Monitoring Drug-Protein Interactions in the Bacterial Periplasm by Solution Nuclear Magnetic Resonance Spectroscopy.

The rapid spread of antimicrobial resistance across bacterial pathogens poses a serious risk to the efficacy and sustainability of available treatments. This puts pressure on research concerning the development of new drugs. Here, we present an in-cell NMR-based research strategy to monitor the activity of the enzymes located in the periplasmic space delineated by the inner and outer membranes of Gram-negative bacteria. We demonstrate its unprecedented analytical power in monitoring in situ and in real time (i) the hydrolysis of ß-lactams by ß-lactamases, (ii) the interaction of drugs belonging to the ß-lactam family with their essential targets, and (iii) the binding of inhibitors to these enzymes. We show that in-cell NMR provides a powerful analytical tool for investigating new drugs targeting the molecular components of the bacterial periplasm.

The lipopolysaccharide-transporter complex LptB2FG also displays adenylate kinase activity in vitro dependent on the binding partners LptC/LptA.

Lipopolysaccharide (LPS) is an essential glycolipid that covers the surface of gram-negative bacteria. The transport of LPS involves a dedicated seven-protein transporter system called the lipopolysaccharide transport system (Lpt) machinery that physically spans the entire cell envelope. The LptB2FG complex is an ABC transporter that hydrolyzes ATP to extract LPS from the inner membrane for transport to the outer membrane. Here, we extracted LptB2FG directly from the inner membrane with its original lipid environment using styrene-maleic acid polymers. We found that styrene-maleic acid polymers-LptB2FG in nanodiscs display not only ATPase activity but also a previously uncharacterized adenylate kinase (AK) activity, as it catalyzed phosphotransfer between two ADP molecules to generate ATP and AMP. The ATPase and AK activities of LptB2FG were both stimulated by the interaction on the periplasmic side with the periplasmic LPS transport proteins LptC and LptA and inhibited by the presence of the LptC transmembrane helix. We determined that the isolated ATPase module (LptB) had weak AK activity in the absence of transmembrane proteins LptF and LptG, and one mutation in LptB that weakens its affinity for ADP led to AK activity similar to that of fully assembled complex. Thus, we conclude that LptB2FG is capable of producing ATP from ADP, depending on the assembly of the Lpt bridge, and that this AK activity might be important to ensure efficient LPS transport in the fully assembled Lpt system.

Peptidoglycan binding by a pocket on the accessory NTF2-domain of Pgp2 directs helical cell shape of Campylobacter jejuni.

The helical morphology of Campylobacter jejuni, a bacterium involved in host gut colonization and pathogenesis in humans, is determined by the structure of the peptidoglycan (PG) layer. This structure is dictated by trimming of peptide stems by the LD-carboxypeptidase Pgp2 within the periplasm. The interaction interface between Pgp2 and PG to select sites for peptide trimming is unknown. We determined a 1.6 Å resolution crystal structure of Pgp2, which contains a conserved LD-carboxypeptidase domain and a previously uncharacterized domain with an NTF2-like fold (NTF2). We identified a pocket in the NTF2 domain formed by conserved residues and located ∼40 Å from the LD-carboxypeptidase active site. Expression of pgp2 in trans with substitutions of charged (Lys257, Lys307, Glu324) and hydrophobic residues (Phe242 and Tyr233) within the pocket did not restore helical morphology to a pgp2 deletion strain. Muropeptide analysis indicated a decrease of murotripeptides in the deletion strain expressing these mutants, suggesting reduced Pgp2 catalytic activity. Pgp2 but not the K307A mutant was pulled down by C. jejuni Δpgp2 PG sacculi, supporting a role for the pocket in PG binding. NMR spectroscopy was used to define the interaction interfaces of Pgp2 with several PG fragments, which bound to the active site within the LD-carboxypeptidase domain and the pocket of the NTF2 domain. We propose a model for Pgp2 binding to PG strands involving both the LD-carboxypeptidase domain and the accessory NTF2 domain to induce a helical cell shape.

Crystallographic analysis of Staphylococcus aureus LcpA, the primary wall teichoic acid ligase.

Gram-positive bacteria, including major clinical pathogens such as Staphylococcus aureus, are becoming increasingly drug-resistant. Their cell walls are composed of a thick layer of peptidoglycan (PG) modified by the attachment of wall teichoic acid (WTA), an anionic glycopolymer that is linked to pathogenicity and regulation of cell division and PG synthesis. The transfer of WTA from lipid carriers to PG, catalyzed by the LytR-CpsA-Psr (LCP) enzyme family, offers a unique extracellular target for the development of new anti-infective agents. Inhibitors of LCP enzymes have the potential to manage a wide range of bacterial infections because the target enzymes are implicated in the assembly of many other bacterial cell wall polymers, including capsular polysaccharide of streptococcal species and arabinogalactan of mycobacterial species. In this study, we present the first crystal structure of S. aureus LcpA with bound substrate at 1.9 Å resolution and those of Bacillus subtilis LCP enzymes, TagT, TagU, and TagV, in the apo form at 1.6-2.8 Å resolution. The structures of these WTA transferases provide new insight into the binding of lipid-linked WTA and enable assignment of the catalytic roles of conserved active-site residues. Furthermore, we identified potential subsites for binding the saccharide core of PG using computational docking experiments, and multiangle light-scattering experiments disclosed novel oligomeric states of the LCP enzymes. The crystal structures and modeled substrate-bound complexes of the LCP enzymes reported here provide insights into key features linked to substrate binding and catalysis and may aid the structure-guided design of specific LCP inhibitors.

Deciphering the structural attributes of protein-heparan sulfate interactions using chemo-enzymatic approaches and NMR spectroscopy.

Heparan sulfates (HS) is a polysaccharide found at the cell surface, where it mediates interactions with hundreds of proteins and regulates major pathophysiological processes. HS is highly heterogeneous and structurally complex and examples that define their structure-activity relationships remain limited. Here, in order to characterize a protein-HS interface and define the corresponding saccharide-binding domain, we present a chemo-enzymatic approach that generates 13C-labeled HS-based oligosaccharide structures. Nuclear magnetic resonance (NMR) spectroscopy, which efficiently discriminates between important or redundant chemical groups in the oligosaccharides, is employed to characterize these molecules alone and in interaction with proteins. Using chemokines as model system, docking based on NMR data on both proteins and oligosaccharides enable the identification of the structural determinant involved in the complex. This study shows that both the position of the sulfo groups along the chain and their mode of presentation, rather than their overall number, are key determinant and further points out the usefulness of these 13C-labeled oligosaccharides in obtaining detailed structural information on HS-protein complexes.

Studying intact bacterial peptidoglycan by proton-detected NMR spectroscopy at 100 kHz MAS frequency.

The bacterial cell wall is composed of the peptidoglycan (PG), a large polymer that maintains the integrity of the bacterial cell. Due to its multi-gigadalton size, heterogeneity, and dynamics, atomic-resolution studies are inherently complex. Solid-state NMR is an important technique to gain insight into its structure, dynamics and interactions. Here, we explore the possibilities to study the PG with ultra-fast (100 kHz) magic-angle spinning NMR. We demonstrate that highly resolved spectra can be obtained, and show strategies to obtain site-specific resonance assignments and distance information. We also explore the use of proton-proton correlation experiments, thus opening the way for NMR studies of intact cell walls without the need for isotope labeling.

Induced conformational changes activate the peptidoglycan synthase PBP1B.

Bacteria surround their cytoplasmic membrane with an essential, stress-bearing peptidoglycan (PG) layer consisting of glycan chains linked by short peptides into a mesh-like structure. Growing and dividing cells expand their PG layer using inner-membrane anchored PG synthases, including Penicillin-binding proteins (PBPs), which participate in dynamic protein complexes to facilitate cell wall growth. In Escherichia coli, and presumably other Gram-negative bacteria, growth of the mainly single layered PG is regulated by outer membrane-anchored lipoproteins. The lipoprotein LpoB is required to activate PBP1B, which is a major, bi-functional PG synthase with glycan chain polymerising (glycosyltransferase) and peptide cross-linking (transpeptidase) activities. In this work we show how the binding of LpoB to the regulatory UB2H domain of PBP1B activates both activities. Binding induces structural changes in the UB2H domain, which transduce to the two catalytic domains by distinct allosteric pathways. We also show how an additional regulator protein, CpoB, is able to selectively modulate the TPase activation by LpoB without interfering with GTase activation.

Negative Impact of Carbapenem Methylation on the Reactivity of ß-Lactams for Cysteine Acylation as Revealed by Quantum Calculations and Kinetic Analyses.

The Enterococcus faecium l,d-transpeptidase (Ldtfm) mediates resistance to most ß-lactam antibiotics in this bacterium by replacing classical peptidoglycan polymerases. The catalytic Cys of Ldtfm is rapidly acylated by ß-lactams belonging to the carbapenem class but not by penams or cephems. We previously reported quantum calculations and kinetic analyses for Ldtfm and showed that the inactivation profile is not determined by differences in drug binding (KD [equilibrium dissociation constant] values in the 50 to 80 mM range). In this study, we analyzed the reaction of a Cys sulfhydryl with various ß-lactams in the absence of the enzyme environment in order to compare the intrinsic reactivity of drugs belonging to the penam, cephem, and carbapenem classes. For this purpose, we synthesized cyclic Cys-Asn (cCys-Asn) to generate a soluble molecule with a sulfhydryl closely mimicking a cysteine in a polypeptide chain, thereby avoiding free reactive amino and carboxyl groups. Computational studies identified a thermodynamically favored pathway involving a concerted rupture of the ß-lactam amide bond and formation of an amine anion. Energy barriers indicated that the drug reactivity was the highest for nonmethylated carbapenems, intermediate for methylated carbapenems and cephems, and the lowest for penams. Electron-withdrawing groups were key reactivity determinants by enabling delocalization of the negative charge of the amine anion. Acylation rates of cCys-Asn determined by spectrophotometry revealed the same order in the reactivity of ß-lactams. We concluded that the rate of Ldtfm acylation is largely determined by the ß-lactam reactivity with one exception, as the enzyme catalytic pocket fully compensated for the detrimental effect of carbapenem methylation.

Human Macrophage Galactose-Type Lectin (MGL) Recognizes the Outer Core of Escherichia coli Lipooligosaccharide.

Carbohydrate-lectin interactions intervene in and mediate most biological processes, including a crucial modulation of immune responses to pathogens. Despite growing interest in investigating the association between host receptor lectins and exogenous glycan ligands, the molecular mechanisms underlying bacterial recognition by human lectins are still not fully understood. Herein, a novel molecular interaction between the human macrophage galactose-type lectin (MGL) and the lipooligosaccharide (LOS) of Escherichia coli strain R1 is described. Saturation transfer difference NMR spectroscopy analysis, supported by computational studies, demonstrated that MGL bound to the purified deacylated LOSR1 mainly through recognition of its outer core and established crucial interactions with the terminal Galα(1,2)Gal epitope. These results assess the ability of MGL to recognise glycan moieties exposed on Gram-negative bacterial surfaces.

Outer-membrane lipoprotein LpoB spans the periplasm to stimulate the peptidoglycan synthase PBP1B.

Bacteria surround their cytoplasmic membrane with an essential, stress-bearing peptidoglycan (PG) layer. Growing and dividing cells expand their PG layer by using membrane-anchored PG synthases, which are guided by dynamic cytoskeletal elements. In Escherichia coli, growth of the mainly single-layered PG is also regulated by outer membrane-anchored lipoproteins. The lipoprotein LpoB is required for the activation of penicillin-binding protein (PBP) 1B, which is a major, bifunctional PG synthase with glycan chain polymerizing (glycosyltransferase) and peptide cross-linking (transpeptidase) activities. Here, we report the structure of LpoB, determined by NMR spectroscopy, showing an N-terminal, 54-aa-long flexible stretch followed by a globular domain with similarity to the N-terminal domain of the prevalent periplasmic protein TolB. We have identified the interaction interface between the globular domain of LpoB and the noncatalytic UvrB domain 2 homolog domain of PBP1B and modeled the complex. Amino acid exchanges within this interface weaken the PBP1B-LpoB interaction, decrease the PBP1B stimulation in vitro, and impair its function in vivo. On the contrary, the N-terminal flexible stretch of LpoB is required to stimulate PBP1B in vivo, but is dispensable in vitro. This supports a model in which LpoB spans the periplasm to interact with PBP1B and stimulate PG synthesis.

Acyl acceptor recognition by Enterococcus faecium L,D-transpeptidase Ldtfm.

In Mycobacterium tuberculosis and ampicillin-resistant mutants of Enterococcus faecium, the classical target of ß-lactam antibiotics is bypassed by L,D-transpeptidases that form unusual 3 → 3 peptidoglycan cross-links. ß-lactams of the carbapenem class, such as ertapenem, are mimics of the acyl donor substrate and inactivate l,d-transpeptidases by acylation of their catalytic cysteine. We have blocked the acyl donor site of E. faecium L,D-transpeptidase Ldt(fm) by ertapenem and identified the acyl acceptor site based on analyses of chemical shift perturbations induced by binding of peptidoglycan fragments to the resulting acylenzyme. An nuclear magnetic resonance (NMR)-driven docking structure of the complex revealed key hydrogen interactions between the acyl acceptor and Ldt(fm) that were evaluated by site-directed mutagenesis and development of a cross-linking assay. Three residues are reported as critical for stabilisation of the acceptor in the Ldt(fm) active site and proper orientation of the nucleophilic nitrogen for the attack of the acylenzyme carbonyl. Identification of the catalytic pocket dedicated to the acceptor substrate opens new perspectives for the design of inhibitors with an original mode of action that could act alone or in synergy with ß-lactams.

Membrane and chaperone recognition by the major translocator protein PopB of the type III secretion system of Pseudomonas aeruginosa.

The type III secretion system is a widespread apparatus used by pathogenic bacteria to inject effectors directly into the cytoplasm of eukaryotic cells. A key component of this highly conserved system is the translocon, a pore formed in the host membrane that is essential for toxins to bypass this last physical barrier. In Pseudomonas aeruginosa the translocon is composed of PopB and PopD, both of which before secretion are stabilized within the bacterial cytoplasm by a common chaperone, PcrH. In this work we characterize PopB, the major translocator, in both membrane-associated and PcrH-bound forms. By combining sucrose gradient centrifugation experiments, limited proteolysis, one-dimensional NMR, and ß-lactamase reporter assays on eukaryotic cells, we show that PopB is stably inserted into bilayers with its flexible N-terminal domain and C-terminal tail exposed to the outside. In addition, we also report the crystal structure of the complex between PcrH and an N-terminal region of PopB (residues 51-59), which reveals that PopB lies within the concave face of PcrH, employing mostly backbone residues for contact. PcrH is thus the first chaperone whose structure has been solved in complex with both type III secretion systems translocators, revealing that both molecules employ the same surface for binding and excluding the possibility of formation of a ternary complex. The characterization of the major type III secretion system translocon component in both membrane-bound and chaperone-bound forms is a key step for the eventual development of antibacterials that block translocon assembly.

Profiling sulfation/epimerization pattern of full-length heparan sulfate by NMR following cell culture 13C-glucose metabolic labeling.

Through its ability to interact with proteins, heparan sulfate (HS) fulfills a large variety of functions. Protein binding depends on the level of HS sulfation and epimerization which are cell specific and dynamically regulated. Characterization of this molecule, however, has been restricted to oligosaccharide fragments available in large amount for structural investigation or to sulfate distribution through compositional analysis. Here we developed a (1)H-(13)C 2D NMR-based approach, directly performed on HS isolated from (13)C-labeled cells. By integrating the peak volumes measured at different chemical shifts, this non-destructive analysis allows us to determine both the sulfation and the iduronic/glucuronic profiles of the polysaccharide. Applied to wild-type and N-deacetylase/N-sulfotransferase-deficient fibroblasts as well as to epithelial cells differentiation, it also gives insights into the functional relationships existing between HS biosynthetic enzymes. This approach should be of significant interest to better understand HS changes that occur through physiologic regulations or during pathological development.

A gp41 MPER-specific llama VHH requires a hydrophobic CDR3 for neutralization but not for antigen recognition.

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10.

Atomic model of a cell-wall cross-linking enzyme in complex with an intact bacterial peptidoglycan.

The maintenance of bacterial cell shape and integrity is largely attributed to peptidoglycan, a highly cross-linked biopolymer. The transpeptidases that perform this cross-linking are important targets for antibiotics. Despite this biomedical importance, to date no structure of a protein in complex with an intact bacterial peptidoglycan has been resolved, primarily due to the large size and flexibility of peptidoglycan sacculi. Here we use solid-state NMR spectroscopy to derive for the first time an atomic model of an l,d-transpeptidase from Bacillus subtilis bound to its natural substrate, the intact B. subtilis peptidoglycan. Importantly, the model obtained from protein chemical shift perturbation data shows that both domains-the catalytic domain as well as the proposed peptidoglycan recognition domain-are important for the interaction and reveals a novel binding motif that involves residues outside of the classical enzymatic pocket. Experiments on mutants and truncated protein constructs independently confirm the binding site and the implication of both domains. Through measurements of dipolar-coupling derived order parameters of bond motion we show that protein binding reduces the flexibility of peptidoglycan. This first report of an atomic model of a protein-peptidoglycan complex paves the way for the design of new antibiotic drugs targeting l,d-transpeptidases. The strategy developed here can be extended to the study of a large variety of enzymes involved in peptidoglycan morphogenesis.

A peptidoglycan fragment triggers ß-lactam resistance in Bacillus licheniformis.

To resist to ß-lactam antibiotics Eubacteria either constitutively synthesize a ß-lactamase or a low affinity penicillin-binding protein target, or induce its synthesis in response to the presence of antibiotic outside the cell. In Bacillus licheniformis and Staphylococcus aureus, a membrane-bound penicillin receptor (BlaR/MecR) detects the presence of ß-lactam and launches a cytoplasmic signal leading to the inactivation of BlaI/MecI repressor, and the synthesis of a ß-lactamase or a low affinity target. We identified a dipeptide, resulting from the peptidoglycan turnover and present in bacterial cytoplasm, which is able to directly bind to the BlaI/MecI repressor and to destabilize the BlaI/MecI-DNA complex. We propose a general model, in which the acylation of BlaR/MecR receptor and the cellular stress induced by the antibiotic, are both necessary to generate a cell wall-derived coactivator responsible for the expression of an inducible ß-lactam-resistance factor. The new model proposed confirms and emphasizes the role of peptidoglycan degradation fragments in bacterial cell regulation.

MAS NMR experiments of corynebacterial cell walls: Complementary 1H- and CPMAS CryoProbe-enhanced 13C-detected experiments.

Bacterial cell walls are gigadalton-large cross-linked polymers with a wide range of motional amplitudes, including rather rigid as well as highly flexible parts. Magic-angle spinning NMR is a powerful method to obtain atomic-level information about intact cell walls. Here we investigate sensitivity and information content of different homonuclear 13C13C and heteronuclear 1H15N, 1H13C and 15N13C correlation experiments. We demonstrate that a CPMAS CryoProbe yields ca. 8-fold increased signal-to-noise over a room-temperature probe, or a ca. 3-4-fold larger per-mass sensitivity. The increased sensitivity allowed to obtain high-resolution spectra even on intact bacteria. Moreover, we compare resolution and sensitivity of 1H MAS experiments obtained at 100 kHz vs. 55 kHz. Our study provides useful hints for choosing experiments to extract atomic-level details on cell-wall samples.

Molecular recognition of Escherichia coli R1-type core lipooligosaccharide by DC-SIGN.

Due to their ability to recognize carbohydrate structures, lectins emerged as potential receptors for bacterial lipopolysaccharides (LPS). Despite growing interest in investigating the association between host receptor lectins and exogenous glycan ligands, the molecular mechanisms underlying bacterial recognition by human lectins are still not fully understood. We contributed to fill this gap by unveiling the molecular basis of the interaction between the lipooligosaccharide of Escherichia coli and the dendritic cell-specific intracellular adhesion molecules (ICAM)-3 grabbing non-integrin (DC-SIGN). Specifically, a combination of different techniques, including fluorescence microscopy, surface plasmon resonance, NMR spectroscopy, and computational studies, demonstrated that DC-SIGN binds to the purified deacylated R1 lipooligosaccharide mainly through the recognition of its outer core pentasaccharide, which acts as a crosslinker between two different tetrameric units of DC-SIGN. Our results contribute to a better understanding of DC-SIGN-LPS interaction and may support the development of pharmacological and immunostimulatory strategies for bacterial infections, prevention, and therapy.

Solid-state NMR on bacterial cells: selective cell wall signal enhancement and resolution improvement using dynamic nuclear polarization.

Dynamic nuclear polarization (DNP) enhanced solid-state nuclear magnetic resonance (NMR) has recently emerged as a powerful technique for the study of material surfaces. In this study, we demonstrate its potential to investigate cell surface in intact cells. Using Bacillus subtilis bacterial cells as an example, it is shown that the polarizing agent 1-(TEMPO-4-oxy)-3-(TEMPO-4-amino)propan-2-ol (TOTAPOL) has a strong binding affinity to cell wall polymers (peptidoglycan). This particular interaction is thoroughly investigated with a systematic study on extracted cell wall materials, disrupted cells, and entire cells, which proved that TOTAPOL is mainly accumulating in the cell wall. This property is used on one hand to selectively enhance or suppress cell wall signals by controlling radical concentrations and on the other hand to improve spectral resolution by means of a difference spectrum. Comparing DNP-enhanced and conventional solid-state NMR, an absolute sensitivity ratio of 24 was obtained on the entire cell sample. This important increase in sensitivity together with the possibility of enhancing specifically cell wall signals and improving resolution really opens new avenues for the use of DNP-enhanced solid-state NMR as an on-cell investigation tool.

Staphylococcus aureus sacculus mediates activities of M23 hydrolases.

Peptidoglycan, a gigadalton polymer, functions as the scaffold for bacterial cell walls and provides cell integrity. Peptidoglycan is remodelled by a large and diverse group of peptidoglycan hydrolases, which control bacterial cell growth and division. Over the years, many studies have focused on these enzymes, but knowledge on their action within peptidoglycan mesh from a molecular basis is scarce. Here, we provide structural insights into the interaction between short peptidoglycan fragments and the entire sacculus with two evolutionarily related peptidases of the M23 family, lysostaphin and LytM. Through nuclear magnetic resonance, mass spectrometry, information-driven modelling, site-directed mutagenesis and biochemical approaches, we propose a model in which peptidoglycan cross-linking affects the activity, selectivity and specificity of these two structurally related enzymes differently.