1 Service de Conformation de Macromolécules Biologiques, Centre de Biologie Structurale et Bioinformatique, Université Libre de Bruxelles,av. F.D. Roosevelt 50, CP160/16, B-1050 Brussels, Belgium and 2 European Bioinformatics Institute (EBI), Genome Campus, Hinxton, Cambridge CB10 1SD, UK
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Abstract |
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Keywords: B-factor/cluster analysis/conserved water molecules/MHC class-I
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Introduction |
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The tertiary structures of an increasing number of MHC-class-I (Bjorkman et al., 1987a,b
; Madden et al., 1992
; Collins et al., 1995
; Wang et al., 1995
; Reid et al., 1996
; Smith et al., 1996a
,b
; Glithero et al., 1999
; Speir et al., 1999
, 2001
; Tormo et al., 1999
; Maenaka et al., 2000
; Hillig et al., 2001
) and class-II (Stern et al., 1994
; Ghosh et al., 1995
; Fremont et al., 1996
, 1998
; Murthy and Stern, 1997
; Scott et al., 1998
) molecules have been determined by X-ray diffraction. The October 2001 release of the Protein Data Bank (RCSB-PDB) (Berman et al., 2000
), contains 68 entries for MHC class-I molecules, comprising nine allele types from human, four from mouse and one from rat. Valuable information on the atomic interactions between the MHC molecule and bound peptide antigens and on those in the ternary complex of MHC with the peptide and the T-cell receptor molecules has been obtained from the tertiary structures of the corresponding complexes (Garboczi et al., 1996
). The peptide binding groove in the MHC class-I molecules has been described as featuring six pockets that interact with the side chains of the bound peptide and are lined by amino acid residues, which differ between the allele types (Bjorkman et al., 1987a
; Saper et al., 1991
; Matsumura et al., 1992
). Following this description, these six pockets have been classified on the basis of their selective recognition of specific amino acid types in residues of the bound peptide (Zhang et al., 1998
).
Water positions conserved in different crystal structures of the same protein, in particular completely buried positions that are isolated from the bulk solvent, have been analyzed by several authors (Rashin et al., 1986; Williams et al., 1994
). Several studies were also devoted to the analysis of conserved water positions in crystal structures of related proteins such as the serine protease family (Sreenivasan and Axelsen, 1992
) or the microbial RNases (Loris et al., 1999
). These and other studies showed that conserved water molecules can occur in enzyme active sites, where they seem to play a role in ligand binding (see, for example, Kryger et al., 2000
) or catalysis. They can also be found in polar cavities located inside proteins, where they are believed to enhance protein stability. Conserved water positions in protein crystal structures may therefore correspond to water molecules that play structurally or functionally important roles.
The interactions of water molecules with the MHC proteins have also received attention, primarily in relation to peptide binding. They have been analyzed in X-ray structures of MHC class-I peptide complexes (Madden et al., 1992; Fremont et al., 1995
; Smith et al., 1996a
) or in the context of molecular dynamics simulations of these complexes (Meng et al., 1997
, 2000
). These studies mainly focused on the variation in bound water in the peptide binding groove and its role in determining peptide-binding propensities.
In this work, we took the converse approach and analyzed water positions conserved across the crystal structure of MHC class-Ipeptide complexes from a recent release of the PDB. To identify such positions we applied an automatic clustering procedure to water molecules in 11 high-resolution well refined MHCpeptide complexes, after superposition of the corresponding protein backbones. This led to the identification of a total of 67 clusters of water molecules. Correlations between various properties of these clusters, such as the number of water molecules in each cluster, their conservation across the different crystal structures, their normalized B-factors and the average distance between molecules in the cluster, were established. In addition, we identified three very highly conserved water molecules, located at key positions in the MHC protein. The particular roles that these molecules may play in maintaining the stability of the free and peptide-bound states of MHC are discussed.
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Materials and methods |
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From the total of 68 entries for class-I MHCpeptide complexes in the October 2001 release of the PDB, 12 high-quality, high-resolution (better than 2.65 Å) complexes, with many identified water molecules were selected (Table I). The H-2Db allele has four entries in the PDB, 1BZ9, 1CE6, 1HOC and 1QLF. Of those, 1HOC has the highest resolution (2.4 Å), but since the number of water molecules in this entry is smaller than in 1QLF, a structure of somewhat lower resolution, the latter structure was used instead. We also included the murine H-2M3 molecule (RCSB-PDB code 1MHC) which is class-Ib and as such is not polymorphic: it binds only N-formylated peptides. Its conformation is, however, very similar to those of the other MHC molecules in our data set, therefore qualifying as a valid example in our study.
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The first step in our approach was to transform the atomic coordinates of all 12 selected structures into the same reference frame. To that end, the backbones of the 1 and
2 domains of the MHC molecules were superimposed on to those of HLA-B3501 (1A1N). Next, water molecules distant by <5 Å from any protein atom of the
1 and
2 domains in each individual crystal structure were selected and their coordinates were transformed into the same reference frame. These coordinates were then subjected to the cluster analysis.
This analysis was performed using the Average Group method (Sneath and Sokal, 1973; Kendall, 1975
), with the Euclidean distance between water molecules as the metric. The distance cutoff for defining clusters was set to 1.5 Å. This particular value was used to ensure that clusters do not contain several water molecules belonging to the same crystal structure, which may be within hydrogen boding distance of one another.
Clusters grouping water molecules from at last six crystal structures (conservation ratio 0.5) were retained for further analysis and the average coordinate of the oxygen atoms in each cluster was computed.
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Results and discussion |
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Our cluster analysis identified a total of 67 clusters of conserved water positions in the 12 crystal structures of MHC class-I molecules listed in Table I. Average coordinates of water molecules in each cluster and the members of the cluster from the different MHC molecules are listed in Table II
.
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These observations suggest that the pattern in the number of highly conserved water molecules in the MHC class-I structures is probably not related to the species from which the molecules originate and may therefore be representative of MHC structures in general.
The positions of the identified clusters of conserved molecules are shown in Figure 1 together with the ribbon drawing of the reference MHC molecule (HLA-B3501, RCSB-PDB code, 1A1N). The views shown are from the directions of the
3-domain (Figure 1a
) and the bound TCR (Figure 1b
), respectively. The clusters are displayed as spheres whose radii are proportional to the average distance between water molecules in each cluster, representing a measure of the spatial spread of the cluster. The color of each sphere reflects how well the water positions in the cluster are conserved across the analyzed crystal structures.
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Interestingly, we see that clusters containing highly conserved water positionsthose from nine or more structuresalso tend to have smaller radii. The smallest average distance between water positions (0.35 Å) is observed for the cluster with the highest conservation ratio (1.0) amongst all 67 clusters. Analysis of the parent 3D structures shows that the water molecule whose positions are grouped in this tight cluster forms three hydrogen bonds with the nearby main chain atoms. Inspection of the superimposed backbones shows, moreover, that the main chain conformation in this region is well conserved in the 12 MHC molecules (see below).
In contrast, several other clusters in the vicinity of this tight cluster highlighted by a yellow circle in Figure 1a have a comparatively larger radius. Inspection of the superimposed MHC backbones in this region reveals that these display appreciable conformational variability. These more variable clusters are located near the beginning of the
2 helix. The backbone structure in this region also displays appreciable variability across the 12 MHC class-I molecules examined. The variability in the water positions of these clusters therefore probably relates to the variability in local backbone conformation.
The purple circle in Figure 1a highlights a cluster (No. 23) with a large dispersion in water positions (0.9 Å) and rather low conservation ratio (0.67). This cluster partially overlaps with three other large clusters that also have relatively low conservation ratios. This indicates that the water positions in this region of the MHC molecule are rather variable and that our clustering procedure had some difficulty in clustering them.
Figure 2 shows the relationship between the average normalized B-factors of the water positions in each cluster and two other parameters of the clusters, the conservation ratio and the average distance between the positions. The normalized B-factor can be taken as an indicator of the accuracy with which the water positions in the cluster have been determined in the crystallographic experiment (Buerger, 1960
). It was computed as
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These results taken together indicate that the more accurately a water position is defined in a given protein crystal structure, the more likely it is to be identified in a similar position in a different crystal structure of the same or closely related polypeptide. This agrees with findings made previously in studies of conserved water positions of other protein systems (Loris et al., 1999).
Detailed analysis of specific water positions
Of the total of 67 water positions conserved in more than half of the structures (Figure 1), three of the most conserved positions (labeled 1, 2 and 3 in Table II
) warrant detailed analysis. Their location relative to the MHC backbone is depicted in Figure 3
, which displays the MHC molecule with the TCR binding side facing the viewer.
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In the following we analyze in detail the environment of these three water molecules and discuss their putative role in the context of the MHCpeptide complex.
The most highly conserved water position and its putative structural role.
The most highly conserved water position, present in all 12 MHC structures, is represented by cluster No. 1. Its hydrogen bonding interactions with the main chain atoms in the structure of HLA-B3501 (1A1N) (see Figure 4) are as follows: O waterO Ile94 (2.94 Å), O waterOE1 Gln96 (2.89 Å) and O waterN Gly120 (2.77 Å).
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A third interesting observation can be made concerning the interactions of water in this conserved position with the protein backbone. Inspection of the backbone conformation in this region reveals that the polypeptide segment spanning residues 118121 adopts a twisted ß-turn conformation (Figures 4 and 5), with the following backbone torsion angles: (
118,
118) = (127.8°, 122.3°), (
119,
119) = (50.7°, 35.8°), (
120,
120) = (80.5°, 11.9°) and (
121,
121) = (126.4°, 140.9°). This segment is located near the C- and N-termini of
-helices in
1- and
2-domains, respectively. The high degree of conservation in our structure set of both the water position and the twisted ß-turn conformation of the backbone segment with which it interacts suggests that the water molecule plays a role in stabilizing this conformation.
To verify this hypothesis a database comprising 510 451 pentapeptide fragments from protein structures in the PDB with <90% sequence identity (Ogata and Umeyama, 1998) was searched for fragments with a similar twisted ß-turn structure. This search was performed by superimposing all the pentapeptides from the database on to the 118121 segment of the 1A1N entry and selecting those which superimposed with an r.m.s.d.
1.0 Å. Water molecules within a 5.0 Å distance from the selected fragments in the parent structures were extracted in turn and their coordinates were transformed into the common reference frame. From those, water molecules within 1.5 Å of the conserved water position in MHC were retained and their interactions examined.
This search produced 59 fragments from different protein structures including six MHC class-I molecules; 43 of those, including four fragments from MHC molecules (located elsewhere than residues 118122) have a water molecule near the main chain of residue n + 3 of the ß-turn. These are shown superimposed on to the 118122 fragment of MHC together with the corresponding water molecules in Figure 6. This shows that twisted ß-turn structures in very different protein structures tend to have a water molecule interacting with the backbone of residue n + 3, suggesting that this water molecule plays an important role in maintaining this conformation. Its conservation in the MHC class-I molecules observed here can hence also be attributed to a structural role of a rather general nature.
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These observations and considerations suggest that when position 63 is occupied by a polar residue Glu, Gln or Asn, a structural water molecule plays a part in modulating the interactions of the MHC molecules with the P1 and P2 sites of the peptide. It is interesting to see in this regard that, unlike for position 63, the side chains at positions 7 and 59 (both tyrosines) with which the conserved water molecule also forms H-bonds are completely conserved in type and conformation in our sample of MHC molecules, as illustrated in Figure 8.
Conserved water position at the first -helix kink in the
2-domain.
The third most conserved water position, corresponding to cluster No. 3, occurs in 10 out of the 12 analyzed structures (Table II
). Figure 9
illustrates its environment in the HLA-B3501 (1A1N) structure. The H-bonds made by this water molecule are O waterNE1 Trp133 (3.05 Å), O waterO Trp147 (3.04 Å) and O waterN Asn153 (2.93 Å).
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To illustrate this point, Figure 10 displays the backbones of residues 152 and 153, the full Trp147 residue and the positions of conserved water molecules in all the MHC structures of our sample, after their backbones have been superimposed. The main chain atoms of P
-1 in the peptide moieties are also displayed. We see that the displacement of the backbone oxygen of Trp147 is significantly larger than those of main chain atoms of residues 152 and 153 and reaches 2.61 Å. On the other hand, this displacement seems to follow closely that of main chain atoms of P
-1 in the peptide. Furthermore, the hydrogen bond made by the backbone oxygen of Trp 147 to the conserved water molecule is maintained in all the MHC structures.
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Conclusion |
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The structural and functional roles of the three most conserved water positions have been discussed in detail. One appears to play an important structural role. Not only does it appear to stabilize a twisted ß-turn structure that occurs in all the MHC structures of our sample, but similar twisted ß-turn motifs, containing an equivalent water position, are also identified in many other protein structures of the PDB. This water molecule therefore seems to be a universal structural determinant of this specific and recurrent twisted ß-turn motif.
The two other water molecules are suggested to play a role in modulating, directly or indirectly, the positions and orientation of residues lining pockets in the binding groove of MHC class-I molecules, which are important for peptide recognition. One water molecule forms a hydrogen bond with an MHC residue influencing the positions of (MHC or peptide) residues in pocket B. The other water molecule bridges two segments of a kinked -helix in the
2 domain, whose relative orientation and position are likely to modulate the size of pocket F, thereby presumably indirectly influencing peptide binding.
The information provided here on the conserved water positions and their structural and functional roles should be very valuable for the prediction and modeling of MHCpeptide interactions, a topic with many useful practical applications. Our findings on the commonly occurring twisted ß-turn with its associated water molecule in proteins should have useful general applications in protein structure predictions.
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Notes |
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Acknowledgments |
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References |
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Received August 7, 2001; revised April 11, 2002; accepted April 18, 2002.