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

Ramos, Joao ; Laux, Valerie ; Mason, Sax A. ; Lemée, Marie Hélène ; Bowler, Matthew W. ; Diederichs, Kay ; Haertlein, Michael ; Forsyth, V. Trevor LU ; Mossou, Estelle and Larsen, Sine , et al. (2025) In Structure 33(1). p.3-148
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,... (More)

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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organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
active site, atomic resolution, deuteration, H-bonds, H/D exchange, hen egg-white lysozyme, neutron crystallography, protonation states, X-ray crystallography
in
Structure
volume
33
issue
1
pages
3 - 148
publisher
Cell Press
external identifiers
  • pmid:39577430
  • scopus:85211337604
ISSN
0969-2126
DOI
10.1016/j.str.2024.10.030
language
English
LU publication?
yes
id
a563e8e4-3a59-4f4c-959e-697058a59f8c
date added to LUP
2025-02-26 14:51:30
date last changed
2025-07-03 01:54:42
@article{a563e8e4-3a59-4f4c-959e-697058a59f8c,
  abstract     = {{<p>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 D<sub>2</sub>O 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.</p>}},
  author       = {{Ramos, Joao and Laux, Valerie and Mason, Sax A. and Lemée, Marie Hélène and Bowler, Matthew W. and Diederichs, Kay and Haertlein, Michael and Forsyth, V. Trevor and Mossou, Estelle and Larsen, Sine and Langkilde, Annette E.}},
  issn         = {{0969-2126}},
  keywords     = {{active site; atomic resolution; deuteration; H-bonds; H/D exchange; hen egg-white lysozyme; neutron crystallography; protonation states; X-ray crystallography}},
  language     = {{eng}},
  number       = {{1}},
  pages        = {{3--148}},
  publisher    = {{Cell Press}},
  series       = {{Structure}},
  title        = {{Structure and dynamics of the active site of hen egg-white lysozyme from atomic resolution neutron crystallography}},
  url          = {{http://dx.doi.org/10.1016/j.str.2024.10.030}},
  doi          = {{10.1016/j.str.2024.10.030}},
  volume       = {{33}},
  year         = {{2025}},
}