Purification, Crystallization and Preliminary X-ray Analysis of Penicillin Binding Protein 4 fromEschericha coli,a Protein Related to Class A β-Lactamases

Thunnissen, Marjolein M.G.M.; Boer, Bijtske de; Dijkstra, Bauke W.

doi:10.1006/jmbi.1994.0128

Cited by 15 publications

(9 citation statements)

References 40 publications

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“…Substrate binding can be a key determinant in the localization of proteins [18,65,66]. To test whether PBP4 substrate binding is needed to localize at division sites we expressed the active site mutants PBP4(S62G) and (S62A) [61] that fails to bind b-lactams and therefore is not able to hydrolyze the PG stem peptide (Fig. S2 and S3).…”

Section: Pbp4 Localizes Between the Peptidoglycan Layer And The Outer Membranementioning

confidence: 99%

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Midcell localization of PBP4 ofEscherichia colimodulates the timing of divisome assembly

Verheul

Lodge

Yau

et al. 2020

Preprint

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Insertion of new material into the Escherichia coli peptidoglycan (PG) sacculus between the cytoplasmic membrane and the outer membrane requires a well-organized balance between synthetic and hydrolytic activities to maintain cell shape and avoid lysis. The hydrolytic enzymes outnumber the enzymes that insert new PG by far and very little is known about their specific function. Here we show that the DD-carboxy/endopeptidase PBP4 localizes in a PBP1A/LpoA and FtsEX dependent fashion at midcell during septal PG synthesis. Midcell localization of PBP4 requires its non-catalytic domain 3 of unknown function, but not the activity of PBP4 or FtsE. Domain 3 is also needed for the interaction of PBP4 with NlpI, but not for its interactions with PBP1A or LpoA. Microscale thermophoresis with isolated proteins shows that domain 3 is needed for the interaction with NlpI, but not PBP1A or LpoA. In vivo crosslinking experiments confirm the interaction of PBP4 with PBP1A and LpoA. We propose that PBP4 functions together with the amidases AmiA and B to create denuded glycan strands to attract the initiator of septal PG synthesis, FtsN. Consistent with this model, we found that the arrival of FtsN and other cell division proteins at midcell was significantly delayed in cells lacking PBP4.

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Section: Pbp4 Localizes Between the Peptidoglycan Layer And The Outer Membranementioning

confidence: 99%

“…A non-catalytical domain of unknown function (domain 2) is inserted into the transpeptidase domain 1, and a third domain (domain 3) is inserted into domain 2. Domain 3 is positioned above the active side with its catalytic serine 62, which resides in domain 1, and might be involved in substrate binding or regulation (56,57) (Fig. 1A).…”

Section: Introductionmentioning

confidence: 99%

Midcell localization of PBP4 ofEscherichia colimodulates the timing of divisome assembly

Verheul

Lodge

Yau

et al. 2020

Preprint

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“…X-ray structures of membrane-bound penicillin-binding proteins may soon appear, because water-soluble forms are being crystallized (12,22,36,86). In the meantime, given the similarity of the ␤-lactamase structures with the X-ray structure of the penicillin-inhibited DD-peptidase (25,46), one might speculate on the structural basis of mutations discovered in the low-affinity penicillin-binding proteins (24,81,82).…”

Section: Altered Penicillin-binding Proteinsmentioning

confidence: 99%

Extended-spectrum and inhibitor-resistant TEM-type beta-lactamases: mutations, specificity, and three-dimensional structure

Knox

1995

Antimicrob Agents Chemother

301

339

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“…A non-catalytical domain of unknown function (domain 2), which is inserted into the transpeptidase domain 1, and a third domain (domain 3), which is inserted into domain 2. Domain 3 is positioned above the active site (Serine 62, in domain 1), and might be involved in substrate binding or regulation [ 58 , 59 ] ( Fig 1A ). Site directed mutagenesis suggested that residues from domain 1 (R361) and 2 (D155) are important for the endopeptidase activity [ 60 ].…”

Section: Introductionmentioning

confidence: 99%

Early midcell localization of Escherichia coli PBP4 supports the function of peptidoglycan amidases

et al. 2022

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Insertion of new material into the Escherichia coli peptidoglycan (PG) sacculus between the cytoplasmic membrane and the outer membrane requires a well-organized balance between synthetic and hydrolytic activities to maintain cell shape and avoid lysis. Since most bacteria carry multiple enzymes carrying the same type of PG hydrolytic activity, we know little about the specific function of given enzymes. Here we show that the DD-carboxy/endopeptidase PBP4 localizes in a PBP1A/LpoA and FtsEX dependent fashion at midcell during septal PG synthesis. Midcell localization of PBP4 requires its non-catalytic domain 3 of unknown function, but not the activity of PBP4 or FtsE. Microscale thermophoresis with isolated proteins shows that PBP4 interacts with NlpI and the FtsEX-interacting protein EnvC, an activator of amidases AmiA and AmiB, which are needed to generate denuded glycan strands to recruit the initiator of septal PG synthesis, FtsN. The domain 3 of PBP4 is needed for the interaction with NlpI and EnvC, but not PBP1A or LpoA. In vivo crosslinking experiments confirm the interaction of PBP4 with PBP1A and LpoA. We propose that the interaction of PBP4 with EnvC, whilst not absolutely necessary for mid-cell recruitment of either protein, coordinates the activities of PBP4 and the amidases, which affects the formation of denuded glycan strands that attract FtsN. Consistent with this model, we found that the divisome assembly at midcell was premature in cells lacking PBP4, illustrating how the complexity of interactions affect the timing of cell division initiation.

show abstract

Purification, Crystallization and Preliminary X-ray Analysis of Penicillin Binding Protein 4 fromEschericha coli,a Protein Related to Class A β-Lactamases

Cited by 15 publications

References 40 publications

Midcell localization of PBP4 ofEscherichia colimodulates the timing of divisome assembly

Midcell localization of PBP4 ofEscherichia colimodulates the timing of divisome assembly

Extended-spectrum and inhibitor-resistant TEM-type beta-lactamases: mutations, specificity, and three-dimensional structure

Early midcell localization of Escherichia coli PBP4 supports the function of peptidoglycan amidases

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