Allostery, Recognition of Nascent Peptidoglycan, and Cross-linking of the Cell Wall by the Essential Penicillin-Binding Protein 2x of <i>Streptococcus pneumoniae</i>

Bernardo-García, N.; Mahasenan, Kiran V.; Batuecas, M.T.; Lee, Mijoon; Hesek, Dušan; Petráčková, Denisa; Doubravová, Linda; Branný, Pavel; Mobashery, Shahriar; Hermoso, J.A.

doi:10.1021/acschembio.7b00817

Cited by 31 publications

(47 citation statements)

References 37 publications

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“…To investigate the inferred crystal structure location of S. pyogenes PBP2x mutations relative to the S. pneumoniae orthologue, S. pyogenes PBP2x sequence variations were plotted onto the S. pneumoniae PBP2x crystal structure bound to oxacillin (PDB: 5OIZ) (11). Sequence conservation as determined by the frequency (for S. pyogenes ) and percentage (for S. pneumoniae ) of variant amino acids compared to the consensus was rendered onto the PBP2x crystal structure using UCSF Chimera (12).…”

Section: Methodsmentioning

confidence: 99%

Restricted sequence variation inStreptococcus pyogenespenicillin binding proteins

Hayes

Lacey

Morris

et al. 2020

Preprint

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AbstractA recent clinical report has linked Streptococcus pyogenes β-lactam antibiotic resistance to mutations in the Penicillin Binding Protein PBP2x. To determine whether this is an isolated case or reflects a broader prevalence of mutations that might confer reduced β-lactam susceptibility, we investigated the relative frequency of penicillin binding protein (PBP) sequence variation within a global database of 9,667 S. pyogenes isolates. We found that mutations in S. pyogenes PBPs (PBP2x, PBP1a, PBP1b and PBP2a) occur infrequently across this global database with less than 3 amino acid changes differing between >99% of the global population. Only 4 of the 9,667 strains contained mutations near transpeptidase active sites. The reported PBP2x T553K substitution was not identified. These findings are in contrast to those of 2,520 S. pneumococcus sequences where PBP mutations are relatively frequent and are often located in key β-lactam binding pockets. These data, combined with the general lack of penicillin resistance reported in S. pyogenes worldwide, suggests that extensive, unknown, constraints restrict S. pyogenes PBP sequence plasticity. These findings imply that while heavy antibiotic pressure may select for mutations in the PBPs, there is currently no evidence of such mutations becoming fixed in the S. pyogenes population nor that mutations are being sequentially acquired in the PBPs.ImportancePenicillin is the first line therapeutic option for Streptococcus pyogenes infections. Despite the global high prevalence of S. pyogenes infections and widespread use of penicillin, reports of resistance to penicillin have been incredibly rare. Recently, penicillin resistance was detected in two clinical S. pyogenes isolates with accompanying mutations in the active site of the penicillin binding protein PBP2x, raising concerns that penicillin resistance may become more widespread. We screened a global database of S. pyogenes genome sequences to investigate the frequency of penicillin binding protein (PBP) mutations, identifying that PBP mutations are uncommon relative to Streptococcus pneumoniae. These findings support clinical observations that penicillin resistance is rare in S. pyogenes, and suggest that there are considerable constraints on S. pyogenes PBP sequence variation.

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Section: Methodsmentioning

confidence: 99%

Restricted sequence variation inStreptococcus pyogenespenicillin binding proteins

Hayes

Lacey

Morris

et al. 2020

Preprint

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“…It is possible that the PASTA domains strengthen the interaction between DivIB and the transpeptidase domain, but alternatively the PASTA domains interact with another region of DivIB. Our results show that PASTA domains can have distinct functions in similar proteins – whereas the PASTA domains in S. pneumoniae clearly function to allosterically activate the transpeptidase activity of the protein (12), their function in B. subtilis PBP2B is to stabilize an important protein-protein interaction in the divisome.…”

Section: Discussionmentioning

confidence: 88%

“…In PBP2x, loss of the PASTA domains abolishes the binding of Bocillin-FL, a fluorescent penicillin derivative (11) and localization of PBP2x to the division site (9), suggesting that PASTA domains mediate the interaction with peptidoglycan. This was nicely illustrated recently by a series of crystal structures that revealed that the PBP2x PASTA domains form an allosteric binding site for a pentapeptide stem in a nascent peptidoglycan strand, which positions another peptide stem on the same strand in the active site so that it can be cross-linked (12). The allosteric binding site is formed at the interface of the two PASTA domains and the transpeptidase domain and comprises the entire first and part of the second PASTA domain.…”

Section: Introductionmentioning

confidence: 94%

“…The allosteric binding site is formed at the interface of the two PASTA domains and the transpeptidase domain and comprises the entire first and part of the second PASTA domain. Binding of the terminal D-Ala-D-Ala of the stempeptide at this side displaces a ‘gatekeeper’ Arginine residue on the transpeptidase domain, which subsequently forms salt bridges with an Aspartate and a Glutamate residue on the first PASTA domain which opens up the active site so that the donor stempeptide for transpeptidation on the same glycan strand can bind (12). The PASTA domains of B. subtilis PBP2B lack all the residues required for this allosteric activation.…”

Section: Introductionmentioning

confidence: 99%

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The PASTA domains of Bacillus subtilis PBP2B stabilize the interaction of PBP2B with DivIB

Angeles

Macia-Valero

Bohorquez

et al. 2019

Preprint

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23Bacterial cell division is mediated by a protein complex known as the divisome. Many protein-24 protein interactions in the divisome have been characterized. In this report, we analyse the role 25 of the PASTA (Penicillin binding protein And Serine Threonine kinase Associated)-domains 26 of Bacillus subtilis PBP2B. PBP2B itself is essential and cannot be deleted, but removing the 27 PBP2B PASTA domains results in impaired cell division and a heat sensitive phenotype. This 28 resembles the deletion of divIB, a known interaction partner of PBP2B. Bacterial two hybrid 29 and co-immunoprecipitation analyses show that the interaction between PBP2B and DivIB is 30 weakened when the PBP2B PASTA domains are removed. Combined, our results show that 31 the PBP2B PASTA domains are required to stabilize the interaction between PBP2B and 32DivIB. 33 34 Protein stability 129Membranes from strains 4132 and 4133 grown at 30°C on CH with 0.2% (w/v) xylose were 130 isolated. Cells were grown until exponential phase and spun down (3 000 rpm, 7 min, 4 °C). 131Pellets were washed in PBS and then cells were lysed by sonication. Membranes were collected 132 by centrifugation (45,000 rpm, 4°C, 50 min) and resuspended in PBS. The protein 133 concentration was equalised for the two strain samples and aliquots of membrane material of 134 the same volume were prepared. Aliquots were incubated at 30 or 48 °C for 5 min, 20 min, 1 135 hr, 2 hr and 14 hr. Then, Bocillin 650/665 (5 µg/ml) was added to each sample, and samples 136 were further incubated at RT for 10 min. After incubation, sample buffer was added to each 137 sample to stop further protein degradation, and samples were run in SDS (10 %) gel. GFP and 138Bocillin were detected using a Typhoon FLA950 (GE Healthcare). For GFP, the 473 nm laser 139 and the LPB (Long Pass Blue) filter were used, and for Bocillin the 635 nm laser and the LPR 140 (Long Pass Red) were used. 141After imaging, the same gels were used for immunoblotting. Proteins were transferred to a 142 PVDF membrane. Primary antibodies were anti-GFP (Thermofisher). Anti-Rabbit IgG alkaline 143 phosphatase conjugated secondary antibodies (Sigma Aldrich) were used. Blots were 144 developed using CDP-Star (Roche) and chemiluminescence was detected using a Fujifilm LAS 145 4000 imager (GE Healthcare).

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“…We discern simple insight and design our next experiment with attention to the singular rather than the plurality of Ockham's razor . The allosteric control of S. aureus PBP2a (and perhaps of other PBPs) may well be to synchronize loop opening of the active site with protein translocation, but this explanation is almost certainly an oversimplification. The narrative presented here conceptualizes a two‐dimensional pathway for the peptidoglycan construction and deconstruction by P. aeruginosa when the realities of both AmpR regulation and cell‐wall biosynthesis are multidimensional .…”

Section: Resultsmentioning

confidence: 99%

Constructing and deconstructing the bacterial cell wall

Fisher

Mobashery

2019

Protein Science

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The history of modern medicine cannot be written apart from the history of the antibiotics. Antibiotics are cytotoxic secondary metabolites that are isolated from Nature. The antibacterial antibiotics disproportionately target bacterial protein structure that is distinct from eukaryotic protein structure, notably within the ribosome and within the pathways for bacterial cell‐wall biosynthesis (for which there is not a eukaryotic counterpart). This review focuses on a pre‐eminent class of antibiotics—the β‐lactams, exemplified by the penicillins and cephalosporins—from the perspective of the evolving mechanisms for bacterial resistance. The mechanism of action of the β‐lactams is bacterial cell‐wall destruction. In the monoderm (single membrane, Gram‐positive staining) pathogen Staphylococcus aureus the dominant resistance mechanism is expression of a β‐lactam‐unreactive transpeptidase enzyme that functions in cell‐wall construction. In the diderm (dual membrane, Gram‐negative staining) pathogen Pseudomonas aeruginosa a dominant resistance mechanism (among several) is expression of a hydrolytic enzyme that destroys the critical β‐lactam ring of the antibiotic. The key sensing mechanism used by P. aeruginosa is monitoring the molecular difference between cell‐wall construction and cell‐wall deconstruction. In both bacteria, the resistance pathways are manifested only when the bacteria detect the presence of β‐lactams. This review summarizes how the β‐lactams are sensed and how the resistance mechanisms are manifested, with the expectation that preventing these processes will be critical to future chemotherapeutic control of multidrug resistant bacteria.

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Allostery, Recognition of Nascent Peptidoglycan, and Cross-linking of the Cell Wall by the Essential Penicillin-Binding Protein 2x of Streptococcus pneumoniae

Cited by 31 publications

References 37 publications

Restricted sequence variation inStreptococcus pyogenespenicillin binding proteins

Restricted sequence variation inStreptococcus pyogenespenicillin binding proteins

The PASTA domains of Bacillus subtilis PBP2B stabilize the interaction of PBP2B with DivIB

Constructing and deconstructing the bacterial cell wall

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