The active site of hen egg-white lysozyme: flexibility and chemical bonding

Jeanette Held,Sander Van Smaalen

doi:10.1107/s1399004714001928

Abstract

Chemical bonding at the active site of hen egg-white lysozyme (HEWL) is analyzed on the basis of Bader's quantum theory of atoms in molecules [QTAIM; Bader (1994), Atoms in Molecules: A Quantum Theory. Oxford University Press] applied to electron-density maps derived from a multipole model. The observation is made that the atomic displacement parameters (ADPs) of HEWL at a temperature of 100 K are larger than ADPs in crystals of small biological molecules at 298 K. This feature shows that the ADPs in the cold crystals of HEWL reflect frozen-in disorder rather than thermal vibrations of the atoms. Directly generalizing the results of multipole studies on small-molecule crystals, the important consequence for electron-density analysis of protein crystals is that multipole parameters cannot be independently varied in a meaningful way in structure refinements. Instead, a multipole model for HEWL has been developed by refinement of atomic coordinates and ADPs against the X-ray diffraction data of Wang and coworkers [Wang et al. (2007), Acta Cryst. D63, 1254-1268], while multipole parameters were fixed to the values for transferable multipole parameters from the ELMAM2 database [Domagala et al. (2012), Acta Cryst. A68, 337-351] . Static and dynamic electron densities based on this multipole model are presented. Analysis of their topological properties according to the QTAIM shows that the covalent bonds possess similar properties to the covalent bonds of small molecules. Hydrogen bonds of intermediate strength are identified for the Glu35 and Asp52 residues, which are considered to be essential parts of the active site of HEWL. Furthermore, a series of weak C-H...O hydrogen bonds are identified by means of the existence of bond critical points (BCPs) in the multipole electron density. It is proposed that these weak interactions might be important for defining the tertiary structure and activity of HEWL. The deprotonated state of Glu35 prevents a distinction between the Phillips and Koshland mechanisms.

Highlights

The three-dimensional structure of the antibacterial hen eggwhite lysozyme (HEWL) was uncovered in 1965 (Blake et al, 1965), heralding enzyme crystallography
Following two earlier reports on amino acids (Mondal et al, 2012; Prathapa et al, 2013), we present here the dynamic electron density of the independent atom model (IAM) and multipole model of HEWL, along with an analysis of their topological properties
Since negative electron densities are nonphysical, their presence in sMtaPt(r) indicates a shortcoming of the model, which may be the result of using values of the multipole parameters from the ELMAM2 database instead of using their true values

Summary

Introduction

The three-dimensional structure of the antibacterial hen eggwhite lysozyme (HEWL) was uncovered in 1965 (Blake et al, 1965), heralding enzyme crystallography. Lysozyme resides among the most intensively studied enzymes and its function is well known. According to the Phillips mechanism (Phillips, 1966), the two residues Glu (glutamic acid) and Asp (aspartic acid) (Fig. 1) play an essential role. The terminal proton of Glu is transferred to the O atom of the glycosidic bond between two. D70, 1136–1146 research papers neighbouring sugar residues, leading to the cleavage of the glycosidic bond and the formation of a carbenium ion. The positive charge of this carbenium ion is stabilized by the negative charge of Asp until a hydroxyl ion binds to the positive C atom and Glu is reprotonated

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Conclusion