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. 2002 Sep;11(9):2125–2137. doi: 10.1110/ps.0213502

A structurally conserved water molecule in Rossmann dinucleotide-binding domains

Christopher A Bottoms 1, Paul E Smith 2, John J Tanner 1
PMCID: PMC2373605  PMID: 12192068

Abstract

A computational comparison of 102 high-resolution (≤1.90 Å) 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 α-helix "A" oriented perpendicular to the β-sheet. Thus, the conserved water molecule appears to be an inherent structural feature of the classic Rossmann dinucleotide-binding domain.

Keywords: NAD, NADP, FAD, dinucleotide-protein interactions, Rossmann fold, structurally conserved water molecule, molecular recognition

Flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), and nicotinamide adenine dinucleotide phosphate (NADP) serve as enzyme cofactors in many essential biologic processes, such as glycolysis (NAD), the citric acid cycle (FAD and NAD), and photosynthesis (NADP) (Fig. 1 ▶).

Fig. 1.

Fig. 1.

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

Because of the central role played by adenine dinucleotides in biologic redox chemistry, many dinucleotide-dependent enzymes are potential drug design targets for the treatment of cancer (Konno et al. 1991; Nagai et al. 1991; Franchetti et al. 1998; Faig et al. 2001), bacterial infections (Zhang et al. 1999), and trypanosomal diseases (Verlinde et al. 1994; Verlinde and Hol 1994; Van Calenbergh et al. 1995; Aronov et al. 1998; Bressi et al. 2001). Knowledge of the structural and dynamic features that govern dinucleotide recognition by enzymes is important for understanding the many biochemical roles of dinucleotides and for designing analogs for therapeutic applications.

The Rossmann fold was first identified in dinucleotide-binding proteins (Rossmann et al. 1974) and remains one of the most thoroughly studied dinucleotide-binding folds. The Rossmann fold, or mononucleotide-binding motif, is a single βαβαβ motif that binds a mononucleotide. The fold that binds NAD and NADP consists of two mononucleotide-binding motifs that are structurally related by a pseudo twofold rotation, with the most N-terminal strands adjacent to each other (Fig. 2 ▶). Together, the two βαβαβ motifs form a six-stranded parallel β-sheet flanked by α-helices, with relative strand order 321456. The fold that binds FAD typically contains one mononucleotide-binding motif with two additional parallel β-strands, giving a relative strand order of 32145 (Murzin et al. 1995).

Fig. 2.

Fig. 2.

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

Although dinucleotide-binding domains show very low overall sequence homology, large portions of their protein backbones superimpose very well (Branden and Tooze 1991). However, there are two common sequence features. First, a glycine-rich phosphate-binding loop connects the C-terminus of β1 with the N-terminus of αA (Wierenga et al. 1985) (Fig. 2 ▶). Typically, this loop contains three conserved glycine residues arranged in patterns such as GXGXXG. Mutations in the conserved glycine residues of the loop have been correlated with attenuation or elimination of enzyme activity (Rescigno and Perham 1994; Nishiya and Imanaka 1996; Eschenbrenner et al. 2001) and also with disease (van Grunsven et al. 1998). The second feature is a side chain interaction with the adenine ribose group of the dinucleotide. In NAD- and FAD-binding Rossmann fold proteins, the carboxylate of Asp or Glu interacts with the adenine ribose hydroxyls. In contrast, NADP-binding proteins typically have Arg interacting with the monophosphate at the O2` position of adenine ribose (Branden and Tooze 1991).

The conserved characteristics of dinucleotide-binding proteins have been described in several studies (Rossmann et al. 1974; Lesk 1995); however, the role that structural water plays in dinucleotide recognition has not been extensively analyzed. Five years ago, Carugo and Argos (1997) reported a study of NAD(P)–protein interactions using a data set of 32 NAD(P)–enzyme complexes representing 19 enzymes. The resolution of these structures ranged from 1.6 to 3.20 Å, averaging 2.30 Å, and only eight structures had 1.9 Å resolution or better. Since their work, many high-resolution (≤1.90 Å) structures have become available for study. Here we describe the results of a survey of 102 high-resolution enzyme/dinucleotide complexes that display the Rossmann dinucleotide-binding fold. These higher resolution structures allow an accurate description of the role of solvent in dinucleotide recognition. We find that bridging water molecules contributes significantly to the dinucleotide-binding interface in all of the enzymes studied. Moreover, we identified a structurally conserved water molecule that links, through hydrogen bonding, the glycine-rich loop and the dinucleotide pyrophosphate moiety. We assert that this conserved water molecule is an integral characteristic of dinucleotide-binding Rossmann fold domains, and thus it contributes significantly to dinucleotide recognition.

Results

Data set of structures analyzed

All crystal structures (as of January 2002) in the Protein Data Bank (PDB) (Berman et al. 2000) with NAD(P) or FAD (Fig. 1 ▶) bound to a Rossmann fold motif and having a resolution of 1.9 Å or better were selected for analysis. The resulting data set consists of 102 structures, and represents 43 enzymes and 40 species (Tables 1, 2, and 3). The crystallographic resolution ranges from 1.15 to 1.90 Å, averaging 1.70 Å. Notwithstanding our conservative resolution cutoff, this study is one of the largest comparisons of dinucleotide-binding proteins performed to date.

Table 1.

NAD-binding proteins

Enzyme Species PDB codes and references Sequence pattern Wat
(G...XGXXG)
Alcohol dehydrogenase Equus caballus 1EE2 (Adolph et al. 2000); 1HET, 1HEU (Meijers et al. 2001); 20HX (AI-Karadaghi et al. 1994); 3BTO (Cho et al. 1997) G...LGGVG ✓
3-Dehydroquinate synthase Aspergillus nidulans 1DQS(Carpenter et al. 1998) G...GGVIG
GAPDH Bacillus stearothermophilus 1GD1 (Skarzynski et al. 1987) G...PGRIG ✓
Escherichia coli 1GAD (Duee et al. 1996) G...PGRIG ✓
l-3-Hydroxyacyl-CoA dh Homo sapiens 1F0Y (Barycki et al. 2000) G...GGLMG ✓
d-2-Hydroxyisocaproate dh Lactobacillus casei 1DXY (Dengler et al. 1997) G...TGHIG ✓
Lactate dehydrogenase Plasmodium falciparum 1LDG (Dunn et al. 1996) G...SGMIG ✓
Phenylalanine dh Rhodococcus sp, M4 1BW9 (Vanhooke et al. 1999); 1C1D, 1C1X (Brunhuber et al. 2000) G...LGAVG ✓
(G..XXGXXG)
dTDP-Glucose 4,6- dehydratase Escherichia coli 1BXK (Hegeman et al. 2001) G..GAGFIG ✓
Malate dehydrogenase Escherichia coli 1EMD (Hall and Banaszak 1993) G..AAGGIG ✓
Thermus flavus 1BMD (Kelly et al. 1993) G..AAGQIG ✓
Sulfolipid biosynthesis Arabidopsis thaliana 1QRR (Mulichak et al. 1999) G..GDGYCG ✓
UDP-Galactose 4-epimerase Escherichia coli 1NAH (Thoden et al. 1996a); 1UDA, 1UDB, 1UDC (Thoden et al. 1997); 1XEL (Thoden et al. 1996c); 2UDP (Thoden et al. 1996b) G..GSGYIG ✓
Homo sapiens 1EK5, 1EK6 (Thoden et al. 2000); 1HZJ (Thoden et al. 2001) G..GAGYIG ✓
(G..XXXGXG)
7-α-Hydroxysteroid dh Escherichia coli 1FMC (Tanaka et al. 1996b) G..AGAGIG ✓
d-Glucose-1-dehydrogenase Bacillus megaterium 1GCO (Yamamoto et al. 2001) G..SSTGLG ✓
l-3-Hydroxyacyl-CoA dh Rattus norvegicus 1E6W (Powell et al. 2000) G..GASGLG ✓
meso-2,3-Butanediol dh Klebsiella pneumoniae 1GEG (Otagiri et al. 2001) G..AGQGIG ✓
(GXXXXXXXG)
Enoyl ACP reductase Brassica napus 1D7O (Roujeinikova et al. 1999); 1ENO (Rafferty et al. 1995) GIADDNGYG ✓
Escherichia coli 1QG6 (Ward et al. 1999); 1QSG (Stewart et al. 1999) GVASKLSIA ✓
NMN adenylyltransferase Methanobacterium thermoautotrophicum 1EJ2 (Saridakis et al. 2001) GRMQPFHRG
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Table 2.

NADP-binding proteins

Enzyme Species PDB codes and references Sequence pattern Wat
(G...XGXXG)
Acetohydroxy acid isomeroreductase Spinacia oleracea 1YVE(Biou et al. 1997) G...WGSQA ✓
Adrenodoxin reductase Bos taurus 1E1M(Ziegler and Schulz 2000) G...QGNVA ✓
Glutathione reductase Homo sapiens 1GRB(Karplus and Schulz 1989) G...AGYIA ✓
NADP(H) transhydrogenase Bos taurus 1D4O(Prasad et al. 1999) G...YGLCA
Biliverdin-IX β reductase Homo sapiens 1HDO, 1HE2, 1HE3, 1HE4, 1HE5(Pereira et al. 2001) (G..XXGXXG) G..ATGQTG ✓
Coenzyme F420H2: NADP+- oxidoreductase (Fno) Archaeoglobus fulgidus 1JAY(Warkentin et al. 2001) G..GTGNLG ✓
GDP 4-keto-6-deoxy-d- mannose epimerase rd Escherichia coli 1E6U(Rosano et al. 2000) G..HRGMVG ✓
l-Lactate/malate dh Methanococcus jannaschil 1HYE(Lee et al. 2001) G..ASGRVG ✓
1,3,6,8-Tetrahydroxynaphthalene reductase Magnaporthe grisea 1JA9(Liao et al. 2001) (G..XXXGXG) G..AGRGIG ✓
Carbonyl reductase Mus musculus 1CYD(Tanaka et al. 1996a) G..AGKGIG ✓
Mannitol dehydrogenase Agaricus bisporus 1H5Q(Horer et al. 2001) G..GNRGIG ✓
Nitric-oxide synthase Rattus norvegicus 1F20(Zhang et al. 2001) G..PGTG(IA)
Sepiapterin reductase Mus musculus 1OAA(Auerbach et al. 1997) G..ASRGFG ✓
Tropinone reductase-II Datura stramonium 2AE2(Yamashita et al. 1999) G..GSRGIG ✓
(G..XXXXXG)
Methylenetetrahydrofolate dh Homo sapiens 1A4I(Allaire et al. 1998) G..RSKIVG ✓
Pteridine reductase Leishmania major 1E7W(Gourley et al. 2001) G..AAKRLG ✓
Other
Dihydrofolate reductase Candida albicans 1AI9, 1AOE(Whitlow et al. 1997); 1IA1, 1IA2, 1IA3, 1IA4(Whitlow et al. 2001) GRKT
Escherichia coli 1RA2, 1RA3, 1RA4, 1RC4, 1RX2, 1RX9(Sawaya and Kraut 1997) GGGRV
Gallus gallus 8DFR(Matthews et al. 1985) GKKT
Lactobacillus casei 3DFR(Bolin et al. 1982) GRRT
Myobacterium tuberculosis 1DF7, 1DG7(Li et al. 2000) GRRT
Pneumocystis carinii 1DYR(Champness et al. 1994); 2CD2(Cody et al. 1999) GRKT
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Table 3.

FAD-binding proteins

Enzyme Species PDB codes and references Sequence pattern Wat
(G...XGXXG)
Adrenodoxin reductase Bos taurus 1CJC(Ziegler et al. 1999); 1E1M(Ziegler and Schulz 2000) G...SGPAG ✓
Alkyl hydroperoxide rd Escherichia coli 1FL2(Bieger and Essen 2001) G...SGPAG ✓
Cholesterol oxidase Brevibacterium sterolicum 1COY, 3COX (Li et al. 1993) G...SGYGG ✓
Streptomyces sp. 1B4V(Yue et al. 1999) G...TGYGA ✓
d-Amino acid oxidase Rhodotorula gracilis 1C0K, 1C0L, 1C0P(Umhau et al. 2000) G...SGVIG ✓
Dihydropyrimidine dh Sus scrofa 1H7W(Dobritzsch et al. 2001) G...AGPAS ✓
Flavocytochrome C3 Shewanella frigidimarina 1QJD(Taylor et al. 1999) G...SGGAG ✓
Glucose oxidase Aspergillus niger 1CF3(Wohlfahrt et al. 1999) G...GGLTG ✓
Penicillium amagasakiense 1GPE(Wohlfahrt et al. 1999) G...GGLTG ✓
Glutathione reductase Escherichia coli 1GER(Mittl and Schulz 1994) G...GGSGG ✓
Homo sapiens 1DNC, 1GSN(Becker et al. 1998); 1GRB (Karplus and Schulz 1989); 3GRS (Karplus and Schulz 1987) G...CGSGG ✓
p-Hydroxybenzoate hydroxylase Pseudomonas fluorescens 1PBE(Schreuder et al. 1989) G...AGPGC ✓
Polyamine oxidase Zea mays 1B37, 1B5Q(Binda et al. 1999); 1H82, 1H83(Binda et al. 2001) G...AGMSG ✓
Sarcosine oxidase Bacillus sp. 1EL5, 1EL7, 1EL8(Wagner et al. 2000) G...AGSMG ✓
Trypanothione reductase Crithidia fasciculata 1FEC(Strickland and Karplus 1995) G...AGSGG ✓
Other
Quinone reductase Homo sapiens 1D4A(Faig et al. 2000); 1H69(Falg et al. 2001); 1KBQ(Winski et al. 2001) AHSERTSFNY
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Tables 1, 2, and 3 show alignments of the glycine-rich pyrophosphate-binding sequences for the enzymes studied. The majority of enzymes in our data set display the pyrophosphate-binding loop sequence patterns GXGXXG, GXXGXXG, or GXXXGXG, where G is Gly and X is any amino acid residue. Two enzymes have the sequence pattern GXXXXXG, and two have the pattern GXXXXXXXG. Note that sometimes Ala substitutes for the last Gly, and, in one case, dihydropyrimidine dehydrogenase, Ser substitutes for the last Gly. Note that all the FAD-binding proteins in our data set except one have the phosphate-binding loop sequence GXGXXG. Quinone reductase does not have a glycine-rich loop, although it is still classified as a Rossmann fold protein.

Identification of a structurally conserved water molecule

All the structures in our database have several water molecules in the interface between the dinucleotide and the protein. On average, FAD-, NADP-, and NAD-binding proteins accommodate about 12, 11, and 9 interfacial (within 3.75Å of the protein and dinucleotide) water molecules per dinucleotide-binding site, respectively. As expected, many of these interfacial water molecules hydrogen bond to both the dinucleotide and the protein, thus forming water-mediated hydrogen bonds. Figure 3 ▶ shows the average numbers of direct and water-mediated hydrogen bonds formed between the protein and the five groups of each of the dinucleotides. Water-mediated hydrogen bonds comprise 29, 32, and 29% of the protein/dinucleotide hydrogen bonds for NAD, NADP, and FAD, respectively. The pyrophosphate group forms almost as many water-mediated hydrogen bonds (2.7 for NAD, 2.4 for NADP, and 3.5 for FAD) as direct hydrogen bonds (2.8 for NAD, 3.2 for NADP, and 4.1 for FAD) to the protein. The remaining groups average one or fewer water-mediated hydrogen bonds, with the exception of adenine ribose in NADP. This group averages more than two water-mediated hydrogen bonds, a result due to its monophosphate. Thus, bridging water molecules are concentrated around the pyrophosphate and monophosphate groups, although they are commonly found associated with the rest of the dinucleotide as well.

Fig. 3 .

Fig. 3 .

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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.

The presence of numerous water-mediated hydrogen bonds between protein and dinucleotides implicates water as a significant component of dinucleotide recognition. Superimposition of the structures allowed us to identify whether any of the bridging water molecules bound in structurally conserved sites. The crystal structures were superimposed using the coordinates of only three alpha carbon atoms within the glycine-rich loop, as described in Materials and Methods. This analysis revealed a structurally conserved water molecule located at the N-terminus of the phosphate binding helix (i.e., αA) in 77 structures, which represents 37 of 43 enzymes studied. The structures displaying the conserved water molecule are indicated by the checkmark in Tables 1–3. Figure 4A ▶ shows the superimposed phosphate binding loops, cofactors, and structurally conserved water molecules of these 37 enzymes. Note the almost perfect superimposition of the phosphate moiety, the glycine-rich loop, and the structurally conserved water molecule of these different structures. In contrast, adenine, nicotinamide, and flavin do not superimpose as well. Figure 4B ▶ shows the structurally conserved water molecule from 77 structures after superposition onto the alcohol dehydrogenase structure 1HET, as described in Materials and Methods. Also shown are secondary structurally elements and NADH of 1HET. These water molecules have an RMSD of 0.5 Å from a reference water molecule, which indicates that the water molecules occupy essentially the same location in their respective structures. As will be discussed below, the six enzymes that do not bind this water molecule show significant deviations from the classic Rossmann fold motif.

Fig. 4.

Fig. 4.

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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).

Hydrogen bonds formed by the structurally conserved water molecule

The structurally conserved water molecule typically makes four hydrogen bonds. Two of the four bonds are invariant, and two vary according to the sequence pattern of the glycine-rich phosphate-binding loop. The hydrogen-bonding pattern of the conserved water molecule is shown schematically in Figure 5A ▶, and an example of this hydrogen bond coordination is shown in Figure 5B ▶. A table detailing the hydrogen bonding partners of the structurally conserved water molecule in each dinucleotide-binding site is provided in the electronic supplement.

Fig. 5.

Fig. 5.

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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.

The two invariant hydrogen bonds formed by the conserved water molecule involve pyrophosphate and the amine of the last conserved Gly (Fig. 5 ▶). Thus, the water molecule always links the pyrophosphate to the glycine-rich loop. Moreover, the interaction with the pyrophosphate is stereospecific. Although the pyrophosphate is not chiral, the oxygen atoms are distinct stereochemically (Schultze and Feigon 1997). Almost without exception, the pyrophosphate atom that interacts with the structurally conserved water molecule is O2N in the case of NAD- or NADP-binding proteins. In FAD-binding proteins, however, it is always O1P (i.e., equivalent to O1N of NAD[P]), which interacts with the structurally conserved water molecule. Interestingly, the only two structures in our study that have both FAD and NADP bound are also the only ones in which NADP binds to the structurally conserved water molecule via its pyrophosphate atom O1N (see 1E1M and 1GRB).

The partners for the other two hydrogen bonds formed by the conserved water molecule depend upon the sequence of the glycine-rich loop. One sequence-dependent hydrogen bond involves either the first or second conserved Gly. This bond occurs in structures with the sequence patterns GXGXXG, GXXGXXG, and GXXXXXXXG. It also occurs in seven of the eight structures with the sequence pattern GXXXGXG, but does not occur in structures with the sequence pattern GXXXXXG. In structures with the sequence GXGXXG, the water molecule forms a hydrogen bond with the amino group of the second conserved Gly. An example of this hydrogen-bonding pattern is shown in Figure 5B ▶. In structures with the sequence patterns GXXGXXG, GXXXGXG, and GXXXXXXXG, the water molecule instead forms a hydrogen bond with the carbonyl group of the first conserved Gly.

The second sequence-dependent hydrogen bond involves a C-terminal residue of β4. In structures with the pattern GXGXXG and GXXGXXG, this hydrogen bond usually involves the backbone carbonyl of either a small residue or a hydrophobic residue (i.e., Ala, Cys, Gly, Leu, Phe, Ser, or Val for the structures studied). However, occasionally it will involve the hydroxyl of Ser or Thr. In structures with the sequence pattern GXXXGXG, the C-terminal residue of β4 usually donates a hydrogen bond via the side chain of an Asn. In structures with the sequence pattern GXXXXXXXG, the C-terminal residue of β4 donates a hydrogen bond via a Ser hydroxyl.

The hydrogen bonding patterns observed in structures with the sequence GXXXXXG are minor variations of the paradigm described above. There are two such structures in our data set, 1A4I (human methylenetetrahydrofolate dehydrogenase) and 1E7W (Leishmania major pteridine reductase). The water molecule of 1A4I does not superimpose as well as the others, as is evident from its 1.1-Å separation from the others in Figure 4 ▶. The conserved water molecule of 1A4I displays a hydrogen-bonding pattern identical to that of GXGXXG structures, with Ser of its GRSKIVG sequence playing the role of the second conserved Gly. On the other hand, the hydrogen bond coordination in 1E7W is very similar to that of proteins with the sequence GXXXGXG. The only exception is that the first conserved glycine residue interacts with the structurally conserved water molecule via another water molecule inside the phosphate-binding loop. To our knowledge, this is the only case in which the structurally conserved water molecule forms a hydrogen bond with another water molecule.

Finally, the conserved water molecule sometimes forms a fifth hydrogen bond. For example, in structures with the sequence pattern GXXXGXG, the conserved water molecule forms a fifth hydrogen bond with the carbonyl of the third "X" residue. It is also common to see the methylene of the second or third conserved Gly within hydrogen bonding distance of the structurally conserved water molecule. Several studies have mentioned the presence of CH . . . O hydrogen bonds in NAD(P) interfaces (Carugo and Argos 1997; Chu and Hwang 1998). Although CH . . . O hydrogen bonds are somewhat unconventional, the methylene of glycine can theoretically form a significant hydrogen bond. Such a bond would have about half the energy of a typical hydrogen bond from water (Scheiner et al. 2001).

Structures lacking the conserved water molecule

Six enzymes (3-dehydroquinate synthase, NMN adenylyltransferase, NADP[H] transhydrogenase, nitric-oxide synthase, dihydrofolate reductase, quinone reductase) lack the structurally conserved water molecule (Tables 1–3). These enzymes show significant deviations from the standard Rossmann fold motif in terms of their sequence and/or structure. Thus, the conserved water molecule is found only in classic Rossmann fold structures.

Some of the unusual features of these enzymes are obvious. For example, 3-dehydroquinate synthase has a nonstandard topology in which the phosphate-binding loop connects β4 and αD instead of β1 and αA. No other enzyme in our data set has this unusual topology. In NADP(H) transhydrogenase and dihydrofolate reductase, the long axis of αA runs perpendicular to the β-sheet rather than parallel to it as in all the other structures. NMN adenylyltransferase differs from the other enzymes in our data set because NAD is a product of catalysis rather than a redox cofactor. The pyrophosphate binds near the beginning of the phosphate-binding loop rather than at the end of the loop near the N-terminus of αA as occurs in the classic Rossmann fold structures. Thus, the pyrophosphate interacts with the Rossmann fold in a fundamentally unique way that is related to the nature of the reaction catalyzed.

3-Dehydroquinate synthase, NADP(H) transhydrogenase, and nitric-oxide synthase show a subtle, but very important, deviation from the standard Rossmann fold structure concerning the hydrogen bonding potential of the last residue of their phosphate-binding sequences. In the classic Rossmann fold, this residue occurs at the beginning of αA (Fig. 2 ▶) and its amino group donates one of the invariant hydrogen bonds to the structurally conserved water molecule (Fig. 5A ▶). However, in 3-dehydroquinate synthase, NADP(H) transhydrogenase, and nitric-oxide synthase, this residue cannot hydrogen bond to a water molecule because it is the fifth or later residue of the helix, and its amino group must form an "n + 4" α-helix hydrogen bond with the carbonyl four residues preceding it in the sequence.

Lastly, dihydrofolate reductase and quinone reductase deviate from the classic Rossmann fold because of the length and sequence, respectively, of their phosphate binding loops. The dihydrofolate reductases have short phosphate-binding loops. Their loops consist of four to five residues compared to six to nine residues in the other structures (Tables 1–3). The resulting loop between β1 and αA is not wide enough to accommodate a water molecule. Quinone reductase, on the other hand, has no glycine residues in its phosphate-binding loop. This is a critical feature, because, in the classic Rossmann fold motif, the first Gly of the phosphate-binding loop always occupies the first quadrant (i.e., φ > 0, ξ > 0) of the Ramachandran plot. This region of φ–ξ space is typically inaccessible to nonglycine residues. Thus, the phosphate-binding loop of quinone reductase cannot adopt the backbone conformations typically observed in classic Rossmann fold proteins.

Discussion

Bridging water molecules

NAD, NADP, and FAD have extraordinary hydrogen bonding potential. They have 19, 22, and 22 polar nitrogen and oxygen atoms, respectively. Therefore, it is not surprising that NAD, NADP, and FAD form about 16, 19, and 23 hydrogen bonds with the protein. However, the observation that water molecules mediate about 30% of these hydrogen bonds is a novel result, and indicates that bridging water is an important component of dinucleotide recognition.

More generally, our results demonstrate a pitfall of ignoring water molecules when analyzing protein crystal structures. In the present case, neglecting water molecules would have given the incorrect impression that Rossmann fold domains greatly underutilize the hydrogen bonding potential of dinucleotides. This issue is critical for ligand docking calculations and structure-based drug design and optimization (Marrone et al. 1997; Raymer et al. 1997).

The result that water molecules are important in dinucleotide recognition is perhaps not surprising, because solvent is important in protein recognition of small molecules, DNA, and protein. For example, about 40% of all protein–DNA hydrogen bonds are water mediated (Luscombe et al. 2001), and protein–protein interfaces contain an average of about 22 water molecules and 11 water-mediated hydrogen bonds (Janin 1999).

Structurally conserved water molecule

We discovered a water molecule that bridges the glycine-rich loop and the pyrophosphate in 77 of the 102 structures studied. The structurally conserved water molecule binds at the N-terminus of αA and it displays a conserved hydrogen-bonding pattern (Fig. 4 ▶). Structurally conserved water molecules have also been identified in fatty acid binding proteins (Likic et al. 2000) and serine proteases (Sreenivasan and Axelsen 1992). Water molecules of ligand-binding sites have also been found to be conserved between pairs of homologous proteins from different species (Poornima and Dean 1995). Several authors have noted the presence of a water molecule that we refer to as the structurally conserved water molecule, but they did not realize its ubiquity in Rossmann fold domains or its conserved hydrogen-bonding pattern (Lamzin et al. 1994; Dessen et al. 1995; Rafferty et al. 1995; Dengler et al. 1997; Adolph et al. 2000; Thoden et al. 2000; Horer et al. 2001; Pantano et al. 2002). In NAD-binding proteins, we found the water molecule to be more highly conserved than the carboxylate interaction with the adenine-ribose diol. Not only do the structures in our data set represent a significant cross section of enzymes, but they also represent a variety of species and even biologic kingdoms. Such structural conservation argues strongly for an important functional role.

The structurally conserved water molecule typically forms hydrogen bonds with two of the three conserved glycine residues, a C-terminal residue of β4, and the dinucleotide pyrophosphate. The water molecule always forms hydrogen bonds to both the pyrophosphate and the last conserved Gly, which is part of helix αA. This water-mediated hydrogen bond is significant, because previous studies have noted that direct hydrogen bonding between αA and the pyrophosphate is not optimal. Based on this observation, a favorable electrostatic interaction between the helix dipole of αA and the pyrophosphate has been considered an important factor in dinucleotide recognition (Wierenga et al. 1985). This conclusion, however, was based on analyses of crystal structures that generally lacked water molecules; therefore, water-mediated hydrogen bonds could not be considered. Including water-mediated hydrogen bonds almost doubles the number of observed hydrogen bonds between the protein and pyrophosphate. Due to the high solvent content about the pyrophosphate and the several water-mediated hydrogen bonds, we wonder if the helix dipole would be an important contributing factor to pyrophosphate binding. Additional studies would be needed to resolve this issue.

Structures that exhibit the classic Rossmann fold motif bind the structurally conserved water molecule, whereas structures that have major deviations from the standard Rossmann fold do not. The following classic features are shared by all the structures bearing the conserved water molecule. The β-sheet topology is 321456 for NAD(P)- and 32145 for FAD-binding domains, and αA is parallel to the β-sheet. The phosphate-binding loop is at least six residues long, and it contains Gly at the first position. The last residue of the phosphate-binding sequence is located at the beginning of αA where its amino group is available to hydrogen bond to the water.

The conserved water molecule appears to be a structural feature inherent to the classic Rossmann dinucleotide-binding fold itself, because this water molecule is also present in apo structures. Examples of such apo structures include the FAD utilizing enzyme l-aspartate oxidase, 1CHU (Mattevi et al. 1999), the NAD enzyme malate dehydrogenase, 1MLD (Gleason et al. 1994), and the NADP enzyme secondary alcohol dehydrogenase, 1PED (Korkhin et al. 1998). Some of the FAD-containing structures in our study also contained NADP-binding domains that lacked NADP. All of these apo-NADP domains also contained the structurally conserved water molecule.

There are several potential functional roles for the conserved water molecule in dinucleotide recognition. First, it could help maintain the unique conformation of the glycine-rich phosphate-binding loop. This role is suggested by the presence of the water molecule in apo structures of dinucleotide-binding enzymes, and by the fact that at least two residues of the glycine-rich loop hydrogen bond to the water molecule (Fig. 5A ▶). These residues interact with the water molecule through their backbone carbonyl or amide groups; thus, these protein–water hydrogen bonds appear to stabilize the backbone conformation of the loop by maintaining these residues in specific phi and psi ranges. A second functional role for the water molecule is that it helps to maintain the cofactor in an extended conformation. Experimental (Zens et al. 1976) and computational studies (Smith and Tanner 1999,Smith and Tanner 2000) of NAD+ in various solvents suggest that NAD+ is folded in aqueous solution such that the two bases stack against each other at a distance of about 4–5 Å. Thus, NAD+ must unfold into an extended conformation during complexation to the enzyme. The extent to which the enzyme facilitates the unfolding of the cofactor is unknown; however, the protein presumably provides interactions that encourage and maintain the extended geometry. The hydrogen bonding and steric interactions provided by the conserved water molecule could help constrain the dihedral angles of the pyrophosphate group to the values observed in enzyme/dinucleotide complexes. A third possible functional role for the conserved water molecule is that the sequence independent hydrogen bond between the dinucleotide pyrophosphate and the water molecule (Fig. 5 ▶) presumably provides a favorable enthalpic contribution to the free energy of binding. Future studies will explore these potential roles to evaluate their possible contributions to dinucleotide recognition and binding.

Materials and methods

Selection and preparation of structures

Crystal structures of enzyme/dinucleotide complexes that contained at least one Rossmann fold were selected for analysis. This selection was restricted to structures having resolutions of 1.90 Å or better to ensure reliable solvent structure. Excluded were any structures having mutations other than for expression purposes and any structures having chemical modifications in the active site. One hundred two structures, representing 43 enzymes, were included in the study (Tables 1–3). Some of the proteins had representatives in multiple species.

To ensure an accurate accounting of interactions, including those involving solvent positions near subunit interfaces, the biologically relevant oligomeric forms of the enzyme were used. Typically, we used the "Likely Quaternary Structure" obtained from the European Bioinformatics Institute Macromolecular Structure Database (EBI-MSD) via the OCA-browser interface (http://oca.ebi.ac.uk/oca-bin/ocamain). Using symmetry operators, the EBI generates oligomeric structures from coordinates deposited in the PDB. Each structure downloaded from the EBI-MSD was superimposed onto its parent structure from the PDB using CNS (Brunger et al. 1998) and visualized in O (Jones et al. 1991) to verify the accuracy of the symmetry expansion and to inspect the dinucleotide binding site. In a few cases, the symmetry expansion was incorrect or incomplete, and CNS was used to create the correct quaternary structures from the corresponding PDB structures. In other cases, visual inspection revealed that a few subunits contained incompletely modeled NAD+ molecules. These subunits were omitted from the subsequent analysis. Bound sulfate ions present due to the crystallization medium were removed.

To facilitate comparison, all the structures were superimposed onto a common structure using CNS. Each protein was superimposed onto the ADH structure of Meijers et al. (2001) (1HET) using the α-carbon coordinates of the first, third from last, and last residue of the phosphate-binding loop. The molecular visualization programs O and Protein Explorer (Martz 2001) were used to examine the structures.

Hydrogen bonding calculations

Hydrogen bonds found in biologic molecules typically have an acceptor–donor distance of 2.7–3.2 Å (Jeffrey 1997). For the purposes of our study, the functional definition of a hydrogen bond was the presence of an acceptor and a donor within 3.2 Å of each other. Only nitrogen and oxygen atoms were considered for our hydrogen bonding calculations (i.e., CH. . .O hydrogen bonds were not included in Fig. 3 ▶). Due to the difficult nature of assigning hydrogen atom positions for water molecules and hydroxyl groups, as well as the absence of explicit hydrogen atoms in the crystal structures under study, an angle cutoff was not included in our definition of a hydrogen bond. Based on our functional definition, X-PLOR (Brunger 1992) was used to identify hydrogen bonds.

The data set of structures used in this study contains some redundancy; therefore, hydrogen-bonding calculations for each structure were weighted to avoid biasing the results toward enzymes heavily represented in our data set. Each enzyme contributed equally to the overall average by assigning each structure a weight equal to the inverse of the product of the number of sources per enzyme and the number of structures per source. For example, Table 1 lists three structures of UDP-galactose-4-epimerase from Homo sapiens and six structures from Escherichia coli. Thus, each H. sapiens UDP-galactose-4-epimerase structure would receive a weight of 1/6, while each E. coli UDP-galactose-4-epimerase structure would receive a weight of 1/12. Likewise, because there is only one source and one structure representing 7α-hydroxysteroid dehydrogenase (1FMC), it would receive a weight of 1. In addition, most of the structures used in this analysis are oligomers; therefore, the hydrogen bonding data were averaged over all the subunits within each structure. Each subunit contributed equally to the average for its structure.

Electronic supplemental material

A table listing the atoms within 3.4 Å of the structurally conserved water molecules of the structures surveyed is included as a supplement available at www.proteinscience.org. (Filename: supp.doc. This was created in Microsoft Word for PC.)

Acknowledgments

We thank the Interdisciplinary Plant Group and the Genetics Area Program of the University of Missouri–Columbia for providing predoctoral support for CAB. This project was partially supported by a Big 12 Faculty Fellowship Award to J.J.T.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

Abbreviations

  • A, adenine

  • ACP, acyl carrier protein

  • ADH, alcohol dehydrogenase

  • CNS, crystallography and NMR system

  • CoA, coenzyme A

  • dh, dehydrogenase

  • dTDP, deoxythymidine diphosphate

  • EBI-MSD, European Bioinformatics Institute Macromolecular Structure Database

  • FAD, flavin adenine dinucleotide

  • GAPDH, d-glyceraldehyde-3-phosphate dehydrogenase

  • GDP, Guanosine diphosphate

  • N, nicotinamide

  • NAD, nicotinamide adenine dinucleotide

  • NADP, nicotinamide adenine dinucleotide phosphate

  • NMN, nicotinamide adenine mononucleotide

  • P, pyrophosphate

  • PDB, Protein Data Bank

  • Ra, adenine ribose

  • rd, reductase

  • RMSD, root-mean-square deviation

  • Rn, nicotinamide ribose

  • UDP-galactose, uridine 5`-diphosphate galactose

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0213502.

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