A method to search for similar protein local structures at ligand-binding sites and its application to adenine recognition

We have developed a method of searching for similar spatial arrangements of atoms around a given chemical moiety in proteins that bind a common ligand. The first step in this method is to consider a set of atoms that closely surround a given chemical moiety. Then, to compare the spatial arrangements of such surrounding atoms in different proteins, they are translated and rotated so that the chemical moieties are superposed on each other. Spatial arrangements of surrounding atoms in a pair of proteins are judged to be similar, when there are many corresponding atoms occupying similar spatial positions. Because the method focuses on the arrangements of surrounding atoms, it can detect structural similarities of binding sites in proteins that are dissimilar in their amino acid sequences or in their chain folds. We have applied this method to identify modes of nucleotide base recognition by proteins. An all-against-all comparison of the arrangements of atoms surrounding adenine moieties revealed an unexpected structural similarity between protein kinases, cAMP-dependent protein kinase (cAPK), and casein kinase-1 (CK1), and D-Ala:D-Ala ligase (DD-ligase) at their adenine-binding sites, despite a lack of similarity in their chain folds. The similar local structure consists of a four-residue segment and three sequentially separated residues. In particular the four-residue segments of these enzymes were found to have nearly identical conformations in their backbone parts, which are involved in the recognition of adenine. This common local structure was also found in substrate-free three-dimensional structures of other proteins that are similar to DD-ligase in the chain fold and of other protein kinases. As the proteins with different folds were found to share a common local structure, these proteins seem to constitute a remarkable example of convergent evolution for the same recognition mechanism. Received: 9 December 1996 / Accepted: 7 February 1997     

On the molecular discrimination between adenine and guanine by proteins

The molecular recognition and discrimination of adenine and guanine ligand moieties in complexes with proteins have been studied using empirical observations on carefully selected crystal structures. The distribution of protein folds that bind these purines has been found to differ significantly from that across the whole PDB, but the most populated architectures and folds are also the most common in three genomes from the three different domains of life. The protein environments around the two nucleic acid bases were significantly different, in terms of the propensities of amino acid residues to be in the binding site, as well as their propensities to form hydrogen bonds to the bases. Plots of the distribution of protein atoms around the two purines clearly show different clustering of hydrogen bond donors and acceptors opposite complimentary acceptors and donors in the rings, with hydrophobic areas below and above the rings. However, the clustering pattern is fuzzy, reflecting the variety of ways that proteins have evolved to recognise the same molecular moiety. Furthermore, an analysis of the conservation of residues in the protein chains binding guanine shows that residues in contact with the base are in general better conserved than the rest of the chain.     

Enzyme-mononucleotide interactions: three different folds share common structural elements for ATP recognition

Three ATP-dependent enzymes with different folds, cAMP-dependent protein kinase, D-Ala:D-Ala ligase and the alpha-subunit of the alpha2beta2 ribonucleotide reductase, have a similar organization of their ATP-binding sites. The most meaningful similarity was found over 23 structurally equivalent residues in each protein and includes three strands each from their beta-sheets, in addition to a connecting loop. The equivalent secondary structure elements in each of these enzymes donate four amino acids forming key hydrogen bonds responsible for the common orientation of the "AMP" moieties of their ATP-ligands. One lysine residue conserved throughout the three families binds the alpha-phosphate in each protein. The common fragments of structure also position some, but not all, of the equivalent residues involved in hydrophobic contacts with the adenine ring. These examples of convergent evolution reinforce the view that different proteins can fold in different ways to produce similar structures locally, and nature can take advantage of these features when structure and function demand it, as shown here for the common mode of ATP-binding by three unrelated proteins.     

"Acceptor-donor-acceptor" motifs recognize the Watson-Crick, Hoogsteen and Sugar "donor-acceptor-donor" edges of adenine and adenosine-containing ligands

Nucleotides are among the most extensively exploited chemical moieties in nature and, as a part of a handful of different protein ligands, nucleotides play key roles in energy transduction, enzymatic catalysis and regulation of protein function. We have previously reported that in many proteins with different folds and functions a distinctive adenine-binding motif is involved in the recognition of the Watson-Crick edge of adenine. Here, we show that many proteins do have clear structural motifs that recognize adenosine (and some other nucleotides and nucleotide analogs) not only through the Watson-Crick edge, but also through the sugar and Hoogsteen edges. Each of the three edges of adenosine has a donor-acceptor-donor (DAD) pattern that is often recognized by proteins via a complementary acceptor-donor-acceptor (ADA) motif, whereby three distinct hydrogen bonds are formed: two conventional N-H...O and N-H...N hydrogen bonds, and one weak C-H...O hydrogen bond. The local conformation of the adenine-binding loop is betabetabeta or betabetaalpha and reflects the mode of nucleotide binding. Additionally, we report 21 proteins from five different folds that simultaneously recognize both the sugar edge and the Watson-Crick edge of adenine. In these proteins a unique beta-loop-beta supersecondary structure grasps an adenine-containing ligand between two identical adenine-binding motifs as part of the betaalphabeta-loop-beta fold.     

Molecular determinants for ATP-binding in proteins: a data mining and quantum chemical analysis

Adenosine 5'-triphosphate (ATP) plays an essential role in all forms of life. Molecular recognition of ATP in proteins is a subject of great importance for understanding enzymatic mechanism and for drug design. We have carried out a large-scale data mining of the Protein Data Bank (PDB) to analyze molecular determinants for recognition of the adenine moiety of ATP by proteins. Non-bonded intermolecular interactions (hydrogen bonding, pi-pi stacking interactions, and cation-pi interactions) between adenine base and surrounding residues in its binding pockets are systematically analyzed for 68 non-redundant, high-resolution crystal structures of adenylate-binding proteins. In addition to confirming the importance of the widely known hydrogen bonding, we found out that cation-pi interactions between adenine base and positively charged residues (Lys and Arg) and pi-pi stacking interactions between adenine base and surrounding aromatic residues (Phe, Tyr, Trp) are also crucial for adenine binding in proteins. On average, there exist 2.7 hydrogen bonding interactions, 1.0 pi-pi stacking interactions, and 0.8 cation-pi interactions in each adenylate-binding protein complex. Furthermore, a high-level quantum chemical analysis was performed to analyze contributions of each of the three forms of intermolecular interactions (i.e. hydrogen bonding, pi-pi stacking interactions, and cation-pi interactions) to the overall binding force of the adenine moiety of ATP in proteins. Intermolecular interaction energies for representative configurations of intermolecular complexes were analyzed using the supermolecular approach at the MP2/6-311 + G* level, which resulted in substantial interaction strengths for all the three forms of intermolecular interactions. This work represents a timely undertaking at a historical moment when a large number of X-ray crystallographic structures of proteins with bound ATP ligands have become available, and when high-level quantum chemical analysis of intermolecular interactions of large biomolecular systems becomes computationally feasible. The establishment of the molecular basis for recognition of the adenine moiety of ATP in proteins will directly impact molecular design of ATP-binding site targeted enzyme inhibitors such as kinase inhibitors.     

NADP-Dependent enzymes. II: Evolution of the mono- and dinucleotide binding domains

Nicotinamide adenine dinucleotides [NAD and NADP with both referred to as NAD(P)] are among the more diffuse redox cofactors. Despite their stereochemical similarity where the only difference is a phosphomonoester on the ribose near the adenine of NADP, they show different biochemical reactivities with NAD behaving as an oxidant and NADP as a reductant. NAD(P)-dependent enzymes generally share a common open α/β fold with few exceptions only recently structurally characterized. This study of the molecular evolution of the NAD(P) binding domains, possible given the large number of known molecular structures, addresses two main questions: 1) can a common fold exist in different biological systems (divergent evolution) and 2) does a relationship exist among similar biological systems that display different folds (convergent evolution)? Both the structures of mono- and dinucleotide binding domains have been classified by cluster analysis based on the similarity evaluated by their main chain Cα superposition. Moreover, the cofactor conformations and the stereochemical characteristics of their pockets have also been classified by analogous methods on the basis of the published tertiary structures. Two primary results appear: 1) the classification of the mononucleotide binding domains is different from that of the dinucleotide binding folds and 2) both divergent and convergent evolutionary pathways can be hypothesized, the latter less frequently observed and less pronounced but nevertheless evident. The generally accepted hypothesis that dinucleotide binding domains have evolved by gene duplication of primordial genes coding for the smaller mononucleotide binding domains is acceptable but the two halves of the resulting dinucleotide binding domains are evolutionarly uncorrelated. The NH₂-terminal mononucleotide binding domain is less variable than the COOH-terminal half, probably because it involves the binding of the ADP moiety of NAD(P) invariant in all examined systems. There is evidence to postulate that evolutionary pathways for NAD(P)-dependent enzymes are both divergent and convergent. In fact, nearly all combinations of similarity/dissimilarity in overall fold, cofactor conformation, and cofactor binding pocket structural characteristics for each enzyme pair examined are possible. The NAD(P)-dependent enzymes apparently provide a canonical example of an evolutionary principle that “anything goes.” © 1997 Wiley-Liss Inc.     

Adenine recognition is a key checkpoint in the energy release mechanism of phage T4 DNA packaging motor

ATP is the source of energy for numerous biochemical reactions in all organisms. Tailed bacteriophages use ATP to drive powerful packaging machines that translocate viral DNA into a procapsid and compact it to near-crystalline density. Here we report that a complex network of interactions dictates adenine recognition and ATP hydrolysis in the pentameric phage T4 large "terminase" (gp17) motor. The network includes residues that form hydrogen bonds at the edges of the adenine ring (Q138 and Q143), base-stacking interactions at the plane of the ring (I127 and R140), and cross-talking bonds between adenine, triphosphate, and Walker A P-loop (Y142, Q143, and R140). These interactions are conserved in other translocases such as type I/type III restriction enzymes and SF1/SF2 helicases. Perturbation of any of these interactions, even the loss of a single hydrogen bond, leads to multiple defects in motor functions. Adenine recognition is therefore a key checkpoint that ensures efficient ATP firing only when the fuel molecule is precisely engaged with the motor. This may be a common feature in the energy release mechanism of ATP-driven molecular machines that carry out numerous biomolecular reactions in biological systems.     

Mutational analysis of ATP-grasp residues in the two ATP sites of Saccharomyces cerevisiae carbamoyl phosphate synthetase

The ATP-grasp fold is found in enzymes that catalyze the formation of an amide bond and occurs twice in carbamoyl phosphate synthetase. We have used site-directed mutagenesis to further define the relationship of these ATP folds to the ATP-grasp family and to probe for distinctions between the two ATP sites. Mutations at D265 and D810 severely diminished activity, consistent with consensus ATP-grasp roles of facilitating the transfer of the gamma-phosphate group of ATP. H262N was inactive whereas H807N, the corresponding mutation in the second ATP domain, exhibited robust activity, suggesting that these residues were not involved in the ATP-grasp function common to both domains. Mutations at I316 were somewhat catalytically impaired and were structurally unstable, consistent with a consensus role of interaction with the adenine and/or ribose moiety of ATP. L229G was too unstable to be purified and characterized. S228A showed essentially wild-type behavior.     

Crystal structure of a lysine biosynthesis enzyme,LysX, from Thermus thermophilus HB8

The thermophilic bacterium Thermus thermophilus synthesizes lysine through the alpha-aminoadipate pathway, which uses alpha-aminoadipate as a biosynthetic intermediate of lysine. LysX is the essential enzyme in this pathway, and is believed to catalyze the acylation of alpha-aminoadipate. We have determined the crystal structures of LysX and its complex with ADP at 2.0A and 2.38A resolutions, respectively. LysX is composed of three alpha+beta domains, each composed of a four to five-stranded beta-sheet core flanked by alpha-helices. The C-terminal and central domains form an ATP-grasp fold, which is responsible for ATP binding. LysX has two flexible loop regions, which are expected to play an important role in substrate binding and protection. In spite of the low level of sequence identity, the overall fold of LysX is surprisingly similar to that of other ATP-grasp fold proteins, such as D-Ala:D-Ala ligase, PurT-encoded glycinamide ribonucleotide transformylase, glutathione synthetase, and synapsin I. In particular, they share a similar spatial arrangement of the amino acid residues around the ATP-binding site. This observation strongly suggests that LysX is an ATP-utilizing enzyme that shares a common evolutionary ancestor with other ATP-grasp fold proteins possessing a carboxylate-amine/thiol ligase activity.     

Protein fold and structure in the truncated (2/2) globin family

Analysis of amino acids sequences and protein folds has recently unraveled the structural bases and details of several proteins from the recently discovered "truncated hemoglobin" family. The analysis here presented, in agreement with previous surveys, shows that truncated hemoglobins can be classified in three main groups, based on their structural properties. Crystallographic analyses have shown that all three groups adopt a 2-on-2 alpha-helical sandwich fold, resulting from apparent editing of the classical 3-on-3 alpha-helical sandwich of vertebrate and invertebrate conventional globins. Specific structural features distinguish each of the three groups. Among these, a protein matrix tunnel system is typical of group I, a Trp residue at the G8 topological site is conserved in groups II and III, and TyrB10 is almost invariant through the three groups. A strongly intertwined network of hydrogen bonds stabilizes the heme bound ligand, despite variability of the heme distal residues observed in the different proteins considered. Details of ligand recognition in the three groups are discussed at the light of residue conservation and of differing ligand diffusion pathways to the heme. Based on structural analyses of the family-specific fold, we endorse a recent proposal of leaving the "truncated hemoglobins" term, that does not represent properly the observed 2-on-2 alpha-helical sandwich fold, and adopting the simple "2/2Hb" term to concisely address this protein family.     

Adenine recognition: a motif present in ATP-, CoA-, NAD-, NADP-, and FAD-dependent proteins.

Adenosine triphosphate (ATP) plays an essential role in energy transfer within the cell. In the form of NAD, adenine participates in multiple redox reactions. Phosphorylation and ATP-hydrolysis reactions have key roles in signal transduction and regulation of many proteins, especially enzymes. In each cell, proteins with many different functions use adenine and its derivatives as ligands; adenine, of course, is present in DNA and RNA. We show that an adenine binding motif, which differs according to the backbone chain direction of a loop that binds adenine (and in one variant by the participation of an aspartate side-chain), is common to many proteins; it was found from an analysis of all adenylate-containing protein structures from the Protein Data Bank. Indeed, 224 protein-ligand complexes (86 different proteins) from a total of 645 protein structure files bind ATP, CoA, NAD, NADP, FAD, or other adenine-containing ligands, and use the same structural elements to recognize adenine, regardless of whether the ligand is a coenzyme, cofactor, substrate, or an allosteric effector. The common adenine-binding motif shown in this study is simple to construct. It uses only (1) backbone polar interactions that are not dependent on the protein sequence or particular properties of amino acid side-chains, and (2) nonspecific hydrophobic interactions. This is probably why so many different proteins with different functions use this motif to bind an adenylate-containing ligand. The adenylate-binding motif reported is present in "ancient proteins" common to all living organisms, suggesting that adenine-containing ligands and the common motif for binding them were exploited very early in evolution. The geometry of adenine binding by this motif mimics almost exactly the geometry of adenine base-pairing seen in DNA and RNA.     

RNA recognition: towards identifying determinants of specificity.

Members of a family of proteins containing a conserved approximately 80-amino acid RNA recognition motif (RRM) bind specifically to a wide variety of RNA molecules. Structural studies, in combination with sequence alignments, indicate the structural context of both conserved and non-conserved elements in the motif. These analyses suggest that all RRM proteins share a common fold and a similar protein-RNA interface, and that non-conserved residues contribute additional contacts for sequence-specific RNA recognition.     

Structure of nicotinamide mononucleotide adenylyltransferase: a key enzyme in NAD(+) biosynthesis

BACKGROUND: Nicotinamide adenine dinucleotide (NAD(+)) is an essential cofactor involved in fundamental processes in cell metabolism. The enzyme nicotinamide mononucleotide adenylyltransferase (NMN AT) plays a key role in NAD(+) biosynthesis, catalysing the condensation of nicotinamide mononucleotide and ATP, and yielding NAD(+) and pyrophosphate. Given its vital role in cell life, the enzyme represents a possible target for the development of new antibacterial agents. RESULTS: The structure of NMN AT from Methanococcus jannaschii in complex with ATP has been solved by X-ray crystallography at 2.0 A resolution, using a combination of single isomorphous replacement and density modification techniques. The structure reveals a hexamer with 32 point group symmetry composed of alpha/beta topology subunits. The catalytic site is located in a deep cleft on the surface of each subunit, where one ATP molecule and one Mg(2+) are observed. A strictly conserved HXGH motif (in single-letter amino acid code) is involved in ATP binding and recognition. CONCLUSIONS: The structure of NMN AT closely resembles that of phosphopantetheine adenylyltransferase. Remarkably, in spite of the fact that the two enzymes share the same fold and hexameric assembly, a striking difference in their quaternary structure is observed. Moreover, on the basis of structural similarity including the HXGH motif, we identify NMN AT as a novel member of the newly proposed superfamily of nucleotidyltransferase alpha/beta phosphodiesterases. Our structural data suggest that the catalytic mechanism does not rely on the direct involvement of any protein residues and is likely to be carried out through optimal positioning of substrates and transition-state stabilisation, as is proposed for other members of the nucleotidyltransferase alpha/beta phosphodiesterase superfamily.     

Sequence-structure analysis of FAD-containing proteins

We have analyzed structure-sequence relationships in 32 families of flavin adenine dinucleotide (FAD)-binding proteins, to prepare for genomic-scale analyses of this family. Four different FAD-family folds were identified, each containing at least two or more protein families. Three of these families, exemplified by glutathione reductase (GR), ferredoxin reductase (FR), and p-cresol methylhydroxylase (PCMH) were previously defined, and a family represented by pyruvate oxidase (PO) is newly defined. For each of the families, several conserved sequence motifs have been characterized. Several newly recognized sequence motifs are reported here for the PO, GR, and PCMH families. Each FAD fold can be uniquely identified by the presence of distinctive conserved sequence motifs. We also analyzed cofactor properties, some of which are conserved within a family fold while others display variability. Among the conserved properties is cofactor directionality: in some FAD-structural families, the adenine ring of the FAD points toward the FAD-binding domain, whereas in others the isoalloxazine ring points toward this domain. In contrast, the FAD conformation and orientation are conserved in some families while in others it displays some variability. Nevertheless, there are clear correlations among the FAD-family fold, the shape of the pocket, and the FAD conformation. Our general findings are as follows: (a) no single protein 'pharmacophore' exists for binding FAD; (b) in every FAD-binding family, the pyrophosphate moiety binds to the most strongly conserved sequence motif, suggesting that pyrophosphate binding is a significant component of molecular recognition; and (c) sequence motifs can identify proteins that bind phosphate-containing ligands.     

A novel ADP- and zinc-binding fold from function-directed in vitro evolution

A great challenge to biologists is to create proteins with novel folds and tailored functions. As an alternative to de novo protein design, we investigated the structure of a randomly generated protein targeted to bind ATP. The crystal structure reveals a novel alpha/beta fold bound to its ligand, representing both the first protein structure derived from in vitro evolution and the first nucleotide-binding protein stabilized by a zinc ion.     

Novel CalphaNN structural motif for protein recognition of phosphate ions

Phosphate is one of the most frequently exploited chemical moieties in nature, present in a wide range of naturally occurring and critically important small molecules. Several phosphate group recognition motifs have been found for a few narrow groups of proteins, but for many protein families and folds the mode of phosphate recognition remains unclear. Here, we have analyzed the structures of all fold-representative protein-ligand complexes listed in the FSSP database, regardless of whether the bound ligand included a phosphate group. Based on a phosphate-binding motif that we identified in pyridoxal phosphate binding proteins, we have identified a new anion-binding structural motif, CalphaNN, common to 104 fold-representative protein structures that belong to 62 different folds, of which 86% of the fold-representative structures (51 folds) bind phosphate or lone sulfate ions. This motif leads to a precise mode for phosphate group recognition forming a structure where atoms of the phosphate group occupy the most favorable stabilizing positions. The anion-binding CalphaNN motif is based only on main-chain atoms from three adjacent residues, has a conservative betaalphaalpha or betaalphabeta geometry, and recognizes the free phosphate (sulfate) ion as well as one or more phosphate groups in nucleotides and in a variety of cofactors. Moreover, the CalphaNN motif is positioned in functionally important regions of protein structures and often residues of the motif directly participate in the function of the protein.     

Functional attributes of the phosphate group binding cup of pyridoxal phosphate-dependent enzymes

Twenty-four structures of pyridoxal-5'-phosphate (PLP)-dependent enzymes that represent five different folds are shown to share a common recognition pattern for the phosphate group of their PLP-ligands. All atoms that interact with the phosphate group of PLP in these proteins are organized within a two-layer structure so that the first interacting layer contains from five to seven atoms and parallel with this is a second layer containing from three to seven interacting atoms. In order to identify features of the phosphate-binding site common to PLP-dependent enzymes, a simple procedure is described that assigns relative positions to all interacting atoms unambiguously, such that the networks of interactions for different proteins can be compared. On the basis of these diagrams for 24 enzyme-cofactor complexes, a detailed comparison of the two-layer structures of PLP-dependent enzymes, with both similar and different folds, was made. A majority of the structurally defined PLP-dependent proteins use the same atom types in analogous "key" positions to bind their PLP-ligands. In some instances, proteins use water molecules when a key position is unoccupied. A similar two-layer recognition pattern extends to protein recognition of at least one other, non-PLP ligand, glucosamine 6-phosphate. We refer to this three-dimensional recognition pattern as the phosphate-binding cup. In general, the phosphate-binding cup provides a very stable anchoring point for PLP. When numerous water molecules occur within the cup, however, then the phosphate group of PLP participates directly in the enzymatic reactions with inorganic phosphate replacing the water molecules of the cup. With glucosamine-6-phosphate synthase, the water molecules of the phosphate-binding cup facilitate the entry of substrate and the exit of product.     

Folding and binding integrity of variants of a prototype ligand-binding module from the LDL receptor possessing multiple alanine substitutions

The LA repeats that comprise the ligand-binding domain of the LDL receptor are among the most common autonomously structured extracellular modules found in the nonredundant protein sequence database. Here, we investigate the information content of the amino acid sequence of a typical LA module by constructing sequences with alanine residues at nonconserved positions in the module. Starting with the sequence of the fifth ligand-binding repeat of the LDL receptor (LA5), we created generic LA modules with alanine substitutions of nonconserved residues in only the N-terminal lobe, only the C-terminal lobe, and throughout both lobes of the module. LA variants with alanine residues at as many as 18 of 37 positions fold to a preferred disulfide isomer in the presence of calcium. Indeed, the six cysteines, the C-terminal calcium coordinating residues, two hydrophobic residues involved in packing, two glycines, and five other residues that form side chain-intramodule hydrogen bonds are alone sufficient to specify the fold of an LA module when alanine residues are present at all other positions. The LA variants with multiple alanines in either the N- or C-terminal lobe were then exploited to identify residues of LA5 that contribute to the binding of apoE-containing ligands in LDL receptor-derived "minireceptors", implicating nonconserved residues of the N-terminal lobe of LA5 in recognition of apoE-DMPC. Our library of LA modules with multiple alanine substitutions should be generally useful for probing the roles of nonconserved side chains in ligand recognition by proteins of the LDL receptor family.     

Two "unrelated" families of ATP-dependent enzymes share extensive structural similarities about their cofactor binding sites.

Two proteins, D-alanine:D-alanine ligase and cAMP-dependent protein kinase, share a remarkable degree of structural convergence despite having different three-dimensional folds and different enzymatic functions. Here we report that as many as 103 residues from 10 segments form two identical super-secondary structures between which the cofactor ATP is bound. The cofactor, two bound metal cations, and several water molecules form a large network of electrostatic and hydrophobic interactions common to both enzymes, and these are mediated by the similar placement of equivalent amino acids within the common supersecondary structures.     

SAM (dependent) I AM: the S-adenosylmethionine-dependent methyltransferase fold

The S-adenosylmethionine-dependent methyltransferase enzymes share little sequence identity, but incorporate a highly conserved structural fold. Surprisingly, residues that bind the common cofactor are poorly conserved, although the binding site is localised to the same region of the fold. The substrate-binding region of the fold varies enormously. Over the past two years, there has been a significant increase in the number of structures that are known to incorporate this fold, including several uncharacterized proteins and two proteins that lack methyltransferase activity.