range (Å)

34.78–2.67

39.24–2.66

48.54–2.70

48.72–2.85

Rwork/Rfree

0.18/0.23

0.19/ 0.23

0.22/0.28

0.17/0.23

No. Atoms Protein

5131

5057

5022

5230

Water

229

212

100

41

Ligand

–

32

61

61

R.m.s. deviations Bond length (Å)

0.016

0.019

0.028

0.015

Bond angles (°)

1.75

1.99

2.59

1.75

Ramachandran Favored/outliers (%) Monomers per AU

92.89/1.36 1

89.72/2.3 1

87.85/ 1.7 1

89.67/2.69 1

PDB code

5OAU

5OJ0

5OIZ

5OJ1

*Values between parentheses correspond to the highest resolution shells

Cloning and purification of PBP2x. The cloning and purification of PBP2x from non-encapsulated Streptococcus pneumoniae RX1 was done as indicated below. To prepare the recombinant PBP2x, the gene encoding PBP2x was fused with a GST tag (pET-GSTPBP2X) and transformed into Escherichia coli Rosetta 2 (DE3) pLysS (BL21). The recombinant protein was purified from bacterial lysate by affinity chromatography using a GST column (Glutathione resin). Subsequently, the GST tag of the protein was digested by enterokinase, the protein was dialyzed and purified by FPLC (HiLoad 16/600 Sephadex 75 prep grade column). The protein purity was checked by SDSPAGE. The final protein was lyophilized. Crystallization. The purified PBP2x was constituted at 10 mg mL–1 (75 mM Tris-HCl, pH 7.8). Good quality crystals were obtained in 0.1 M sodium acetate pH 4.5, 2.3–3.0 M NaCl, using the sitting drop vapour-diffusion technique. Drops were prepared mixing 1 µL of protein

with 1 µL of precipitant solution. Crystals for the complexes were prepared as follows: (i) for the PBP2x:cefepime complex, crystals were soaked in a solution with 20 mM cefepime, 0.1 M sodium acetate pH 4.5, 3.2 M NaCl; (ii) for the PBP2x:oxacillin complex, crystals were soaked in a solution containing 20 mM oxacillin, 0.1 M sodium acetate pH 4.5, 3.2 M NaCl; (iii) for the PBP2x:oxacillin:5 complex crystals were soaked in a solution with 20 mM oxacillin, 20 mM compound 5, 0.1 M sodium acetate pH 4.5, 3.0 M NaCl. All crystals were cryoprotected in 70% (v/v) Paratone/ 30% Paraffine prior to vitrification. All data sets were collected on beamline BL13-XALOC at ALBA Synchrotron (Barcelona, Spain) with 0.25º oscillation range at a resolution of 2.67 Å (PBP2x), 2.66 Å (PBP2x:cefepime), 2.70 Å (PBP2x:oxacillin), and 2.85 Å (PBP2x:oxacillin:5). Data sets were processed using XDS32 and scaled used AIMLESS from CCP433 program suite. PBP2x crystallized in the trigonal P312 space



group, with unit cell parameters a = 100.16 Å, b = 100.16 Å, c = 189.82 Å and one monomer in the asymmetric unit. Structure solution and refinement. Protein complex structures were solved using MOLREP34 with the crystal structure of PBP2x from S. pneumoniae (PDB code 1K25). Datasets were analysed using the XTRIAGE program from PHENIX35 suggesting a small fraction (0.001, 0, 0.019 and 0.019 for apo, PBP2x:cefepime, PBP2x:oxacillin and PBP2x:oxacillin:5 respectively) of pseudomerohedral twinning, the proposed twin law (–h, –k, l) was used for further refinements. Intensive model building was performed with Coot.36 The refinement converged to final good statistics by using PHENIX.35 The possibility of two conformations for the loop comprised of amino acids 527-534 was observed for the PBP2x:oxacillin:5 complex. Refinement, including partial occupancy for both conformations, did not improve the quality of the electron density map. We chose to keep the conformation producing the better electron density map and refinement statistics. Final model quality and validation were assessed using MolProbity.37 Table 1 shows the crystallographic data and the most valuable information regarding refinement statistics for all the structures solved in this work. Enzyme-substrate/product modeling and moleculardynamics simulations. The apo and PBP2x:cefepime Xray structure coordinates reported in this work were prepared for computational calculations. The protein preparation wizard program of the Schrödinger Suite (v 2015, Schrödinger Inc, NY) was used to add hydrogen atoms and bond orders. The missing regions of the X-ray protein coordinates were modeled with the Prime program (v 4.0, Schrödinger Inc, NY). The protein model was energy-minimized with the OPLS2005 forcefield. The octasaccharide coordinates were prepared from our previously reported solution NMR structure of the peptidoglycan.15 The PBP2x:cefepime X-ray structure was used for modeling peptidoglycan strand complexes with PBP2x since this unique structure with unfolded β3-β4 loop showed a groove that would allow sufficient unhindered space to accommodate the peptidoglycan atoms. Initially, the acyl-D-Ala-D-Ala segment of the pentapeptide in the third MurNAc was docked (Glide program, v. 5.6; Schrödinger Inc, NY) to the catalytic site using the information of the acylated X-ray structure (PBP2x:oxacillin complex; present work) by the protocol that we reported previously.19 The remaining regions of the pentapeptide were modeled, assisted by additional molecular docking of the fragment. The symmetry of the previously reported NMR-based 3D-structure of the peptidoglycan cell wall15 guided the reach of another peptide stem, the one at the first MurNAc, towards the allosteric site. The residue Arg426 undergo rotation as evident from the X-ray structure conformation (PDB ID: 1QMF) as well as demonstrated during our MD simulation of the apo X-ray structure (See Supplementary movie 1). Hence, Arg426 was computationally modeled in an open conformation. The modeling of the acyl-d-Ala-D-Ala terminus of the peptide stem at the allosteric site, was accomplished by another molecular docking calculation. The knowledge

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of the β-lactam ring occupancy as demonstrated in the Xray structure of cefuroxime (Figure 1B) bound to the allosteric site of PBP2x (Figure 2B; PDB ID: 1QMF)6 assisted this docking calculation. A similar docking protocol was used to model the crosslinked product on the PBP2x X-ray structure. The binding pose of the second strand (strand 2, the acceptor strand) of the peptidoglycan peptide stem was modeled based on our previous studies,14, 19 while the position of the sugar backbone of the strand was aided by previously reported 3D-structure of the peptidoglycan.15 The complexes were energy-minimized, equilibrated and subjected to molecular-dynamics simulation for 40 ns with PMEMD module of AMBER1613 program applying previously reported protocol. 19 Atomic charges for the octasaccharide were derived quantum-mechanically at HF/6–31G(d) level of theory and were fit with RESP methodology.38 Forcefield parameters were obtained from Amber ff12SB forcefield and general amber forcefield (GAFF).13 Accession codes. PBP2x (5OAU), PBP2x:Cefepime complex (5OJ0), PBP2x:Oxacillin complex (5OIZ), PBP2x:Oxacillin:5 complex (5OJ1).

ASSOCIATED CONTENT Supporting Information Available Methods of cloning and purification, X-ray crystallography, movies and additional figures. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *S.M.: E-mail, [email protected]; Phone, +1–574–631–2933 *J.A.H.: E-mail, [email protected]; Phone, +34–91–745–9538

Author Contributions §The first two authors contributed equally to this work. Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work the NIH grants AI104987 and GM61629 (to SM), by grant BFU2014-59389-P from the Spanish Ministry of Economy and Competitiveness (to JAH) and grants P207/12/1568 and Institutional Research Concept RVO 61388971 (to PB).

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(19) Mahasenan, K. V., Molina, R., Bouley, R., Batuecas, M. T., Fisher, J. F., Hermoso, J. A., Chang, M., and Mobashery, S. (2017) Conformational Dynamics in Penicillin-Binding Protein 2a of Methicillin-Resistant Staphylococcus aureus, Allosteric Communication Network and Enablement of Catalysis. J. Am. Chem. Soc. 139, 2102-2110. (20) Bui, N. K., Eberhardt, A., Vollmer, D., Kern, T., Bougault, C., Tomasz, A., Simorre, J. P., and Vollmer, W. (2012) Isolation and analysis of cell wall components from Streptococcus pneumoniae. Anal. Biochem. 421, 657-666. (21) Garcia-Bustos, J., and Tomasz, A. (1990) A biological price of antibiotic resistance: major changes in the peptidoglycan structure of penicillin-resistant pneumococci. Proc. Natl. Acad. Sci. U. S. A. 87, 5415-5419. (22) Vollmer, W. (2007) Structure and biosynthesis of the pneumococcal cell wall. In Molecular Biology of Streptococci (Hakenbeck, R., Chhatwal, S., Ed.), pp 83-117, Horizon Bioscience, Wymondham, U.K. (23) Typas, A., Banzhaf, M., Gross, C. A., and Vollmer, W. (2011) From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nat. Rev. Microbiol. 10, 123-136. (24) Mohammadi, T., van Dam, V., Sijbrandi, R., Vernet, T., Zapun, A., Bouhss, A., Diepeveen-de Bruin, M., NguyenDisteche, M., de Kruijff, B., and Breukink, E. (2011) Identification of FtsW as a transporter of lipid-linked cell wall precursors across the membrane. EMBO J. 30, 1425-1432. (25) Fraipont, C., Alexeeva, S., Wolf, B., van der Ploeg, R., Schloesser, M., den Blaauwen, T., and Nguyen-Disteche, M. (2011) The integral membrane FtsW protein and peptidoglycan synthase PBP3 form a subcomplex in Escherichia coli. Microbiology 157, 251-259. (26) Morlot, C., Bayle, L., Jacq, M., Fleurie, A., Tourcier, G., Galisson, F., Vernet, T., Grangeasse, C., and Di Guilmi, A. M. (2013) Interaction of Penicillin-Binding Protein 2x and Ser/Thr protein kinase StkP, two key players in Streptococcus pneumoniae R6 morphogenesis. Mol. Microbiol. 90, 88-102. (27) Tsui, H. T., Boersma, M. J., Vella, S. A., Kocaoglu, O., Kuru, E., Peceny, J. K., Carlson, E. E., VanNieuwenhze, M. S., Brun, Y. V., Shaw, S. L., and Winkler, M. E. (2014) Pbp2x localizes separately from Pbp2b and other peptidoglycan synthesis proteins during later stages of cell division of Streptococcus pneumoniae D39. Mol. Microbiol. 94, 21-40. (28) Peters, K., Schweizer, I., Beilharz, K., Stahlmann, C., Veening, J. W., Hakenbeck, R., and Denapaite, D. (2014) Streptococcus pneumoniae PBP2x mid-cell localization requires the C-terminal PASTA domains and is essential for cell shape maintenance. Mol. Microbiol. 92, 733-755. (29) Barendt, S. M., Sham, L. T., and Winkler, M. E. (2011) Characterization of mutants deficient in the L,D-carboxypeptidase (DacB) and WalRK (VicRK) regulon, involved in peptidoglycan maturation of Streptococcus pneumoniae serotype 2 strain D39. J. Bacteriol. 193, 2290-2300. (30) Morlot, C., Noirclerc-Savoye, M., Zapun, A., Dideberg, O., and Vernet, T. (2004) The D,D-carboxypeptidase PBP3 organizes the division process of Streptococcus pneumoniae. Mol. Microbiol. 51, 1641-1648. (31) Meisel, J. E., Fisher, J. F., Chang, M., and Mobashery, S. (2018) Allosteric Inhibition of Bacterial Targets: An Opportunity for Discovery of Novel Antibacterial Classes. In Topics in Medicinal Chemistry (Fisher, J. F., Mobashery, Shahriar, Miller, Marvin J., Ed.), pp 83-117, Springer, Berlin, Heidelberg. (32) Kabsch, W. (2010) Xds. Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 125-132. (33) Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., Keegan, R. M., Krissinel, E. B., Leslie, A. G., McCoy, A., McNicholas, S. J., Murshudov, G. N., Pannu, N. S., Potterton, E. A., Powell, H. R., Read, R. J., Vagin, A., and



Wilson, K. S. (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr., Sect. D: Biol. Crystallogr. 67, 235-242. (34) Vagin, A., and Teplyakov, A. (2010) Molecular replacement with MOLREP. Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 22-25. (35) Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L. W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and Zwart, P. H. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 213-221.

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(36) Emsley, P., and Cowtan, K. (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 60, 2126-2132. (37) Chen, V. B., Arendall, W. B., 3rd, Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S., and Richardson, D. C. (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 12-21. (38) Cornell, W. D., Cieplak, P., Bayly, C. I., and Kollman, P. A. (1993) Application of Resp Charges to Calculate Conformational Energies, Hydrogen-Bond Energies, and Free-Energies of Solvation. J. Am. Chem. Soc. 115, 9620-9631.


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Figure 1. (A) The scheme for the catalytic reaction of PBP2x. The pentapeptide stems interact with the allosteric and active sites of PBP2x. (B) Chemical structures of antibiotics cefuroxime, cefepime and oxacillin highlighting the mimicry of the antibiotic backbone (blue atoms) to the peptide terminus of the natural substrate, the peptidoglycan stem peptide (boxed). The chemical structure of compound 5 is shown. 140x207mm (600 x 600 DPI)



Figure 2. (A) Stereo view of the ribbon representation of the apo structure of PBP2x showing the PASTA (pink) and transpeptidase (green) domains. The two PASTA domains (P1 and P2) are labelled and the position of the catalytic Ser337 is indicated by an arrow (2 o’clock). (B) The surface representation of the Xray structures showing the PASTA (pink) and transpeptidase (green) domains. The previously reported X-ray structure on the left shows binding of two molecules of cefuroxime (spheres); one covalently to the active site and another non-covalently to the allosteric site. The PBP2x:cefepime X-ray structure (present work) is shown on the right, where the allosteric site exists in a closed conformation 151x168mm (300 x 300 DPI)


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Figure 3. The PBP2x active site in the apo, pre-acylated and acylated states. (A) Stereo view showing the catalytic Ser337 oriented towards the core of the protein. (B) Stereo view for the active site of the noncovalent PBP2x:cefepime complex with the antibiotic shown in capped sticks and color coded by atom types (gray for carbon). Polar interactions are represented by dotted lines. (C) Stereo view showing the active site of the covalent PBP2x:oxacillin complex. 118x180mm (300 x 300 DPI)



Figure 4. Recognition of the pentapeptide stem at the allosteric and active sites of PBP2x. The energyminimized pre-acylation complex of PBP2x (gray surface) with peptidoglycan strand (strand 1, the donor strand, depicted in capped sticks with green for carbons) shows the reach of the D-Ala terminus at the allosteric site of the PASTA domain. The insert on the left, a zoomed-in view of the allosteric site, shows interaction of the peptide terminus to the PBP2x residues. The residue Arg426 is in ‘open’ conformation (see Supplementary Movie 1) allowing direct interaction of the D-Ala terminus carboxylate group with a saltbridge. A network of residues (labelled) interacts with Arg426 via hydrogen bonds. During the MD simulation of the complex, lysine of the peptide stem formed additional interactions with Glu695 and Asp440, thus demonstrating a pocket ideal for the recognition of a peptidoglycan peptide terminus. The insert of the right, a 60° rotated view of the structure in the surface representation displayed in the background, shows interaction of the peptide terminus at the active site. Hydrogen bonds are displayed as yellow broken lines. The peptide stem at the allosteric and active sites are labeled. In addition, MurNAc residues which attaches these two peptide stems are also labeled. The protein structure is based on the PBP2x:cefepime X-ray structure complex, which showed an extended β3-β4 loop (reported in this work, Figure 2B) and the peptidoglycan model (built as an octasaccharide; see Figure 1A for chemical structure) is based on previously reported solution NMR structure.15 190x158mm (300 x 300 DPI)


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Figure 5. (Upper left) Model of peptidoglycan donor strand (strand 1, atoms in sphere representation), built as an octasaccharide (n = 3 for compound 2 in Figure 1), bound to PBP2x X-ray structure (gray surface) representing the pre-acylation complex. Two distinct stem peptides in strand 1 reach the active (at 2 o’clock) and the allosteric sites (at 9 o’clock). (Upper right) The structure was rotated 60° along the vertical axis to show both subsites for binding of the peptidoglycan for the product complex (showing occupancy of both subsites for peptidoglycan binding). The crosslinked stem peptides span the active site (yellow broken circle). The steps in the catalytic process (species I–IV) are as computed by our previous QM/MM calculation. 14 84x74mm (600 x 600 DPI)



Table of Contents Figure 70x34mm (300 x 300 DPI)


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Allostery, Recognition of Nascent Peptidoglycan, and Cross-linking of

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