A structurally conserved water molecule in Rossmann dinucleotide-binding domains

Abstract

A computational comparison of 102 high-resolution (</=1.90 A) enzyme-dinucleotide (NAD, NADP, FAD) complexes was performed to investigate the role of solvent in dinucleotide recognition by Rossmann fold domains. The typical binding site contains about 9-12 water molecules, and about 30% of the hydrogen bonds between the protein and the dinucleotide are water mediated. Detailed inspection of the structures reveals a structurally conserved water molecule bridging dinucleotides with the well-known glycine-rich phosphate-binding loop. This water molecule displays a conserved hydrogen-bonding pattern. It forms hydrogen bonds to the dinucleotide pyrophosphate, two of the three conserved glycine residues of the phosphate-binding loop, and a residue at the C-terminus of strand four of the Rossmann fold. The conserved water molecule is also present in high-resolution structures of apo enzymes. However, the water molecule is not present in structures displaying significant deviations from the classic Rossmann fold motif, such as having nonstandard topology, containing a very short phosphate-binding loop, or having alpha-helix "A" oriented perpendicular to the beta-sheet. Thus, the conserved water molecule appears to be an inherent structural feature of the classic Rossmann dinucleotide-binding domain.

Fig. 1.

Chemical structures and nomenclature for (A) NAD(P)⁺ and (B) FAD.

Fig. 2.

Classic Rossmann fold topology. The arrows designate β-strands and rectangles denote α-helices. The circles represent conserved glycine residues.

Fig. 3 .

Protein/dinucleotide hydrogen bonds by groups. Hydrogen bonds to each group are divided into direct and water-mediated categories. Their sum represents the total number of hydrogen bonds between the protein and dinucleotide. The various groups of the three dinucleotides are abbreviated as follows: N, nicotinamide; Rn, nicotinamide ribose; P, pyrophosphate; Ra, adenine ribose; A, adenine; F, flavin isoalloxazine; Rtl, flavin ribityl side chain.

Fig. 4.

Superposition of protein structures. (A) Stereoimage showing the dinucleotides, phosphate-binding loops, and structurally conserved water molecules of 37 enzymes. (B) Stereoimage showing the structurally conserved water molecules of 77 structures superimposed according to their glycine-rich loops as described in Materials and Methods. The NADH and protein shown are from alcohol dehydrogenase (1HET). In both (A) and (B), the oxygen atoms of the water molecules are shown as red spheres with 1/5 van der Waals radii. Figures created using MOLSCRIPT (Kraulis 1991) and Raster3D (Merritt and Bacon 1997).

Fig. 5.

Hydrogen bonding patterns of the structurally conserved water molecule. (A) Schematic of the hydrogen bonds formed by the structurally conserved water molecule. The dotted lines denote hydrogen bonds. (B) An example of the structurally conserved water molecule's hydrogen bond coordination as seen in phenylalanine dehydrogenase of Rhodococcus sp. M4 (PDB code 1C1D). Gly184 and Gly187 donate hydrogen bonds via their amide nitrogens, while the carbonyl of Cys238 accepts a hydrogen bond. Only backbone atoms are shown. Distances are in angstroms.