Abstract
In Bacillus subtilis, LytE, LytF, CwlS, and CwlO are vegetative autolysins, DL-endopeptidases in the NlpC/P60 family, and play essential roles in cell growth and separation. IseA (YoeB) is a proteinaceous inhibitor against the DL-endopeptidases, peptidoglycan hydrolases. Overexpression of IseA caused significantly long chained cell morphology, because IseA inhibits the cell separation DL-endopeptidases post-translationally. Here, we report the first three-dimensional structure of IseA, determined by NMR spectroscopy. The structure includes a single domain consisting of three α-helices, one 3(10)-helix, and eight β-strands, which is a novel fold like a "hacksaw." Noteworthy is a dynamic loop between β4 and the 3(10)-helix, which resembles a "blade." The electrostatic potential distribution shows that most of the surface is positively charged, but the region around the loop is negatively charged. In contrast, the LytF active-site cleft is expected to be positively charged. NMR chemical shift perturbation of IseA interacting with LytF indicated that potential interaction sites are located around the loop. Furthermore, the IseA mutants D100K/D102K and G99P/G101P at the loop showed dramatic loss of inhibition activity against LytF, compared with wild-type IseA, indicating that the β4-3(10) loop plays an important role in inhibition. Moreover, we built a complex structure model of IseA-LytF by docking simulation, suggesting that the β4-3(10) loop of IseA gets stuck deep in the cleft of LytF, and the active site is occluded. These results suggest a novel inhibition mechanism of the hacksaw-like structure, which is different from known inhibitor proteins, through interactions around the characteristic loop regions with the active-site cleft of enzymes.
Highlights
IseA from Bacillus subtilis is an inhibitor protein against DL-endopeptidases including synthetic lethal autolysins
The template structure was the structure of lipoprotein Spr from E. coli [39]
One disulfide bond between Cys-40 and Cys-56 was confirmed by their proton nuclear Overhauser effect (NOE) peaks and the chemical shifts of their C atoms, which were 44.59 and 47.33 ppm, respectively [45]
Summary
IseA from Bacillus subtilis is an inhibitor protein against DL-endopeptidases including synthetic lethal autolysins. LytE, LytF, and CwlS are cell separation autolysins (4 – 6), and the lytE cwlO genotype showed synthetic lethality [1, 2] These enzymes have similar domain structures consisting of putative cell wall-binding domains, which are known as LysM repeats in LytE, LytF, and CwlS or an uncharacterized domain in CwlO, in the N termini, and the catalytic domains of DL-endopeptidase in the C termini (30 –70% amino acid identities). The amino acid sequences of Ivy and IseA show no detectable homology, suggesting that the structure and inhibition mechanism of IseA should be different from Ivy. Many bacterial cell wall autolytic enzymes, including DL-endopeptidases and lytic transglycosylases, play essential roles in cell wall turnover, cell growth, and cell division [2, 3, 12, 16]. The structure shows a novel fold with a characteristic loop, and we suggest an inhibition mechanism of IseA against DL-endopeptidases
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