Allosteric cooperation in ß-lactam binding to a non-classical transpeptidase

Mycobacterium tuberculosis peptidoglycan (PG) is atypical as its synthesis involves a new enzyme class, L,D-transpeptidases. Prior studies of L,D-transpeptidases have identified only the catalytic site that binds to peptide moiety of the PG substrate or ß-lactam antibiotics. This insight was leveraged to develop mechanism of its activity and inhibition by ß-lactams. Here we report identification of an allosteric site at a distance of 21 Å from the catalytic site that binds the sugar moiety of PG substrates (hereafter referred to as the S-pocket). This site also binds a second ß-lactam molecule and influences binding at the catalytic site. We provide evidence that two ß-lactam molecules bind co-operatively to this enzyme, one non-covalently at the S-site and one covalently at the catalytic site. This dual ß-lactam binding phenomenon is previously unknown and is an observation that may offer novel approaches for the structure-based design of new ß-lactam antibiotics for M. tuberculosis.


INTRODUCTION 70
Tuberculosis (TB), is a major threat to global health as it claims more human lives 71 than any other bacterial infection (Chakaya et al., 2021). The emergence of multi-(MDR) and

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In the current study, we investigate the interaction of PG substrate and LdtMt2. Using The crystal structure of LdtMt2 was solved at 1.57Å resolution ( Table 1). An electron 161 density was observed in a pocket between the IgD2-YkuD domains, and a glucose molecule 162 could be modeled into the electron density at 1.0 sigma ( Fig. 1A and S1). This glucose 163 molecule is likely to be part of a PG disaccharide moiety originating from the E.coli cell lysate 164 during LdtMt2 purification. The sugar molecule is ensconced at the IgD2-YkuD domain 165 interface in a pocket, which we referred to as the S-pocket, making several electrostatic 166 interactions with residues R209, E168, R371, Y330 and A171 (Fig. 1A). Three residues 167 M157, A171 and L391 stabilize the sugar through hydrophobic interactions. To provide 168 additional evidence for the binding of PG substrates within the S-pocket, we performed 169 ThermoFluor assays with N-Acetylmuramyl-L-alanyl-D-isoglutamine hydrate, a precursor of 170 PG. A higher molar concentration of N-Acetylmuramyl-L-alanyl-D-isoglutamine gradually shifted the melting curve of LdtMt2 indicative of saturable binding behavior. A single R209E 172 mutation in the S-pocket disrupted the binding of N-Acetylmuramyl-L-alanyl-D-isoglutamine 173 with LdtMt2 (Fig. 1B).

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An atomic model of the L,D-transpeptidase from Bacillus subtilis (LdtBS) in complex 175 with nascent PG chain has been reported earlier (PDB ID: 2MTZ) (Schanda et al., 2014).

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Superposition of the structures of the LdtMt2-sugar complex with the LdtBS-PG complex 177 suggests that longer nascent PG chains thread across the S-pocket in between the IgD1-178 YkuD domains of LdtMt2 (Fig. 1C). Based on the structural details of PG binding in LdtBS and

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This inner cavity of the catalytic site is proposed to bind the acceptor tetrapeptide stem, and 184 the outer cavity to bind the donor tetrapeptide stem prior to their 3-3 transpeptide cross-    Table 2). Deletion of the IgD1 domain assessed by the IgD2-YkuD domain 200 fragment resulted in no effect on the ß-lactam hydrolysis. However, deletion of IgD2 from the 201 YkuD domain as assessed by the YkuD fragment alone led to a significant adverse effect on 202 the nitrocefin hydrolysis activity with an increase in the Km value to 129 µM (an ~8-fold 203 increase) and a decline in enzyme turn-over by ~10-fold ( Fig. 2B and

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When we assessed the MD simulated structure of the R209E mutant, no hydrogen 227 bond interaction were found between H336 and the hydroxyl group of the S351 residue; 228 however, the hydrogen bond interaction with carbonyl oxygen of C354 remained conserved.

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Additionally, the hydrogen bond interaction between the H336-Nε1 residue and the carbonyl 230 oxygen of S337 residue was restored. Another W340 residue that resides outside the

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To identify the functional relevance of conformational changes in the catalytic core 238 residues upon MD simulations in both wild-type and R209E mutant structures, we performed in-vitro ß-lactam hydrolysis activity with site-directed mutants of the catalytic triad residues 240 C354, H336 and S337 and other residues namely M303 and S351 that showed significant 241 conformation changes upon R209E mutation in MD experiments . To our surprise, mutation 242 of catalytic triad residue S337 to S337A did not disrupt the ß-lactam hydrolysis activity. The

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S337 residue was earlier reported as an important part of catalytic triad via stabilization of 244 the protonated H336 tautomer (Erdemli et al., 2012). Mutation of C354 to C354A and H336 245 to H336A disrupted ß-lactam hydrolysis as expected. Additionally, mutation of the S351 246 residue to S351A disrupted ß-lactam hydrolysis activity, almost to the same degree as seen 247 in the H336A mutant, with a ~15-fold decrease in enzyme turn-over (Fig. 3B, Table 2). In 248 MD simulation runs with the wild-type structure, the S351 sidechain hydroxyl group forms 249 hydrogen bond interaction with H336-Nε2, and this hydrogen bond interaction is absent in 250 the R209E mutant structure (Fig. 3A)

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As structural changes occur in the catalytic site due to mutations in the S-pocket ( Fig.   300 3), we hypothesized that catalytic site might also demonstrate an interplay with the S-pocket 301 to indirectly influence non-covalent binding of biapenem. Binding studies were performed 302 between biapenem and a catalytic mutant S351A using ThermoFluor assays. Indeed, the 303 S351A mutant showed a significant decrease in its thermal stability (Fig. 4B). Moreover,    Table 3). Ampicillin docked into the S-pocket with a binding energy of -7.1 kcal/mol with its 362 R1-group tail 2-amino-2-phenylacetyl ensconced in the S-pocket through several 363 electrostatic and hydrophobic interactions with the M157, E207, R209, R371 and Y330 364 residues (Fig. 5A). Another penicillin class member, oxacillin (a penicillinase-resistant 365 penicillin), displayed the highest binding energy of -8.3 kcal/mol through its R1 group 5-366 methyl-3-phenyl-1,2-oxazole-4-carbonyl binding in the S-pocket (Fig. S6). Cefotaxime 367 docked to the S-pocket with a binding score of -7.8 kcal/mol through R1-group tail thiozol-4yl 368 (Fig. 5B). The new carbapenem T203 docked to the S-pocket with the least -6.9 kcal/mol 369 binding with its R3 group 2-isopropoxy-2-oxoethyl (Fig. 5C), similar to the biapenem R3 370 group (Fig. 4C). The ß-lactam ring moieties of all of these ß-lactams were found to be free 371 of any interactions with the S-pocket or surrounding residues, similar to biapenem. However, 372 after 18-28 ns of MD simulation trajectory, the pyrrolidine ring of biapenem could make pi-pi 373 interaction with F330 ( Fig. 4C and Fig. S4A), and it is possible that similar late binding 374 interactions may occur similarly with other the ß-lactams.
ThermoFluor assays were also performed to investigate the binding behaviors of 376 these additional ß-lactam class members with LdtMt2. Ampicillin, which has been reported to 377 be readily hydrolyzed by LdtMt2 (Bianchet et al., 2017), showed a saturable binding behavior 378 (Fig. 5A), but to a significantly lower degree than biapenem (Fig. 4B). Surprisingly, with 379 ampicillin the R209E mutation in the S-pocket completely reversed the gradual thermal shift 380 in LdtMt2 towards a higher Tm indicative of an increase in structural stability in the setting of 381 clearly saturable binding (Fig. 5A). We interpret this to be consistent with reversible acylation 382 of the C354 residue by ampicillin in addition to S-pocket binding. In support of this, a

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We conclude that many ß-lactams (despite being weak or strong inhibitors of LtdMt2 activity) 389 bind through the S-pocket with a saturable binding behavior; however, the carbapenem class

Structural basis of allosteric changes between the S-pocket and catalytic site 396
To further understand the high-resolution details of structural changes may that occur 397 in LdtMt2 upon ß-lactam binding, the crystal structure of LdtMt2 was solved in complex with the 398 new carbapenem drug T203 at a 1.7 Å resolution. Electron densities were observed in both 399 the S-pocket and the outer cavity of catalytic pocket in the LdtMt2. Consistent with our docking 400 results of T203 drug with LdtMt2 (Fig. 5C), the 2-oxoethyl side-chain of R3 group from T203 401 could be modelled into the electron density of the S-pocket. A second T203 drug was also 402 modelled into the electron density map of the catalytic pocket. Fig. 6A and 6B show the 2Fo-

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Fc electron density map (contoured at 1.0σ) of T203 modelled in the S-pocket and catalytic 404 site of the LdtMt2 in the crystal structure.

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R371 makes an electrostatic interaction with the oxygen of the 2-oxoethyl moiety. No electron 408 density was observed for the pyrrolidine ring of T203, while its carboxylic group fitted into an electron density making electrostatic interactions with backbone nitrogen of S296 and the 410 guanidium side chain of R371. The modelling results of T203 into the electron density of the 411 S-pocket were similar to the docking results of T203 drug and biapenem that also have their 412 R3 group ensconced into the S-pocket with their pyrrolidine ring remaining free of any 413 interaction with LdtMt2 (Fig. 5).

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In the catalytic site, the T203 carbapenem interacts with the outer cavity at a covalent 415 distance from the S ϒ atom of C354 (Fig. 6B and S7B). The carbonyl oxygen of T203 makes   Figure 6C). In the S-pocket, the M157 side chain moves closer to the drug by 1.5 Å 434 to make a hydrophobic interaction with the 2-oxoethyl tail of T203 drug (Fig. 6D). The R371 435 residue that was making salt bridge with E168 moves towards S296 through a hydrogen 436 bond interaction and makes an additional ionic interaction with the carboxyl group of the T203 437 drug. The Q327 side chain that was previously producing a steric conflict with the carboxyl 438 group of T203 drug moves away by a distance of 1.8 Å to make a water-mediated salt bridge 439 with the hydroxyl group of Y308 that also moves down towards the S-pocket by a distance

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In summary, the evidence supporting the identity of S-pocket as allosteric site 448 includes its distance of 21 Å from the catalytic residue C354, the observation of saturable 449 binding by ß-lactam drugs in addition to covalent binding, and lastly, the substantial 450 conformational alterations between the S-pocket and catalytic site upon ß-lactam binding.

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Bases on our crystal structure and modelling studies, we propose that prior to the 3-

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We find that various ß-lactams bind to the S-pocket of LdtMt2 through their tail regions, 531 either through their R1 or R3 groups. As we found the docking scores of ampicillin, oxacillin, 532 and cefotaxime to be higher than those of carbapenems, the interactions of these R1 or R3

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We thank personnel at Argonne National Laboratory for data collection of protein crystals.