College of Pharmacy, Yeungnam University, Gyeongsan, Korea.
Division of Life Science, Gyeongsang National University, Jinju, Korea.
FEBS J. 2024 Aug;291(16):3723-3736. doi: 10.1111/febs.17197. Epub 2024 Jun 5.
Peptidoglycan DL-endopeptidases locally cleave the peptide stem of peptidoglycan in the bacterial cell wall. This process facilitates bacterial growth and division by loosening the rigid peptidoglycan layer. IseA binds to the active site of multiple DL-endopeptidases and inhibits excessive peptidoglycan degradation that leads to cell lysis. To better understand how IseA inhibits DL-endopeptidase activity, we determined the crystal structure of the peptidoglycan DL-endopeptidase CwlO/IseA complex and compared it with that of the peptidoglycan DL-endopeptidase LytE/IseA complex. Structural analyses showed significant differences between the hydrophobic pocket-binding residues of the DL-endopeptidases (F361 of CwlO and W237 of LytE). Additionally, binding assays showed that the F361 mutation of CwlO to the bulkier hydrophobic residue, tryptophan, increased its binding affinity for IseA, whereas mutation to alanine reduced the affinity. These analyses revealed that the hydrophobic pocket-binding residue of DL-endopeptidases determines IseA-binding affinity and is required for substrate-mimetic inhibition by IseA.
肽聚糖 DL-内肽酶在细菌细胞壁中局部切割肽聚糖的肽主干。通过疏松刚性的肽聚糖层，这个过程促进了细菌的生长和分裂。IseA 结合到多个 DL-内肽酶的活性部位，并抑制导致细胞裂解的过度肽聚糖降解。为了更好地理解 IseA 如何抑制 DL-内肽酶的活性，我们确定了肽聚糖 DL-内肽酶 CwlO/IseA 复合物的晶体结构，并将其与肽聚糖 DL-内肽酶 LytE/IseA 复合物的结构进行了比较。结构分析显示，DL-内肽酶的疏水性口袋结合残基（CwlO 的 F361 和 LytE 的 W237）之间存在显著差异。此外，结合实验表明，CwlO 的 F361 突变为疏水性更大的色氨酸增加了其与 IseA 的结合亲和力，而突变为丙氨酸则降低了亲和力。这些分析表明，DL-内肽酶的疏水性口袋结合残基决定了 IseA 的结合亲和力，是 IseA 模拟底物抑制所必需的。
