Integrative structural biology of the penicillin-binding protein-1 from Staphylococcus aureus, an essential component of the divisome machinery

14 pags., 6 figs., 2 tabs.The penicillin-binding proteins are the enzyme catalysts of the critical transpeptidation crosslinking polymerization reaction of bacterial peptidoglycan synthesis and the molecular targets of the penicillin antibiotics. Here, we report a combined crystallographic, small-angle X-ray scattering (SAXS) in-solution structure, computational and biophysical analysis of PBP1 of Staphylococcus aureus (saPBP1), providing mechanistic clues about its function and regulation during cell division. The structure reveals the pedestal domain, the transpeptidase domain, and most of the linker connecting to the “penicillin-binding protein and serine/threonine kinase associated” (PASTA) domains, but not its two PASTA domains, despite their presence in the construct. To address this absence, the structure of the PASTA domains was determined at 1.5 Å resolution. Extensive molecular-dynamics simulations interpret the PASTA domains of saPBP1 as conformationally mobile and separated from the transpeptidase domain. This conclusion was confirmed by SAXS experiments on the full-length protein in solution. A series of crystallographic complexes with β-lactam antibiotics (as inhibitors) and penta-Gly (as a substrate mimetic) allowed the molecular characterization of both inhibition by antibiotics and binding for the donor and acceptor peptidoglycan strands. Mass-spectrometry experiments with synthetic peptidoglycan fragments revealed binding by PASTA domains in coordination with the remaining domains. The observed mobility of the PASTA domain in saPBP1 could play a crucial role for in vivo interaction with its glycosyltransferase partner in the membrane or with other components of the divisome machinery, as well as for coordination of transpeptidation and polymerization processes in the bacterial divisome.The work in the USA was supported by grants AI104987 (to SM)and AI116548 (to MC) from the NIH. The work in Spain was fundedby a grant from the Spanish Ministry of Science, Innovation and Competitiveness (BFU2017-90030-P and PID2020-115331GB-100 to JAH). We thank the staff from ALBA and Diamond Light Sourcesynchrotrons for help during X-ray and SAXS data collection,respectivel

Molecular basis of the final step of cell division in Streptococcus pneumoniae

Bacterial cell-wall hydrolases must be tightly regulated during bacterial cell division to prevent aberrant cell lysis and to allow final separation of viable daughter cells. In a multidisciplinary work, we disclose the molecular dialogue between the cell-wall hydrolase LytB, wall teichoic acids, and the eukaryotic-like protein kinase StkP in Streptococcus pneumoniae. After characterizing the peptidoglycan recognition mode by the catalytic domain of LytB, we further demonstrate that LytB possesses a modular organization allowing the specific binding to wall teichoic acids and to the protein kinase StkP. Structural and cellular studies notably reveal that the temporal and spatial localization of LytB is governed by the interaction between specific modules of LytB and the final PASTA domain of StkP. Our data collectively provide a comprehensive understanding of how LytB performs final separation of daughter cells and highlights the regulatory role of eukaryotic-like kinases on lytic machineries in the last step of cell division in streptococci.We thank the staff from the ALBA synchrotron facilities for their help during crystallographic data collection. We gratefully thank Pedro Garcia (CIB, Madrid, Spain) for providing us with the plasmid allowing overproduction of GFP-LytB. This work was supported by grants from the CNRS, the University of Lyon, the Agence National de la Recherche (ANR-18-CE11-0017-02 and ANR-19-CE15-0011-01), and the Bettencourt Schueller Foundation to C.G. The work in Spain was supported by grants BFU2017-90030-P and PID2020-115331GB-100 to J.A.H., funded by MCIN/AEI/10.13039/501100011033. The work in the United States was supported by a grant from the National Institutes of Health (GM131685). J.A.H. and C.G. supervised this work and share last authorship.Peer reviewe

Novel Inhibitor Discovery through Virtual Screening against Multiple Protein Conformations Generated via Ligand-Directed Modeling: A Maternal Embryonic Leucine Zipper Kinase Example

Kinase targets have been demonstrated to undergo major conformational reorganization upon ligand binding. Such protein conformational plasticity remains a significant challenge in structure-based virtual screening methodology and may be approximated by screening against an ensemble of diverse protein conformations. Maternal embryonic leucine zipper kinase (MELK), a member of serine-threonine kinase family, has been recently found to be involved in the tumerogenic state of glioblastoma, breast, ovarian, and colon cancers. We therefore modeled several conformers of MELK utilizing the available chemogenomic and crystallographic data of homologous kinases. We carried out docking pose prediction and virtual screening enrichment studies with these conformers. The performances of the ensembles were evaluated by their ability to reproduce known inhibitor bioactive conformations and to efficiently recover known active compounds early in the virtual screen when seeded with decoy sets. A few of the individual MELK conformers performed satisfactorily in reproducing the native protein–ligand pharmacophoric interactions up to 50% of the cases. By selecting an ensemble of a few representative conformational states, most of the known inhibitor binding poses could be rationalized. For example, a four conformer ensemble is able to recover 95% of the studied actives, especially with imperfect scoring function(s). The virtual screening enrichment varied considerably among different MELK conformers. Enrichment appears to improve by selection of a proper protein conformation. For example, several holo and unliganded active conformations are better to accommodate diverse chemotypes than ATP-bound conformer. These results prove that using an ensemble of diverse conformations could give a better performance. Applying this approach, we were able to screen a commercially available library of half a million compounds against three conformers to discover three novel inhibitors of MELK, one from each template. Among the three compounds validated via experimental enzyme inhibition assays, one is relatively potent (<b>15</b>; K_d = 0.37 μM), one moderately active (<b>12</b>; K_d = 3.2 μM), and one weak but very selective (<b>9</b>; K_d = 18 μM). These novel hits may be utilized to assist in the development of small molecule therapeutic agents useful in diseases caused by deregulated MELK, and perhaps more importantly, the approach demonstrates the advantages of choosing an appropriate ensemble of a few conformers in pursuing compound potency, selectivity, and novel chemotypes over using single target conformation for structure-based drug design in general

Unconventional antibacterials and adjuvants

13 pags., 7 figs., 5 schs.-- Published as part of the Accounts of Chemical Research special issue “Bacterial Multi-Drug Resistance”.ConspectusThe need for new classes of antibacterials is genuine in light of the dearth of clinical options for the treatment of bacterial infections. The prodigious discoveries of antibiotics during the 1940s to 1970s, a period wistfully referred to as the Golden Age of Antibiotics, have not kept up in the face of emergence of resistant bacteria in the past few decades. There has been a renewed interest in old drugs, the repurposing of the existing antibiotics and pairing of synergistic antibiotics or of an antibiotic with an adjuvant. Notwithstanding, discoveries of novel classes of these life-saving drugs have become increasingly difficult, calling for new paradigms. We describe, herein, three strategies from our laboratories toward discoveries of new antibacterials and adjuvants using computational and multidisciplinary experimental methods. One approach targets penicillin-binding proteins (PBPs), biosynthetic enzymes of cell-wall peptidoglycan, for discoveries of non-β-lactam inhibitors. Oxadiazoles and quinazolinones emerged as two structural classes out of these efforts. Several hundred analogs of these two classes of antibiotics have been synthesized and fully characterized in our laboratories. A second approach ventures into inhibition of allosteric regulation of cell-wall biosynthesis. The mechanistic details of allosteric regulation of PBP2a of Staphylococcus aureus, discovered in our laboratories, is outlined. The allosteric site in this protein is at 60 Å distance to the active site, whereby ligand binding at the former makes access to the latter by the substrate possible. We have documented that both quinazolinones and ceftaroline, a fifth-generation cephalosporin, bind to the allosteric site in manifestation of the antibacterial activity. Attempts at inhibition of the regulatory phosphorylation events identified three classes of antibacterial adjuvants and one class of antibacterials, the picolinamides. The chemical structures for these hits went through diversification by synthesis of hundreds of analogs. These analogs were characterized in various assays for identification of leads with adjuvant and antibacterial activities. Furthermore, we revisited the mechanism of bulgecins, a class of adjuvants discovered and abandoned in the 1980s. These compounds potentiate the activities of β-lactam antibiotics by the formation of bulges at the sites of septum formation during bacterial replication, which are points of structural weakness in the envelope. These bulges experience rupture, which leads to bacterial death. Bulgecin A inhibits the lytic transglycosylase Slt of Pseudomonas aeruginosa as a likely transition-state mimetic for its turnover of the cell-wall peptidoglycan. Once damage to cell wall is inflicted by a β-lactam antibiotic, the function of Slt is to repair the damage. When Slt is inhibited by bulgecin A, the organism cannot cope with it and would undergo rapid lysis. Bulgecin A is an effective adjuvant of β-lactam antibiotics. These discoveries of small-molecule classes of antibacterials or of adjuvants to antibacterials hold promise in strategies for treatment of bacterial infections.This work was supported by NIH grants GM131685, AI104987, AI148217 (to S.M.), AI116548, AI90818 (to M.C.) and by a grant from the Spanish Ministry of Science and Innovation BFU2017-90030-P (to J.A.H.)

Catalytic Cycle of Glycoside Hydrolase BglX from Pseudomonas aeruginosa and Its Implications for Biofilm Formation

8 pags., 5 figs.BglX is a heretofore uncharacterized periplasmic glycoside hydrolase (GH) of the human pathogen Pseudomonas aeruginosa. X-ray analysis identifies it as a protein homodimer. The two active sites of the homodimer comprise catalytic residues provided by each monomer. This arrangement is seen in <2% of the hydrolases of known structure. In vitro substrate profiling shows BglX is a catalyst for β-(1→2) and β-(1→3) saccharide hydrolysis. Saccharides with β-(1→4) or β-(1→6) bonds, and the β-(1→4) muropeptides from the cell-wall peptidoglycan, are not substrates. Additional structural insights from X-ray analysis (including structures of a mutant enzyme-derived Michaelis complex, two transition-state mimetics, and two enzyme-product complexes) enabled the comprehensive description of BglX catalysis. The half-chair (H) conformation of the transition-state oxocarbenium species, the approach of the hydrolytic water molecule to the oxocarbenium species, and the stepwise release of the two reaction products were also visualized. The substrate pattern for BglX aligns with the [β-(1→2)-Glc] and [β-(1→3)-Glc] periplasmic osmoregulated periplasmic glucans, and possibly with the Psl exopolysaccharides, of P. aeruginosa. Both polysaccharides are implicated in biofilm formation. Accordingly, we show that inactivation of the bglX gene of P. aeruginosa PAO1 attenuates biofilm formation.The work at the University of Notre Dame was supported by grants from the National Institutes of Health (GM61629 and GM131685), and that in Spain by a grant from MICIU Ministry (BFU2017-90030-P). The authors thank the staff from the ALBA (Barcelona, Spain) synchrotron facility for help in X-ray data collection and CRC of the University of Notre Dame for the computing resources. The authors acknowledge Grant P30 DK089507 from the National Institutes of Health for the BglX transposon mutant of P. aeruginosa

Turnover of Bacterial Cell Wall by SltB3, a Multidomain Lytic Transglycosylase of <i>Pseudomonas aeruginosa</i>

A family of 11 lytic transglycosylases in <i>Pseudomonas aeruginosa</i>, an opportunistic human pathogen, turn over the polymeric bacterial cell wall in the course of its recycling, repair, and maturation. The functions of these enzymes are not fully understood. We disclose herein that SltB3 of <i>P. aeruginosa</i> is an exolytic lytic transglycosylase. We characterize its reaction and its products by the use of peptidoglycan-based molecules. The enzyme recognizes a minimum of four sugars in its substrate but can process a substrate comprised of a peptidoglycan of 20 sugars. The ultimate product of the reaction is <i>N-</i>acetylglucosamine-1,6-anhydro-<i>N</i>-acetylmuramic acid. The X-ray structure of this enzyme is reported for the first time. The enzyme is comprised of four domains, arranged within an annular conformation. The polymeric linear peptidoglycan substrate threads through the opening of the annulus, as it experiences turnover