Structure of the essential inner membrane lipopolysaccharide–PbgA complex

Clairfeuille, Thomas; Buchholz, Kerry R.; Li, Qingling; Verschueren, Erik; Liu, Peter; Sangaraju, Dewakar; Park, Summer; Noland, Cameron L.; Storek, Kelly M.; Nickerson, Nicholas N.; Martin, Lynn M.; Vega, Trisha Dela; Miu, Anh; Reeder, Janina; Ruiz-Gonzalez, Maria; Swem, Danielle L.; Han, Guanghui; DePonte, Daniel P.; Hunter, Mark S.; Gati, Cornelius; Shahidi-Latham, Sheerin; Xu, Min; Skelton, Nicholas J.; Sellers, Benjamin D.; Skippington, Elizabeth; Sandoval, Wendy; Hanan, Emily J.; Payandeh, Jian; Rutherford, Steven T.

doi:10.1038/s41586-020-2597-x

Cited by 67 publications

(112 citation statements)

References 66 publications

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“…Thus, LapB interacts with FtsH, as demonstrated earlier, and with LapC. Consistent with our data of physical interaction of LapB with LapC, a recent finding published during the preparation of this manuscript have shown that LapC tagged with the FLAG epitope can pull-down LapB [32], consistent with our demonstration of LapB and LapC co-purification.…”

Section: Discussionsupporting

confidence: 93%

“…However, the most frequent mutation identified is lapC377fs. The F349 amino acid residue is highly conserved and located in the Mg ++ ion-binding pocket in the pseudo-hydrolase domain and in the putative phosphatase active site [32,34]. Comparison of sensitivity of three isogenic lapC mutant derivatives to CHIR090 revealed that lapC190 is extremely sensitive to this LpxC inhibitor, followed by the lapC377fs derivatives at the concentration, when growth of the parental strain is not affected (Figure 7, lanes 1, 3&4).…”

Section: The C-terminal Truncation Of the Periplasmic Globular Domainmentioning

confidence: 97%

“…This mutation causes an insertion of C residue at nt position 1133 resulting into frame-shift after 377 amino acid (lapC377fs), with the addition of 13 aa residues after Thr377, leading into the truncation of C-terminal periplasmic domain. LapC (YejM) structures have been described recently revealing five IM-spanning helices in the N-terminus and a large C-terminal globular domain connected by a linker domain [32,34,47,48].…”

Section: An Extragenic Suppressor Mutation In the Lapc Gene Restores mentioning

confidence: 99%

“…Thus, in the first approach, Tn10-linked point mutations that simultaneously exhibited the elevated rpoEP3 promoter activity, the sensitivity to the LpxC inhibitor CHIR090 to different extents and membrane permeability defects were analyzed leading to the identification of three point mutations: one having a frame-shift mutation resulting in truncation after 377 amino acid residue isolated three times, the second mutation introduces a stop codon after amino acid residue 190, another resulting into a single amino acid F349S substitution, in the essential yejM gene. During the completion of this work, YejM has been speculated to either maintain lipid homeostasis or be involved in the transport of cardiolipin or exhibit a metal-dependent phosphatase activity [31][32][33][34][35][36][37]. We designated this gene lapC based on identified genetic and biochemical interactions of LapC with LapB.…”

Section: Introductionmentioning

confidence: 99%

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Regulation of the First Committed Step in Lipopolysaccharide Biosynthesis Catalyzed by LpxC Requires the Essential Protein LapC (YejM) and HslVU Protease

Biernacka

Gorzelak

Klein

et al. 2020

IJMS

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We previously showed that lipopolysaccharide (LPS) assembly requires the essential LapB protein to regulate FtsH-mediated proteolysis of LpxC protein that catalyzes the first committed step in the LPS synthesis. To further understand the essential function of LapB and its role in LpxC turnover, multicopy suppressors of ΔlapB revealed that overproduction of HslV protease subunit prevents its lethality by proteolytic degradation of LpxC, providing the first alternative pathway of LpxC degradation. Isolation and characterization of an extragenic suppressor mutation that prevents lethality of ΔlapB by restoration of normal LPS synthesis identified a frame-shift mutation after 377 aa in the essential gene designated lapC, suggesting LapB and LapC act antagonistically. The same lapC gene was identified during selection for mutations that induce transcription from LPS defects-responsive rpoEP3 promoter, confer sensitivity to LpxC inhibitor CHIR090 and a temperature-sensitive phenotype. Suppressors of lapC mutants that restored growth at elevated temperatures mapped to lapA/lapB, lpxC and ftsH genes. Such suppressor mutations restored normal levels of LPS and prevented proteolysis of LpxC in lapC mutants. Interestingly, a lapC deletion could be constructed in strains either overproducing LpxC or in the absence of LapB, revealing that FtsH, LapB and LapC together regulate LPS synthesis by controlling LpxC amounts.

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Section: Discussionsupporting

confidence: 93%

Section: The C-terminal Truncation Of the Periplasmic Globular Domainmentioning

confidence: 97%

Section: An Extragenic Suppressor Mutation In the Lapc Gene Restores mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Regulation of the First Committed Step in Lipopolysaccharide Biosynthesis Catalyzed by LpxC Requires the Essential Protein LapC (YejM) and HslVU Protease

Biernacka

Gorzelak

Klein

et al. 2020

IJMS

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“…Which signals activate the LapAB/FtsH regulation and how this regulation mechanism works are still being elucidated. Recent advances in cell envelope biology have shed light on a third protein involved in LpxC regulation, YejM (PbgA), which directly senses periplasmic LPS levels (10)(11)(12)(13)(14). Discovery of this third member in this regulatory cascade to control LpxC levels will help to finally unravel how bacteria balance GPL and lipid A synthesis and respond to changes in each.…”

mentioning

confidence: 99%

Restoring Balance to the Outer Membrane: YejM’s Role in LPS Regulation

Simpson

Douglass

Trent

2020

mBio

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Gram-negative bacteria produce an asymmetric outer membrane (OM) that is particularly impermeant to many antibiotics and characterized by lipopolysaccharide (LPS) exclusively at the cell surface. LPS biogenesis remains an ideal target for therapeutic intervention, as disruption could kill bacteria or increase sensitivity to existing antibiotics. While it has been known that LPS synthesis is regulated by proteolytic control of LpxC, the enzyme that catalyzes the first committed step of LPS synthesis, it remains unknown which signals direct this regulation. New details have been revealed during study of a cryptic essential inner membrane protein, YejM. Multiple functions have been proposed over the years for YejM, including a controversial hypothesis that it transports cardiolipin from the inner membrane to the OM. Strong evidence now indicates that YejM senses LPS in the periplasm and directs proteolytic regulation. Here, we discuss the standing literature of YejM and highlight exciting new insights into cell envelope maintenance.

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Rationalizing the Unprecedented Stereochemistry of an Enzymatic Nitrile Synthesis through a Combined Computational and Experimental Approach

2021

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In this contribution, the unique and unprecedented stereochemical phenomenon of an aldoxime dehydratase‐catalyzed enantioselective dehydration of racemic E‐ and Z‐aldoximes with selective formation of both enantiomeric forms of a chiral nitrile is rationalized by means of molecular modelling, comprising in silico mutations and docking studies. This theoretical investigation gave detailed insight into why with the same enzyme the use of racemic E‐ and Z‐aldoximes leads to opposite forms of the chiral nitrile. The calculated mutants with a larger or smaller cavity in the active site were then prepared and used in biotransformations, showing the theoretically predicted decrease and increase of the enantioselectivities in these nitrile syntheses. This validated model also enabled the rational design of mutants with a smaller cavity, which gave superior enantioselectivities compared to the known wild‐type enzyme, with excellent E‐values of up to E>200 when the mutant OxdRE‐Leu145Phe was utilized.

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Structure of the essential inner membrane lipopolysaccharide–PbgA complex

Cited by 67 publications

References 66 publications

Regulation of the First Committed Step in Lipopolysaccharide Biosynthesis Catalyzed by LpxC Requires the Essential Protein LapC (YejM) and HslVU Protease

Regulation of the First Committed Step in Lipopolysaccharide Biosynthesis Catalyzed by LpxC Requires the Essential Protein LapC (YejM) and HslVU Protease

Restoring Balance to the Outer Membrane: YejM’s Role in LPS Regulation

Rationalizing the Unprecedented Stereochemistry of an Enzymatic Nitrile Synthesis through a Combined Computational and Experimental Approach

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