Hydrogen atoms in protein structures: high-resolution X-ray diffraction structure of the DFPase

Abstract

BackgroundHydrogen atoms represent about half of the total number of atoms in proteins and are often involved in substrate recognition and catalysis. Unfortunately, X-ray protein crystallography at usual resolution fails to access directly their positioning, mainly because light atoms display weak contributions to diffraction. However, sub-Ångstrom diffraction data, careful modeling and a proper refinement strategy can allow the positioning of a significant part of hydrogen atoms.ResultsA comprehensive study on the X-ray structure of the diisopropyl-fluorophosphatase (DFPase) was performed, and the hydrogen atoms were modeled, including those of solvent molecules. This model was compared to the available neutron structure of DFPase, and differences in the protein and the active site solvation were noticed.ConclusionsA further examination of the DFPase X-ray structure provides substantial evidence about the presence of an activated water molecule that may constitute an interesting piece of information as regard to the enzymatic hydrolysis mechanism.

Systematic analysis of residual density suggests that a major limitation in well‐refined X‐ray structures of proteins is the omission of ordered solvent

“…). In H atom‐deleted models, the highest positive residual peak for deleted H atoms was reported to be only at 4.20 σ , which ranks at the 283‐th of all the positive peaks in the residual ED map calculated here. After careful model re‐refinement for 3O4P that has already included H atoms, large extra peaks were observed in the residual ED map outside of the Cβ‐H groups of K210 and E104 and outside of the Cγ‐H group of K210 [Fig.…”

“… Analysis of diisopropyl fluorophosphatase model at 0.85‐Å resolution (3O4P) . (a) Residual positive (green) and negative (red) peaks as a function of peak number.…”

“…Thus, there is no doubt that missing ordered water molecules appears a major obstacle to the completeness of model. This is likely to be the main reason why protein crystallographers have a difficult time to see H atoms in ED maps . Recent interpretations of cryo‐electron microscopy image reconstruction suggest that ionization states affect both X‐ray and electron structure factors .…”

“…This model is one of the most complete protein models in the PDB examined so far (Table I), and has the smallest R-factor gap with R Ratio of 1.22 (see Methods). [4][5][6][7][8][9][10][11][12][13][14][15] Evidence will be provided below that its corresponding residual ED map is the closest to true featurelessness with all the residual peaks contributed mainly by random noise present in the intensity data.…”

What roles do the residue Asp229 and the coordination variation of calcium play of the reaction mechanism of the diisopropyl-fluorophosphatase? A DFT investigation

“…However, a recent report of a high-resolution X-ray structure of DFPase suggests that a third water molecule, bound to the catalytic Ca 2+ , shares a proton with Asp229. Elias et al [22] proposed that this water molecule is partially activated and may play a role in enzyme regeneration. Quantum mechanical/molecular mechanical umbrella sampling simulations displayed that the hydrolysis of diisopropyl fluorophosphate (DFP) and (S)-sarin processes by DFPase presents two different reaction pathways involving nucleophilic attack by Asp229 or an activated water on phosphorus.…”