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

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. [Display omitted] •Structures of lysozyme determined using atomic resolution neutron diffraction data•Comparison between perdeuterated and D2O buffer-exchanged lysozyme structures•Unambiguous assignment of Glu35 and Asp52 protonation in the active site•The role of Asn44, Asn46, and Asn59 in the stabilization of Asp52 by hydrogen bonds Ramos et al. report crystal structures of perdeuterated and hydrogenated hen egg-white lysozyme from atomic resolution neutron and X-ray diffraction data. The unambiguous assignment of hydrogen positions, protonation states, and water networks, based on the neutron scattering length density maps, resolves previous ambiguity of important features of the active site.