Dataset Information

Opposites Attract: Escherichia coli Heptosyltransferase I Conformational Changes Induced by Interactions between the Substrate and Positively Charged Residues.

ABSTRACT: Gram-negative bacterial viability is greatly reduced by the disruption of heptose sugar addition during the biosynthesis of lipopolysaccharide (LPS), an important bacterial outer membrane component. Heptosyltransferase I (HepI), a member of the GT-B structural subclass of glycosyltransferases, is therefore an essential enzyme for the biosynthesis of the LPS. The disruption of HepI also increases the susceptibility of bacteria to hydrophobic antibiotics, making HepI a potential target for drug development. In this work, the structural and dynamic properties of the catalytic cycle of HepI are explored. Previously, substrate-induced stabilization of HepI was observed and hypothesized to be assisted by interactions between the substrate and residues located on dynamic loops. Herein, positively charged amino acids were probed to identify binding partners of the negatively charged phosphates and carboxylates of Kdo2-lipid A and its analogues. Mutant enzymes were characterized to explore changes in enzymatic activities and protein stability. Molecular modeling of HepI in the presence and absence of ligands was then performed with the wild type and mutant enzyme to allow determination of the relative change in substrate binding affinity resulting from each mutation. Together, these studies suggest that multiple residues are involved in mediating substrate binding, and a lack of additivity of these effects illustrates the functional redundancy of these binding interactions. The redundancy of residues mediating conformational transitions in HepI illustrates the evolutionary importance of these structural rearrangements for catalysis. This work enhances the understanding of HepI's protein dynamics and mechanism and is a model for improving our understanding of glycosyltransferase enzymes.

SUBMITTER: Cote JM

PROVIDER: S-EPMC7582807 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Publications

Opposites Attract: <i>Escherichia coli</i> Heptosyltransferase I Conformational Changes Induced by Interactions between the Substrate and Positively Charged Residues.

Cote Joy M JM Hecht Cody J S CJS Patel Kaelan R KR Ramirez-Mondragon Carlos A CA Sham Yuk Y YY Taylor Erika A EA

Biochemistry 20200210 34

Gram-negative bacterial viability is greatly reduced by the disruption of heptose sugar addition during the biosynthesis of lipopolysaccharide (LPS), an important bacterial outer membrane component. Heptosyltransferase I (HepI), a member of the GT-B structural subclass of glycosyltransferases, is therefore an essential enzyme for the biosynthesis of the LPS. The disruption of HepI also increases the susceptibility of bacteria to hydrophobic antibiotics, making HepI a potential target for drug de ...[more]

PMID: 32011131

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Project description:UnlabelledBacterial cell division is an essential and highly coordinated process. It requires the polymerization of the tubulin homologue FtsZ to form a dynamic ring (Z-ring) at midcell. Z-ring formation relies on a group of FtsZ-associated proteins (Zap) for stability throughout the process of division. In Escherichia coli, there are currently five Zap proteins (ZapA through ZapE), of which four (ZapA, ZapB, ZapC, and ZapD) are small soluble proteins that act to bind and bundle FtsZ filaments. In particular, ZapD forms a functional dimer and interacts with the C-terminal tail of FtsZ, but little is known about its structure and mechanism of action. Here, we present the crystal structure of Escherichia coli ZapD and show it forms a symmetrical dimer with centrally located α-helices flanked by β-sheet domains. Based on the structure of ZapD and its chemical cross-linking to FtsZ, we targeted nine charged ZapD residues for modification by site-directed mutagenesis. Using in vitro FtsZ sedimentation assays, we show that residues R56, R221, and R225 are important for bundling FtsZ filaments, while transmission electron microscopy revealed that altering these residues results in different FtsZ bundle morphology compared to those of filaments bundled with wild-type ZapD. ZapD residue R116 also showed altered FtsZ bundle morphology but levels of FtsZ bundling similar to that of wild-type ZapD. Together, these results reveal that ZapD residues R116, R221, and R225 likely participate in forming a positively charged binding pocket that is critical for bundling FtsZ filaments.ImportanceZ-ring assembly underpins the formation of the essential cell division complex known as the divisome and is required for recruitment of downstream cell division proteins. ZapD is one of several proteins in E. coli that associates with the Z-ring to promote FtsZ bundling and aids in the overall fitness of the division process. In the present study, we describe the dimeric structure of E. coli ZapD and identify residues that are critical for FtsZ bundling. Together, these results advance our understanding about the formation and dynamics of the Z-ring prior to bacterial cell division.

Dataset Information

Opposites Attract: Escherichia coli Heptosyltransferase I Conformational Changes Induced by Interactions between the Substrate and Positively Charged Residues.

Publications

Opposites Attract: <i>Escherichia coli</i> Heptosyltransferase I Conformational Changes Induced by Interactions between the Substrate and Positively Charged Residues.

Similar Datasets

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