Rapid determination of hydrogen positions and protonation states of diisopropyl fluorophosphatase by joint neutron and X-ray diffraction refinement

Abstract

Hydrogen atoms constitute about half of all atoms in proteins and play a critical role in enzyme mechanisms and macromolecular and solvent structure. Hydrogen atom positions can readily be determined by neutron diffraction, and as such, neutron diffraction is an invaluable tool for elucidating molecular mechanisms. Joint refinement of neutron and X-ray diffraction data can lead to improved models compared with the use of neutron data alone and has now been incorporated into modern, maximum-likelihood based crystallographic refinement programs like CNS. Joint refinement has been applied to neutron and X-ray diffraction data collected on crystals of diisopropyl fluorophosphatase (DFPase), a calcium-dependent phosphotriesterase capable of detoxifying organophosphorus nerve agents. Neutron omit maps reveal a number of important features pertaining to the mechanism of DFPase. Solvent molecule W33, coordinating the catalytic calcium, is a water molecule in a strained coordination environment, and not a hydroxide. The smallest Ca-O-H angle is 53 degrees, well beyond the smallest angles previously observed. Residue Asp-229, is deprotonated, supporting a mechanism involving nucleophilic attack by Asp-229, and excluding water activation by the catalytic calcium. The extended network of hydrogen bonding interactions in the central water filled tunnel of DFPase is revealed, showing that internal solvent molecules form an important, integrated part of the overall structure.

Fig. 1.

Overall structure of DFPase and backbone H/D exchange characteristics. (A) Top view, with the 2 calcium ions represented as blue spheres. (B) Side view, rotated 90° from a). The occupancy of backbone amide hydrogens is scaled from blue (H, unexchanged) via green and yellow (partially exchanged) to red (D, exchanged).

Fig. 2.

Schematic representation of possible mechanisms for DFPase. (A) Direct nucleophilic attack of Asp-229 on the substrate, with a phosphoenzyme intermediate and a fluoride leaving group. (B) Mechanism involving a calcium-bound hydroxide ion as the active nucleophile.

Fig. 3.

Signature features of nuclear density in proteins. (A) Unexchanged backbone amide hydrogens can be identified by the lack of positive nuclear density for these atoms as shown for Asn-238 (exchanged) and Asn-239 (not exchanged). The sidechain of Asn-239 also shows the weak neutron scattering power of a CH₂ group. Additional information from electron density in joint refinement helps to correctly position the carbon atom. (B) Density for the side-chain amide of Asn-272 allows for unambiguous assignment of the oxygen and nitrogen due to the greater neutron scattering of the NH₂ group and the bean shaped density of W32 allows determining the orientation of that water molecule. Also in this case additional information from electron density in joint refinement helps to correctly position the oxygen atom. (C) Nuclear density for active site residues His-287 and Trp-244 clearly indicating the protonation states of the imidazole and indole ring nitrogen atoms. Hydrogen bonding is indicated by a red line.

Fig. 4.

Schematic representation of hydrogen bonding interactions in the central water tunnel of DFPase. Arrows point from H-bond donor to acceptor. Dashed lines indicate direct metal coordination. Blue circles represent water molecules, amino acids are boxed colored according to the propeller blade they belong to.

Fig. 5.

Active site environment. (A) Catalytic Ca1 with surrounding residues and positive nuclear density. Yellow lines indicate direct calcium coordination. Hydrogen bonding interactions are indicated in red. For the poorly resolved water W140 electron density is displayed (transparent gray sphere) showing the position of the oxygen atom. Nucleophilic Asp-229 is deprotonated. (B) A F_o-F_c nuclear density omit map (blue), with the 2 deuterium atoms of W33 omitted, contoured at 4.0 σ, showing identity of W33 as a water, and not a hydroxide ion. The electron density 2F_o-F_c map at 2.5 σ is superimposed (red), showing the location of the oxygen atom. Distances are shown in Å. The W33 hydrogen–Ca1 distance is indicated as a blue line. The Ca–O–H angle is 53°.