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1 Dipartimento di Scienze Biochimiche "A. Rossi Fanelli," Istituto di Biologia e Patologia Molecolari del Consiglio Nazionale delle Ricerche, and 2 Centro di Ricerca per l’ Analisi dei Modelli e dell’Informazione nei Sistemi Biomedici (CISB), Università La Sapienza, 00185 Roma, Italy
Reprint requests to: Stefano Pascarella, Dipartimento di Scienze Bio-chimiche, Università La Sapienza, P.le A. Moro 5, 00185 Rome, Italy; e-mail: stefano.pascarella{at}uniroma1.it; fax: +0039-06-49917566.
(RECEIVED June 17, 2004; FINAL REVISION July 30, 2004; ACCEPTED August 2, 2004)
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Keywords: PLP-dependent enzymes; remote homology; molecular evolution; conserved hydrophobic contacts; structural stability
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04938104.
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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In an effort to address these questions, the interacting hydrophobic residues conserved at primary and tertiary structure levels have been investigated in the case of fold-type I, pyridoxal-5'-phosphate (PLP)-dependent enzymes. Although there are at least five evolutionarily unrelated superfamilies of PLP-dependent enzymes, each displaying a completely different fold, by far the largest and best-characterized is known as fold-type I, 
family, or aspartate aminotransferase family (Jansonius 1998; Schneider et al. 2000). This large group of enzymes, which are found in all organisms and together cover the whole range of enzymatic activities cataloged by the Enzyme Commission (John 1995), bears several interesting characteristics; its members are highly divergent enzymes that display structural homology with almost undetectable sequence similarity; thanks to the recent massive sequencing of several genomes and advances in protein structure determination, a good wealth of experimentally well-characterized information is now available for this superfamily.
On the basis of such consideration, the present work was aimed at detecting the evolutionarily conserved structural patterns possibly responsible for the maintenance of the fold of this protein superfamily. The analysis was carried out in two steps; initially, a structural study extracted from a nonredundant set of 23 superposed crystallographic structures the features shared by this superfamily of enzymes, that is, the structurally conserved regions (SCRs) and the conserved hydrophobic contacts (CHCs); then, the initial multiple structural alignment was extended by adding sequence homologs to the enzymes whose structure is known, and an evolutionary analysis was undertaken on the final multiple alignment of 921 sequences to detect the most conserved sequence sites. Finally, the structure-based and the sequence-based analyses were compared.
The role played by conserved residues in the stabilization of the native structure and their possible involvement in the mechanism of protein folding was then discussed in light of the most recent studies on PLP-dependent enzymes.
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Results |
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) from different sources comprising the three domains of life, Eukarya, Bacteria, and Archaea. The level of sequence identity between the superimposed structures guaranteed the coverage of values inside the "twilight zone" (Rost 1999), ranging from 6% to 27% (mean 12%, SD ± 3%). Despite the low-sequence identity, this superfamily of enzymes displays a remarkable structural conservation, with a mean secondary structure agreement computed over six states (-helix, 3–10 helix, 
-bridge, extended strand, bend, hydrogen-bonded turn, and loop) of 64% ± 4% and with a maximum pairwise RMSD of 4.2 Å (Table 2).
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3.0 Å, lacking insertions and deletions were detected (Figs. 1, 2A). Positional RMSD values ranged from 0.8 Å in position 65 (residue numbering refers to sites in Fig. 1), to 3.1 Å in position 126 (Table 3). Figure 2A shows that an extensive and evident common structural organization of the main chain around PLP is responsible for the appropriate positioning of key residues previously identified as structural determinants for binding the cofactor (Grishin et al. 1995). Five SCRs are mainly implied in the constitution of this common core as follows: one -helix (3, which displays a mean positional RMSD of 1.59 Å) and four -strands, forming a -sheet (6, 9, 10, and 11, with a mean positional RMSD of 1.76 Å, 1.54 Å, 1.41 Å, and 1.52 Å, respectively).
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The number of sequences retrieved for each structure is shown in Table 1
. The multiple-structure alignment obtained from the superposition of the crystallographic structures was then used as a guide to merge the 23 multiple-sequence alignments comprising 921 nonredundant sequences. A total of 376,573 of the 422,740 pairwise sequence comparisons displayed a sequence identity in the interval 0%–20% (mean 16%, SD ±6%), which suggests that the data set can sample very distant evolutionary events. After obtaining the multiple-sequence alignment, a method for the identification of evolutionarily conserved residues was applied. Because in extensive tests of sequence alignments (Vogt et al. 1995) the BLOsum62, on average, gave superior results compared with most other matrices, it seemed appropriate to adopt this mutational matrix to assign a score for the amino acid exchanges. A weighting scheme based upon sequence similarity was also adopted, to incorporate in the algorithm corrections for sequence evolutionary distance and residue frequency (see Materials and Methods section). The results obtained for the SCRs, expressed in units of SD from the mean conservation value (R), are shown in Table 3. The structural role played by SCRs in maintaining the fold of this superfamily of enzymes is reflected by the high sequence conservation of the corresponding positions of the multiple alignment. Scores displayed by the SCRs are, in fact, all above the mean conservation value, with the only exception being site 14, which obtained a negative score. In particular, residues interacting with the PLP moiety are the most conserved; Asp 67, which is known to interact with the pyridinium nitrogen of PLP (Mehta and Christen 1998), was found in 919 of 921 sequences aligned (the only exceptions are 8-amino-7-oxononanoate synthase from Mesorhizobium loti and Cystathionine -lyase from Bifidobacterium longum, GI 13475018 and GI 23336039, respectively [Holm and Sander 1998], in which Asp was replaced by Gly and Asn, respectively), scoring at a significance of 3.3 SDs from the mean conservation value; a comparable value (R = 3.2) was seen only by the Schiff base-forming lysine, which is placed in a variable loop between SCRs 10 and 11 (Christen and Mehta 2001). Taken together, these two residues represent the major signature of this superfamily of enzymes. Other sites involved in interactions with the cofactor or the substrates are strongly conserved, that is, position 70, interacting with the phenol oxygen of PLP (R = 1.2), the ring moiety stacking on the re side of PLP (data not shown; R = 1.6), the residue stacking on the si side (site 69, R = 1.4), the so-called glycine-rich region (positions 19, 20, and 21; R = 1.6, 1.9, 1.0, respectively), the 5'-phosphate-binding residue in position 77 (R = 1.5), and the Arg residue ion-paired with the carboxyl group of many substrates bound to the fold-type I enzymes (site 133, R = 2.0).
In addition to these positions, other sites not directly involved in any interaction with the cofactor or the substrates show a high degree of sequence conservation, comparable to the conservation measured for functionally important residues (R 
1.0; Table 3). These sites might be grouped in two distinct categories as follows: (1) Gly/Ala-rich sites; (2) positions mainly occupied by residues with a hydrophobic character (position 97, for example, scoring at a significance of 1.7 SDs from the mean conservation value, is almost invariantly occupied by a Leu or an aromatic residue in all of the 921 sequences considered, although it seems not to be implied in any functional role).
The positions mainly occupied by Gly or Ala residues (23, 80, and 92), that show a high degree of sequence conservation (1.9, 1.9, and 1.0, respectively), might play important functions other than binding the PLP moiety or being involved in hydrophobic contacts. For example, two Ala rich sites (23 and 92) are found in the middle of an -helix spine; it was observed that Ala show the strongest preference over any other residue for a middle-helix location (Richardson and Richardson 1989). This, in turn, is due to the structurally unique features shown by Ala, which direct and stabilize the -helix fold (Blaber et al. 1993). The other Gly-rich site (80) was found in 11, where it could be helpful in modulating the curvature of the sheet (Richardson and Richardson 1989).
To test whether the conservation of the physicochemical properties of the second group of positions was driven by selective pressure to maintain the stability of fold-type I, PLP-dependent enzymes through the involvement of the corresponding residues in hydrophobic interactions, an analysis of the conserved hydrophobic contacts (CHCs) was performed on the SCRs previously identified. Previous comparative studies that have been focused on the relationship between sequence conservation of a protein family and the hydrophobic contacts of the corresponding structures available (see, for example, Ptitsyn 1998; Hill et al. 2002; Gromiha et al. 2004; Gunasekaran et al. 2004) have considered two residues to be in contact if the distance between their C atoms or between one atom and any other atom was below an arbitrary threshold. In this work, a different criterion was adopted, which is based on the comparative analysis of the pairwise residue apolar contact areas for every possible pair of residues belonging to the SCRs. CHCs are, therefore, defined as residue hydrophobic contacts involving only apolar atoms (Drabløs 1999), observed in at least two of the structures analyzed. This approach permitted us to quantify the strength of a hydrophobic contact and to assess the correlation between this quantity and the evolutionary conservation of the corresponding sites. The strongest CHCs for each site belonging to the SCRs and the corresponding site involved in the hydrophobic interaction are shown in Table 3.
Figure 3 shows the mean conservation values between pairs of sites involved in CHCs in comparison with their mean hydrophobic contact values. Residues interacting with the cofactor PLP as well as the Ala/Gly-rich sites described above were not plotted, as their high evolutionary conservation reflects functions other than the stabilization of this superfamily fold through the involvement in hydrophobic contacts. A significant linear coefficient (r = 0.70) resulted between the two variables. The statistical significance of r was assessed with the t-test, assuming r = 0 as the null hypothesis. This gave a P-value 
1.7e-53, indicating that there is a statistically significant relationship between the strength of a CHC and the extent of conservation of the involved residues during evolution. At values >16 Å2, the mean conservation grade becomes comparable to the values measured for catalytically important residues (R 1.0). CHCs with the highest values of mean apolar contact area (Table 4) may be grouped in three main clusters (Fig. 2B); a first cluster of CHCs is located at the buried bottom region of the PLP-binding, conserved common core of the major domain constituted by the six SCRs (3 6, L8, 9, 10, and 11); a second small cluster of interacting residues is centered around position 133 of the minor domain (13, 14, 15, 16, and 17); a third cluster of CHCs forms a hinge between SCRs 1 and 12, which are positioned at the beginning and at the end of the major domain, respectively (Fig. 2B). Amino acids belonging to the first cluster of CHCs occur at positions 27, 28, and 31 in 3; 35 and 36 in 4; 42 in 5; 48, 49, and 50 in 6; 58 and 59 in L8; 63, 64, and 65 in 9; 73 in 10; 81 and 84 in 11 (Fig. 2B; Table 4). The five residues participating in the formation of the second cluster (111 in 13, 118 in 14, 125 in 15, 128 in 16, and 134 in 17), are located in proximity of position 133 of 17, which is occupied mainly by an Arg residue (18 of 23 structures analyzed); the carboxyl group of many substrates bound to the fold-type I enzymes is often ion-paired to this arginine (Jansonius 1998). Residues forming the third cluster of CHCs are involved in interhelical contacts in 22 of the 23 structures considered (the only exception is represented by 1BJW
[PDB]
, in which only two CHCs involving site 8 are conserved; Table 4). These residues (positions 4, 8, and 11 of SCR 1; 90 and 97 of SCR 12) form a vertical strip down each side of the helices that delimit the major domain, lying at sites i, i + 4, and i + 7. Site 97, which was described above (R = 1.7), is engaged in the constitution of the two most extensive CHCs measured for the 1–12 hinge (11–97 and 8–97 [Fig. 2B]; the mean apolar contact areas are, according to Table 3, 24.6 Å2 and 17.6 Å2, respectively), and in an additional conserved contact with position 7 (15.3 Å2).
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Discussion |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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Our work focused on the conservation of hydrophobic contacts between the structurally conserved regions resulting from the comparison of 23 distantly related type I PLP-dependent enzymes. The conservation of hydrophobic contacts is the result of the selective pressure exerted during the molecular evolution to maintain a functionally competent fold. We identified three clusters of conserved hydrophobic contacts; the first and the second clusters of CHCs (Fig. 2B) are located in proximity of key residues responsible for the proper positioning of the cofactor and the substrate in the active site. Regarding the first cluster, the separation of residues involved in a functional role (interaction with the PLP moiety and modulation of its activity), all located at the top of the conserved core region (constituted by SCRs 3, 6, 9, 10, and 11) (Fig. 2A,B) and residues involved in a structural role (maintenance of structural stability throughout CHCs), positioned, instead, at the inner bottom core of the same functional unit, is remarkable. This functional and spatial arrangement, comprising a stable scaffold folded around a mutable functional core of residues, can be found in many other evolutionarily successful (Nagano et al. 2002; Selvaraj and Gromiha 2003) structural units exploited by nature during the course of evolution (i.e., TIM-barrel and Ig-like domains), and it seems to provide a suitable way to solve the compromise between three-dimensional stability and plasticity of function, broadening substrate, and reaction specificity, without affecting protein fold and conformation (Todd et al. 2001; Wierenga 2001; Nagano et al. 2002).
In apparent contrast to the two previously described clusters, the helix 1–helix 12 cluster of CHCs (Fig. 2B) seems not to be involved in the proper positioning or stability of any active site residue. Examination of the contact network showed that the CHCs lie along one side of each helix, forming a buried spine at positions i, i + 4, and i + 7. This particular pattern of almost absolutely conserved residue–residue contacts was previously identified by Hill et al. (2002) and Ptitsyn (1998) in the case of the cytokines and c-type cytochromes superfamily of proteins, respectively. In both studies, it was concluded that these residues were of critical importance for protein folding. In the case of PLP-dependent enzymes, previous experimental studies have suggested the presence of three structural nuclei responsible for the proper fold-type I enzymes folding pattern and stability. Herold et al. (1991) demonstrated that the excised PLP-binding domain of aspartate aminotransferase from Escherichia coli, corresponding to the first and third domain in which CHCs are located, is able to fold autonomously both in vivo and in vitro and bind PLP. More recently, Fu et al. (2003) proposed that the folding mechanism of serine hydroxymethyltransferase from E. coli can be divided into two phases, a first fast phase in which two domains, corresponding to the first and second domains in which CHCs are located, have folded into their native state, and a slow final phase in which an interdomain segment, comprising the helix 12, folds into its native conformation, interacting with the N-terminal 1 helix of the major domain. This last step is thought to be involved in PLP binding. The present analysis supports this hypothesis and suggests a possible mechanistic explanation for these experimental studies, serving as a basis for further experiments to establish sequence-structure correlation, and to investigate the role of individual residues and pairwise interactions in the folding and stability of this superfamily of proteins.
A main goal of this work was to determine whether the common structural constraints found for the packing of interacting residues within the protein core of the type-I PLP enzymes was reflected by a sequence conservation pattern observed for the hydrophobic positions in the multiple alignment of the fold-type I superfamily. A plot of the mean conservation grade of two interacting sites of the SCRs against the extent of mean hydrophobic contact value of their apolar fraction can be fit by a linear relationship (r = 0.70). In the present analysis, the mean amino acid pairwise conservation was considered in addition to single-site, positional conservation. A significant advantage of considering pairwise conservation is that it allows one to take into account compensating mutations that may occur in the amino acid sequence during evolution. It should be noted that conserved positions are not invariant; on the contrary, correlated mutations can be detected by comparing different structures. Therefore, it seems that what is really conserved is the three-dimensional location of the hydrophobic interaction and its hydrophobic effect, rather than the specific identity of the side chains participating in a CHC. Although the 23 PLP enzymes taken into consideration are very distantly related, they contain a structural pattern of conserved hydrophobic contacts, whose potential importance in stabilizing the native fold is supported by a preferential conservation throughout the homologous sequences.
Finally, we suggest that the significant correlation between sequence conservation and CHC values and the strategy and the algorithms described to determine it, could be extended to other superfamilies for which suitable sequence and structural information is known, to properly train statistical predictors of protein contact maps (Fariselli and Casadio 2000; Pollastri et al. 2001) and to help in planning protein folding and design experiments.
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Materials and methods |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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An initial multiple alignment was obtained automatically by using the combinatorial extension algorithm, implemented in the program CE (Shindyalov and Bourne 1998). The resulting alignment was utilized as a starting point to build a manually refined structural alignment. Every possible pair of structures was visually inspected and, where necessary, modified to optimize the matching of several structural features, including observed secondary elements, functionally conserved residues known to interact with the PLP moiety and hydrophobic regions, in order to give the most accurate structural alignment. In a few cases of ambiguity, that is, some insertions or deletions in which visual inspection could not discern the optimal matching between two regions, the residue similarity measured by the BLOsum62 (Henikoff and Henikoff 1992) mutational matrix was adopted as a guide criterion. At the end of the manual refinement, structures displaying >30% sequence identity were discharged, leading to a nonredundant ensemble of 23 representatives of fold-type I enzymes with a maximum pairwise sequence identity of 27% and a maximum pairwise RMSD of 4.2 Å.
Identification of the structurally conserved regions
The structural alignment obtained as described above was utilized to identify the common core and the structurally conserved regions between members of this superfamily (SCRs). SCRs were defined as regions displaying similar local conformation, with a mean positional RMSD of the equivalent -carbon positions of every structure superposed 3.0 Å (Hill et al. 2002), lacking indels (insertions and deletions) in all of the structures considered and composed of at least three consecutive residues. A C-language routine was developed to extract from the three-dimensional coordinates of the superimposed structures and their associated multiple alignment the candidate SCRs. For every structurally equivalent position of the multiple structural alignment, the RMSD from the center of mass of the structurally equivalent C atoms was computed. To avoid the presence of SCRs with indels, positions with gaps were not considered. A window of size w = 3 positions was then scrolled through the alignment and used to define seed positions with a mean RMSD 3.0 Å. Each time a seed position was found, w was increased iteratively by one position until the mean score did not raise above 3.0 Å, or until the window reached the end of the alignment.
Identification of the conserved hydrophobic contacts
Computation of conserved hydrophobic contacts (CHCs) performed on the crystallographic structures retrieved is based on the program pdb_np_cont (Drabløs 1999), which computes pairwise atom contact areas between nonpolar atoms from structural protein data in a standard PDB coordinate file. The output of this program was utilized to calculate the pairwise residue contact areas for every possible pair of residues belonging to the SCRs of the structures analyzed. If two positions of the joint multiple structural alignment, x and y, have residues in hydrophobic contact in at least two of the structures, then a candidate CHC was detected. CHCs were then classified on the basis of their strength sxy, defined as:
 | (1) |
where Ai is the apolar contact area of the i-th structure between residues at absolute positions x and y of the structural alignment, and N is the number of superposed structures.
Collection and alignment of sequence homologs
Sequence search was performed against the nonredundant database NRDB (Holm and Sander 1998) with the program BLAST (Altschul et al. 1997), using each of the sequences of the 23 superposed structures as probes. When applicable, the following criteria were adopted to collect or discharge each sequence: (1) Hits were considered to be significant if the E-value was 0.0001—if less than 10 sequences were collected, this value was increased to 0.001; (2) hits were filtered to assure that no sequence with identity >80% or <30% with any other sequence of the multiple alignment was present in the final alignment; (3) hits with sequence length <80% of the query sequence were rejected to avoid the presence of fragmented sequences in the final alignment.
Sequences filtered were aligned to each corresponding query sequence using the program CLUSTALW (Thompson et al. 1994). The 23 multiple alignments were then merged using as a guide the structural alignment of the 23 PLP enzyme sequences (Pascarella and Argos 1992). The final alignment, comprising 973 sequences, was further checked for redundancy. At the end of this final step, a total number of 921 sequences was obtained.
Identification of the evolutionarily conserved positions
To measure the sequence conservation, each position of the final multiple sequence alignment was assigned a score according to:
 | (2) |
where Ok is the score assigned for every position k of the multiple sequence alignment, n is the number of sequences included in the alignment, i and j refers to the i-th and the j-th sequence, respectively, Bscorekij, Bscorekii, and Bscorekjj are the scores assigned to the residue exchange in position k between the i-th and the j-th sequence according to the BLOsum62 mutational matrix, nidij is the number of identical residues, and nalij is the number of aligned residues between the i-th and the j-th sequence, respectively. Therefore, for every possible exchange at a particular position of the multiple alignment, a normalized conservation index is computed, based on the BLOsum62 mutational matrix. Because the BLOsum62 matrix scores for matching the same amino acids vary for different residues, conservation indices for invariant positions of the multiple-sequence alignment would depend on residue type; normalization is used to avoid different conservation scores for invariant positions. The mean 
and the standard deviation (SD) 
for the distribution of Ok values were determined; the significance R of every conservation index of the alignment was then calculated by dividing the difference between Ok and by .
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Acknowledgments |
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This work was supported in part by the Italian "Ministero dell’ Università e della Ricerca" (MIUR). This work will be submitted by A.P. in partial fulfillment of the requirements of the degree of Dottorato di Ricerca at the Università di Roma "La Sapienza." Structural and sequence alignments and the source code of the software developed for the analysis are available on request from the authors.
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