The X-ray structure of lysozyme from bacteriophage lambda (λ lysozyme) in complex with the inhibitor hexa-N-acetylchitohexaose (NAG6) (PDB: 3D3D) has been reported previously showing sugar units from two molecules of NAG6 bound in the active site. One NAG6 is bound with four sugar units in the ABCD sites and the other with two sugar units in the E'F' sites potentially representing the cleavage reaction products; each NAG6 cross links two neighboring λ lysozyme molecules. Here we use NMR and MD simulations to study the interaction of λ lysozyme with the inhibitors NAG4 and NAG6 in solution. This allows us to study the interactions within the complex prior to cleavage of the polysaccharide. 1 HN and 15 N chemical shifts of λ lysozyme resonances were followed during NAG4/NAG6 titrations. The chemical shift changes were similar in the two titrations, consistent with sugars binding to the cleft between the upper and lower domains; the NMR data show no evidence for simultaneous binding of a NAG6 to two λ lysozyme molecules. Six 150 ns MD simulations of λ lysozyme in complex with NAG4 or NAG6 were performed starting from different conformations. The simulations with both NAG4 and NAG6 show stable binding of sugars across the D/E active site providing low energy models for the enzyme-inhibitor complexes. The MD simulations identify different binding subsites for the 5th and 6th sugars consistent with the NMR data. The structural information gained from the NMR experiments and MD simulations have been used to model the enzyme-peptidoglycan complex.

中文翻译：

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噬菌体 lambda 溶菌酶与四-和六-N-乙酰壳六糖复合物的 NMR 和 MD 研究。


先前已报道过来自 lambda 噬菌体的溶菌酶（λ 溶菌酶）与抑制剂六-N-乙酰壳六糖 (NAG6) (PDB: 3D3D) 复合物的 X 射线结构，显示来自结合在活性位点的两个 NAG6 分子的糖单元。一个 NAG6 在 ABCD 位点与四个糖单元结合，另一个在 E'F' 位点与两个糖单元结合，可能代表裂解反应产物；每个 NAG6 交联两个相邻的 λ 溶菌酶分子。在这里，我们使用 NMR 和 MD 模拟来研究溶液中 λ 溶菌酶与抑制剂 NAG4 和 NAG6 的相互作用。这使我们能够在多糖裂解之前研究复合物内的相互作用。 NAG4/NAG6 滴定期间跟踪 λ 溶菌酶共振的 1 HN 和 15 N 化学位移。两次滴定中的化学位移变化相似，与糖与上下结构域之间的裂缝结合一致； NMR 数据显示没有证据表明 NAG6 与两个 λ 溶菌酶分子同时结合。从不同的构象开始，对与 NAG4 或 NAG6 复合的 λ 溶菌酶进行了六次 150 ns MD 模拟。 NAG4 和 NAG6 的模拟显示糖在 D/E 活性位点上的稳定结合，为酶-抑制剂复合物提供了低能量模型。 MD 模拟识别出第五和第六糖的不同结合子位点，与 NMR 数据一致。从 NMR 实验和 MD 模拟中获得的结构信息已用于对酶-肽聚糖复合物进行建模。