Structure and dynamics of the active site of hen egg-white lysozyme from atomic resolution neutron crystallography

IF 4.4 2区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Structure Pub Date : 2024-11-21 DOI:10.1016/j.str.2024.10.030

Joao Ramos, Valerie Laux, Sax A. Mason, Marie-Hélène Lemée, Matthew W. Bowler, Kay Diederichs, Michael Haertlein, V. Trevor Forsyth, Estelle Mossou, Sine Larsen, Annette E. Langkilde

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Abstract

Hen egg-white lysozyme (HEWL) is a widely used model protein in crystallographic studies and its enzymatic mechanism has been extensively investigated for decades. Despite this, the interaction between the reaction intermediate and the catalytic Asp52, as well as the orientation of Asn44 and Asn46 side chains, remain ambiguous. Here, we report the crystal structures of perdeuterated HEWL and D₂O buffer-exchanged HEWL from 0.91 and 1.1 Å resolution neutron diffraction data, respectively. These structures were obtained at room temperature and acidic pH, representing the active state of the enzyme. The unambiguous assignment of hydrogen positions based on the neutron scattering length density maps elucidates the roles of Asn44, Asn46, Asn59, and nearby water molecules in the stabilization of Asp52. Additionally, the identification of hydrogen positions reveals unique details of lysozyme’s folding, hydrogen (H)/deuterium (D) exchange, and side chain disorder.

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通过原子分辨率中子晶体学研究母鸡卵白溶菌酶活性位点的结构和动力学特征

母鸡卵白溶菌酶（HEWL）是晶体学研究中广泛使用的模型蛋白质，几十年来，人们对其酶学机制进行了广泛研究。尽管如此，反应中间体与催化剂 Asp52 之间的相互作用以及 Asn44 和 Asn46 侧链的取向仍然模糊不清。在此，我们分别从 0.91 和 1.1 Å 分辨率的中子衍射数据中报告了氚化 HEWL 和 D2O 缓冲交换 HEWL 的晶体结构。这些结构是在室温和酸性 pH 下获得的，代表了酶的活性状态。基于中子散射长度密度图的氢位置的明确分配阐明了 Asn44、Asn46、Asn59 和附近水分子在稳定 Asp52 中的作用。此外，氢位置的确定揭示了溶菌酶折叠、氢（H）/氘（D）交换和侧链紊乱的独特细节。

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来源期刊

Structure 生物-生化与分子生物学

CiteScore

8.90

自引率

1.80%

发文量

155

审稿时长

3-8 weeks

期刊介绍： Structure aims to publish papers of exceptional interest in the field of structural biology. The journal strives to be essential reading for structural biologists, as well as biologists and biochemists that are interested in macromolecular structure and function. Structure strongly encourages the submission of manuscripts that present structural and molecular insights into biological function and mechanism. Other reports that address fundamental questions in structural biology, such as structure-based examinations of protein evolution, folding, and/or design, will also be considered. We will consider the application of any method, experimental or computational, at high or low resolution, to conduct structural investigations, as long as the method is appropriate for the biological, functional, and mechanistic question(s) being addressed. Likewise, reports describing single-molecule analysis of biological mechanisms are welcome. In general, the editors encourage submission of experimental structural studies that are enriched by an analysis of structure-activity relationships and will not consider studies that solely report structural information unless the structure or analysis is of exceptional and broad interest. Studies reporting only homology models, de novo models, or molecular dynamics simulations are also discouraged unless the models are informed by or validated by novel experimental data; rationalization of a large body of existing experimental evidence and making testable predictions based on a model or simulation is often not considered sufficient.