Defining the Familial Fold of the Vicilin-Buried Peptide Family.

Plants and their seeds have been shown to be a rich source of cystine-stabilized peptides. Recently a new family of plant seed peptides whose sequences are buried within precursors for seed storage vicilins was identified. Members of this Vicilin-Buried Peptide (VBP) family are found in distantly related plant species including the monocot date palm, as well as dicotyledonous species like pumpkin and sesame. Genetic evidence for their widespread occurrence indicates that they are of ancient origin. Limited structural studies have been conducted on VBP family members, but two members have been shown to adopt a helical hairpin fold. We present an extensive characterization of VBPs using solution NMR spectroscopy, to better understand their structural features. Four peptides were produced by solid phase peptide synthesis and shown to favor a helix-loop-helix hairpin fold, as a result of the I-IV/II-III ladderlike connectivity of their disulfide bonds. Interhelical interactions, including hydrophobic contacts and salt bridges, are critical for the fold stability and control the angle at which the antiparallel α-helices interface. Activities reported for VBPs include trypsin inhibitory activity and inhibition of ribosomal function; however, their diverse structural features despite a common fold suggest that additional bioactivities yet to be revealed are likely.

Molecular Basis for the Potent Inhibition of the Emerging Carbapenemase VCC-1 by Avibactam.

In 2016, we identified a new class A carbapenemase, VCC-1, in a nontoxigenic Vibrio cholerae strain that had been isolated from retail shrimp imported into Canada for human consumption. Shortly thereafter, seven additional VCC-1-producing V. cholerae isolates were recovered along the German coastline. These isolates appear to have acquired the VCC-1 gene (blaVCC-1) independently from the Canadian isolate, suggesting that blaVCC-1 is mobile and widely distributed. VCC-1 hydrolyzes penicillins, cephalothin, aztreonam, and carbapenems and, like the broadly disseminated class A carbapenemase KPC-2, is only weakly inhibited by clavulanic acid or tazobactam. Although VCC-1 has yet to be observed in the clinic, its encroachment into aquaculture and other areas with human activity suggests that the enzyme may be emerging as a public health threat. To preemptively address this threat, we examined the structural and functional biology of VCC-1 against the FDA-approved non-ß-lactam-based inhibitor avibactam. We found that avibactam restored the in vitro sensitivity of V. cholerae to meropenem, imipenem, and ertapenem. The acylation efficiency was lower for VCC-1 than for KPC-2 and akin to that of Pseudomonas aeruginosa PAO1 AmpC (k2/Ki = 3.0 × 103 M-1 s-1). The tertiary structure of VCC-1 is similar to that of KPC-2, and they bind avibactam similarly; however, our analyses suggest that VCC-1 may be unable to degrade avibactam, as has been found for KPC-2. Based on our prior genomics-based surveillance, we were able to target VCC-1 for detailed molecular studies to gain early insights that could be used to combat this carbapenemase in the future.

Synergistic activity of fosfomycin, ß-lactams and peptidoglycan recycling inhibition against Pseudomonas aeruginosa.

OBJECTIVES: To evaluate the interconnection between peptidoglycan (PG) recycling, fosfomycin susceptibility and synergy between fosfomycin and ß-lactams in Pseudomonas aeruginosa METHODS: Fosfomycin MICs were determined by broth microdilution and Etest for a panel of 47 PAO1 mutants defective in several components of PG recycling and/or AmpC induction pathways. PAO1 fosfomycin MICs were also determined in the presence of a 5 mM concentration of the NagZ inhibitor PUGNAc. Population analysis of fosfomycin susceptibility and characterization of the resistant mutants that emerged was also performed for selected strains. Finally, fosfomycin, imipenem and fosfomycin + imipenem killing curves were assessed. RESULTS: Mutants defective in AmpG, NagZ or all three AmpD amidases showed a marked increase in fosfomycin susceptibility (at least two 2-fold dilutions with respect to WT PAO1). Moreover, PAO1 fosfomycin MICs were consistently reduced from 48 to 24 mg/L in the presence of a 5 mM concentration of PUGNAc. Fosfomycin hypersusceptibility of the ampG, nagZ and triple ampD mutants was also clearly confirmed in the performed population analysis, although the emergence of resistant mutants, through GlpT mutations, was not avoided. Synergy between fosfomycin and imipenem was evidenced for the WT strain, the AmpC-hyperproducing strain (triple AmpD mutant) and the NagZ and AmpG mutants in killing curves. Moreover, regrowth of resistant mutants was not evidenced for the combination. CONCLUSIONS: PG recycling inhibitors are envisaged as useful adjuvants in the treatment of P. aeruginosa infections with ß-lactams and fosfomycin and therefore further development of these molecules is encouraged.

The ß-lactamase gene regulator AmpR is a tetramer that recognizes and binds the D-Ala-D-Ala motif of its repressor UDP-N-acetylmuramic acid (MurNAc)-pentapeptide.

Inducible expression of chromosomal AmpC ß-lactamase is a major cause of ß-lactam antibiotic resistance in the Gram-negative bacteria Pseudomonas aeruginosa and Enterobacteriaceae. AmpC expression is induced by the LysR-type transcriptional regulator (LTTR) AmpR, which activates ampC expression in response to changes in peptidoglycan (PG) metabolite levels that occur during exposure to ß-lactams. Under normal conditions, AmpR represses ampC transcription by binding the PG precursor UDP-N-acetylmuramic acid (MurNAc)-pentapeptide. When exposed to ß-lactams, however, PG catabolites (1,6-anhydroMurNAc-peptides) accumulate in the cytosol, which have been proposed to competitively displace UDP-MurNAc-pentapeptide from AmpR and convert it into an activator of ampC transcription. Here we describe the molecular interactions between AmpR (from Citrobacter freundii), its DNA operator, and repressor UDP-MurNAc-pentapeptide. Non-denaturing mass spectrometry revealed AmpR to be a homotetramer that is stabilized by DNA containing the T-N11-A LTTR binding motif and revealed that it can bind four repressor molecules in an apparently stepwise manner. A crystal structure of the AmpR effector-binding domain bound to UDP-MurNAc-pentapeptide revealed that the terminal D-Ala-D-Ala motif of the repressor forms the primary contacts with the protein. This observation suggests that 1,6-anhydroMurNAc-pentapeptide may convert AmpR into an activator of ampC transcription more effectively than 1,6-anhydroMurNAc-tripeptide (which lacks the D-Ala-D-Ala motif). Finally, small angle x-ray scattering demonstrates that the AmpR·DNA complex adopts a flat conformation similar to the LTTR protein AphB and undergoes only a slight conformational change when binding UDP-MurNAc-pentapeptide. Modeling the AmpR·DNA tetramer bound to UDP-MurNAc-pentapeptide predicts that the UDP-MurNAc moiety of the repressor participates in modulating AmpR function.

Crystal structure of Arabidopsis thaliana HPPK/DHPS, a bifunctional enzyme and target of the herbicide asulam.

Herbicides are vital for modern agriculture, but their utility is threatened by genetic or metabolic resistance in weeds, as well as regulatory barriers. Of the known herbicide modes of action, 7,8-dihydropterin synthase (DHPS), which is involved in folate biosynthesis, is targeted by just one commercial herbicide, asulam. A mimic of the substrate para-aminobenzoic acid, asulam is chemically similar to sulfonamide antibiotics, and although it is still in widespread use, asulam has faced regulatory scrutiny. With an entire mode of action represented by just one commercial agrochemical, we sought to improve the understanding of its plant target. Here we solve a 2.3 Å resolution crystal structure for Arabidopsis thaliana DHPS that is conjoined to 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), and we reveal a strong structural conservation with bacterial counterparts at the sulfonamide-binding pocket of DHPS. We demonstrate that asulam and the antibiotic sulfamethoxazole have herbicidal as well as antibacterial activity, and we explore the structural basis of their potency by modeling these compounds in mitochondrial HPPK/DHPS. Our findings suggest limited opportunity for the rational design of plant selectivity from asulam and indicate that pharmacokinetic or delivery differences between plants and microbes might be the best ways to safeguard this mode of action.

Antibiotic resistance lessons for the herbicide resistance crisis.

The challenges of resistance to antibiotics and resistance to herbicides have much in common. Antibiotic resistance became a risk in the 1950s, but a concerted global effort to manage it did not begin until after 2000. Widespread herbicide use began during the 1950s and was soon followed by an unabated rise in resistance. Here, we examine what lessons for combatting herbicide resistance could be learnt from the global, coordinated efforts of all stakeholders to avert the antibiotic resistance crisis. © 2021 Society of Chemical Industry.

The Carbapenemase BKC-1 from Klebsiella pneumoniae Is Adapted for Translocation by Both the Tat and Sec Translocons.

The cell envelope of Gram-negative bacteria consists of two membranes surrounding the periplasm and peptidoglycan layer. ß-Lactam antibiotics target the periplasmic penicillin-binding proteins that synthesize peptidoglycan, resulting in cell death. The primary means by which bacterial species resist the effects of ß-lactam drugs is to populate the periplasmic space with ß-lactamases. Resistance to ß-lactam drugs is spread by lateral transfer of genes encoding ß-lactamases from one species of bacteria to another. However, the resistance phenotype depends in turn on these "alien" protein sequences being recognized and exported across the cytoplasmic membrane by either the Sec or Tat protein translocation machinery of the new bacterial host. Here, we examine BKC-1, a carbapenemase from an unknown bacterial source that has been identified in a single clinical isolate of Klebsiella pneumoniae. BKC-1 was shown to be located in the periplasm, and functional in both K. pneumoniae and Escherichia coli. Sequence analysis revealed the presence of an unusual signal peptide with a twin arginine motif and a duplicated hydrophobic region. Biochemical assays showed this signal peptide directs BKC-1 for translocation by both Sec and Tat translocons. This is one of the few descriptions of a periplasmic protein that is functionally translocated by both export pathways in the same organism, and we suggest it represents a snapshot of evolution for a ß-lactamase adapting to functionality in a new host. IMPORTANCE Bacteria can readily acquire plasmids via lateral gene transfer (LGT). These plasmids can carry genes for virulence and antimicrobial resistance (AMR). Of growing concern are LGT events that spread ß-lactamases, particularly carbapenemases, and it is important to understand what limits this spread. This study provides insight into the sequence features of BKC-1 that exemplify the limitations on the successful biogenesis of ß-lactamases, which is one factor limiting the spread of AMR phenotypes by LGT. With a very simple evolutionary adaptation, BKC-1 could become a more effective carbapenemase, underscoring the need to understand the evolution, adaptability, and functional assessment of newly reported ß-lactamases rapidly and thoroughly.

Solution NMR and racemic crystallography provide insights into a novel structural class of cyclic plant peptides.

Head-to-tail cyclic and disulfide-rich peptides are natural products with applications in drug design. Among these are the PawS-Derived Peptides (PDPs) produced in seeds of the daisy plant family. PDP-23 is a unique member of this class in that it is twice the typical size and adopts two ß-hairpins separated by a hinge region. The ß-hairpins, both stabilised by a single disulfide bond, fold together into a V-shaped tertiary structure creating a hydrophobic core. In water two PDP-23 molecules merge their hydrophobic cores to form a square prism quaternary structure. Here, we synthesised PDP-23 and its enantiomer comprising d-amino acids and achiral glycine, which allowed us to confirm these solution NMR structural data by racemic crystallography. Furthermore, we discovered the related PDP-24. NMR analysis showed that PDP-24 does not form a dimeric structure and it has poor water solubility, but in less polar solvents adopts near identical secondary and tertiary structure to PDP-23. The natural role of these peptides in plants remains enigmatic, as we did not observe any antimicrobial or insecticidal activity. However, the plasticity of these larger PDPs and their ability to change structure under different conditions make them appealing peptide drug scaffolds.

A chameleonic macrocyclic peptide with drug delivery applications.

Head-to-tail cyclized peptides are intriguing natural products with unusual properties. The PawS-Derived Peptides (PDPs) are ribosomally synthesized as part of precursors for seed storage albumins in species of the daisy family, and are post-translationally excised and cyclized during proteolytic processing. Here we report a PDP twice the typical size and with two disulfide bonds, identified from seeds of Zinnia elegans. In water, synthetic PDP-23 forms a unique dimeric structure in which two monomers containing two ß-hairpins cross-clasp and enclose a hydrophobic core, creating a square prism. This dimer can be split by addition of micelles or organic solvent and in monomeric form PDP-23 adopts open or closed V-shapes, exposing different levels of hydrophobicity dependent on conditions. This chameleonic character is unusual for disulfide-rich peptides and engenders PDP-23 with potential for cell delivery and accessing novel targets. We demonstrate this by conjugating a rhodamine dye to PDP-23, creating a stable, cell-penetrating inhibitor of the P-glycoprotein drug efflux pump.

The Structure of a Conserved Domain of TamB Reveals a Hydrophobic ß Taco Fold.

The translocation and assembly module (TAM) plays a role in the transport and insertion of proteins into the bacterial outer membrane. TamB, a component of this system spans the periplasmic space to engage with its partner protein TamA. Despite efforts to characterize the TAM, the structure and mechanism of action of TamB remained enigmatic. Here we present the crystal structure of TamB amino acids 963-1,138. This region represents half of the conserved DUF490 domain, the defining feature of TamB. TamB963-1138 consists of a concave, taco-shaped ß sheet with a hydrophobic interior. This ß taco structure is of dimensions capable of accommodating and shielding the hydrophobic side of an amphipathic ß strand, potentially allowing TamB to chaperone nascent membrane proteins from the aqueous environment. In addition, sequence analysis suggests that the structure of TamB963-1138 is shared by a large portion of TamB. This architecture could allow TamB to act as a conduit for membrane proteins.

Conformational flexibility of the glycosidase NagZ allows it to bind structurally diverse inhibitors to suppress ß-lactam antibiotic resistance.

NagZ is an N-acetyl-ß-d-glucosaminidase that participates in the peptidoglycan (PG) recycling pathway of Gram-negative bacteria by removing N-acetyl-glucosamine (GlcNAc) from PG fragments that have been excised from the cell wall during growth. The 1,6-anhydromuramoyl-peptide products generated by NagZ activate ß-lactam resistance in many Gram-negative bacteria by inducing the expression of AmpC ß-lactamase. Blocking NagZ activity can thereby suppress ß-lactam antibiotic resistance in these bacteria. The NagZ active site is dynamic and it accommodates distortion of the glycan substrate during catalysis using a mobile catalytic loop that carries a histidine residue which serves as the active site general acid/base catalyst. Here, we show that flexibility of this catalytic loop also accommodates structural differences in small molecule inhibitors of NagZ, which could be exploited to improve inhibitor specificity. X-ray structures of NagZ bound to the potent yet non-selective N-acetyl-ß-glucosaminidase inhibitor PUGNAc (O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate), and two NagZ-selective inhibitors - EtBuPUG, a PUGNAc derivative bearing a 2-N-ethylbutyryl group, and MM-156, a 3-N-butyryl trihydroxyazepane, revealed that the phenylcarbamate moiety of PUGNAc and EtBuPUG completely displaces the catalytic loop from the NagZ active site to yield a catalytically incompetent form of the enzyme. In contrast, the catalytic loop was found positioned in the catalytically active conformation within the NagZ active site when bound to MM-156, which lacks the phenylcarbamate extension. Displacement of the catalytic loop by PUGNAc and its N-acyl derivative EtBuPUG alters the active site conformation of NagZ, which presents an additional strategy to improve the potency and specificity of NagZ inhibitors.

Selective trihydroxyazepane NagZ inhibitors increase sensitivity of Pseudomonas aeruginosa to ß-lactams.

AmpC ß-lactamase confers resistance to ß-lactam antibiotics in many Gram negative bacteria. Inducible expression of AmpC requires an N-acetylglucosaminidase termed NagZ. Here we describe the synthesis and characterization of hydroxyazepane inhibitors of NagZ. We find that these inhibitors enhance the susceptibility of clinically relevant Pseudomonas aeruginosa to ß-lactams.