0 1994 by “be American Society for THE JOURNAL OF BIOLCGICAL CHEMISTRY Biochemistry and Molecular Biology, Inc. Vol. 269, No. 43, Issue of October 28, pp. 26722-26733, 1994 Printed in U.S.A. Conserved Watersin Legume Lectin Crystal Structures THE IMPORTANCE OF BOUND WATER FOR THE SEQUENCE-STRUCTURE RELATIONSHIP WITHIN THE LEGUME LECTIN FAMILY* (Received for publication, July 8, 1994, and in revised form, August 19, 1994) Remy Loris$§, Philippe P. G. Stasnll, and Lode Wyns$ From the $Laboratorium uoor Ultrastructuur and the lL.aboratorium uoor Cellulaire Zmmunologie, Znstituut voor Moleculaire Biologie, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium An analysis of the water structure in the crystals of different legume lectins has been carried out. Protein hydration is found to be mainly dependent on the de- tailed local surface characteristics of the protein and will adapt upon the limited conformational changes that are the resultant of crystal packing forces and point mutations. Yet a significant portion of the water posi- tions determined by x-ray crystallography appears to be conservedinallindependentcrystalstructures of a given protein. Some of these waters are specific to this one specific protein, while others are conserved in crys- tal structures of homologous proteins as well. For those conserved waters, a clear structural role is often evi- dent. Seven water sites were found to be completely con- served in all legume lectin crystal structures, independ- ent of theirdegree of sequence homology or carbo- hydrate specificity. Of these, four waters are ligands to the manganese and calcium ions, and one water is lo- cated in the saccharide binding site interacting with a conserved Asp and Asn residue. Of the remaining two conserved waters, one of them stabilizes a fi-hairpin, while the other interacts with a P-bulge structure of the back sheet. The role of water in protein structure and function is a much debated but still unresolved question. Completely buried wa- ters are found at conserved sites in homologous protein struc- tures (Edsall and McKenzie, 19831, while genetically engi- neered cavities are rarely found to be occupied by water (Eriksson et al., 1992). Many studies have focused on the gen- eral featuresof protein hydration in order to be able to predict hydration sites, as this gives useful information for molecular dynamics simulations, homology modeling, and protein struc- ture determinationby x-ray crystallography. Moreover, protein hydration is thought to play an important role in the folding process, as is also illustrated by the association of internal waters in proteins with hydrophilic side chains (Sreenivasan and Axelsen, 1992; Rashin et al., 1986). Furthermore, chains of waters between the active site of a protein and the bulk solvent have been suggested to play a role as a “proton wire,” used to move hydrogen atoms in and out the active site during enzy- matic action (Meyer, 1992). * This work was supported by a grant from the Vlaams Actiepro- gramma Biotechnologie project of the Flemish government. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Onderzoek,Belgium. To whom correspondence shouldbeaddressed. 5 Research assistant of the Nationaal Fonds voor Wetenschappelijk Tel.: 32-2-3590209; Fax: 32-2-3590390; E-mail: [email protected]. 11 Financially supported by the Institut voor Wetenschappelyk, Onder- zoek in de Nyverheid en Landbouw. Little is known about the conservation of nonburied waters in related protein structures. Malin and co-workers (Malin et al., 1991) investigated the occurrence of similar hydration sites in four isomorphous ribonuclease T1 structures. This study has recently extended toward ribonuclease T1 inhibitorcomplexes and ribonuclease T1 mutants that crystallize in different space groups (Pletinckx et al., 1994). Inspired by this work and by our observation that the first hydration spheres of lentil lectin and pea lectin are similar (Loris et al., 1993), we decided to look for structurally conserved water sites in the crystal structuresof legume lectins. Structural dataon legume lectins are abundant. The three- dimensional structures of several legume lectins have been determined by x-ray crystallography. These are concanavalin A (Hardman et al., 1982; Naismith et al., 19931, pea lectin (Ein- spahr et al., 1986), Lathyrus lectin (Bourne et al., 1990a), lentil lectin (Loris et al., 1993; 19941, lectin IV from Griffonia sim- plicifolia (GS-IV’; Delbaere et al., 1993), and the lectin from Erythrina corallodendron (Shaanan et al., 1991). Their mono- meric folds are very similar, but the way they associate into dimers or tetramers was found to be quite diverse and con- trolled by the presence of covalently bound carbohydrate (Shaanan et al., 1991) and point mutations (Delbaere et al., 1993). Families of crystal structures of identical and homolo- gous proteins in different liganded states are available, and their multimeric nature frequently gives rise to more than one molecule in the asymmetric unit of the crystal. They are there- fore ideally suited to study which waters form an integral part of the protein and whether and how the solvent structure in the crystal is uniquely determined by the amino acid sequence and three-dimensional structure of the protein. MATERIALS AND METHODS Alignments and Superpositions-All crystallographic coordinates were taken from the latest version of the Protein Data Bank (Bernstein et al., 1977) or were provided to us directly by the authors. The lectin structures considered in this paper, together with the relevant details of their crystallographic refinement, are described in Table I. Secondary structure assignments were performed with the program DSSP (Kabsch and Sander, 1983). The 35 available legume lectin sequences were ex- tracted from the SWISSPROT data base of amino acid sequences. Se- quence alignments were performed manually, taking into account the secondary structure elements of those lectins from which the three- dimensional coordinates are available: lentil lectin, pea lectin, Lathyrus ochrus lectin (LOL), concanavalin A, GS-IV, and E. corallodendron lec- tin. Structure superpositionandcharacterization of protein-protein contacts were performed with X-PLOR (Briinger, 1990). Identification of Conserved Hydration Sites-Conserved hydration sites were identified with the program FIXWAT written byA.Hem- mings (Lisgarten et al., 1993). For two structures that are compared, The abbreviations used are: GS-IV, lectin IV from G. simplicifolia; LOL, L. ochrus lectin; r.m.s., root mean square; LOLOCT, L. ochrus lectin structure complexed with an octasaccharide. 26722
Transcript
0 1994 by “be American Society for THE JOURNAL OF BIOLCGICAL CHEMISTRY
Biochemistry and Molecular Biology, Inc. Vol. 269, No. 43, Issue of October 28, pp. 26722-26733, 1994
Printed in U.S.A.
Conserved Waters in Legume Lectin Crystal Structures THE IMPORTANCE OF BOUND WATER FOR THE SEQUENCE-STRUCTURE RELATIONSHIP WITHIN THE LEGUME LECTIN FAMILY*
(Received for publication, July 8, 1994, and in revised form, August 19, 1994)
Remy Loris$§, Philippe P. G. Stasnll, and Lode Wyns$ From the $Laboratorium uoor Ultrastructuur and the lL.aboratorium uoor Cellulaire Zmmunologie, Znstituut voor Moleculaire Biologie, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium
An analysis of the water structure in the crystals of different legume lectins has been carried out. Protein hydration is found to be mainly dependent on the de- tailed local surface characteristics of the protein and will adapt upon the limited conformational changes that are the resultant of crystal packing forces and point mutations. Yet a significant portion of the water posi- tions determined by x-ray crystallography appears to be conserved in all independent crystal structures of a given protein. Some of these waters are specific to this one specific protein, while others are conserved in crys- tal structures of homologous proteins as well. For those conserved waters, a clear structural role is often evi- dent. Seven water sites were found to be completely con- served in all legume lectin crystal structures, independ- ent of their degree of sequence homology or carbo- hydrate specificity. Of these, four waters are ligands to the manganese and calcium ions, and one water is lo- cated in the saccharide binding site interacting with a conserved Asp and Asn residue. Of the remaining two conserved waters, one of them stabilizes a fi-hairpin, while the other interacts with a P-bulge structure of the back sheet.
The role of water in protein structure and function is a much debated but still unresolved question. Completely buried wa- ters are found at conserved sites in homologous protein struc- tures (Edsall and McKenzie, 19831, while genetically engi- neered cavities are rarely found to be occupied by water (Eriksson et al., 1992). Many studies have focused on the gen- eral features of protein hydration in order to be able to predict hydration sites, as this gives useful information for molecular dynamics simulations, homology modeling, and protein struc- ture determination by x-ray crystallography. Moreover, protein hydration is thought to play an important role in the folding process, as is also illustrated by the association of internal waters in proteins with hydrophilic side chains (Sreenivasan and Axelsen, 1992; Rashin et al., 1986). Furthermore, chains of waters between the active site of a protein and the bulk solvent have been suggested to play a role as a “proton wire,” used to move hydrogen atoms in and out the active site during enzy- matic action (Meyer, 1992).
* This work was supported by a grant from the Vlaams Actiepro- gramma Biotechnologie project of the Flemish government. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Onderzoek, Belgium. To whom correspondence should be addressed. 5 Research assistant of the Nationaal Fonds voor Wetenschappelijk
Tel.: 32-2-3590209; Fax: 32-2-3590390; E-mail: [email protected]. 11 Financially supported by the Institut voor Wetenschappelyk, Onder-
zoek in de Nyverheid en Landbouw.
Little is known about the conservation of nonburied waters in related protein structures. Malin and co-workers (Malin et al., 1991) investigated the occurrence of similar hydration sites in four isomorphous ribonuclease T1 structures. This study has recently extended toward ribonuclease T1 inhibitor complexes and ribonuclease T1 mutants that crystallize in different space groups (Pletinckx et al., 1994). Inspired by this work and by our observation that the first hydration spheres of lentil lectin and pea lectin are similar (Loris et al., 1993), we decided to look for structurally conserved water sites in the crystal structures of legume lectins.
Structural data on legume lectins are abundant. The three- dimensional structures of several legume lectins have been determined by x-ray crystallography. These are concanavalin A (Hardman et al., 1982; Naismith et al., 19931, pea lectin (Ein- spahr et al., 1986), Lathyrus lectin (Bourne et al., 1990a), lentil lectin (Loris et al., 1993; 19941, lectin IV from Griffonia sim- plicifolia (GS-IV’; Delbaere et al., 1993), and the lectin from Erythrina corallodendron (Shaanan et al., 1991). Their mono- meric folds are very similar, but the way they associate into dimers or tetramers was found to be quite diverse and con- trolled by the presence of covalently bound carbohydrate (Shaanan et al., 1991) and point mutations (Delbaere et al., 1993). Families of crystal structures of identical and homolo- gous proteins in different liganded states are available, and their multimeric nature frequently gives rise to more than one molecule in the asymmetric unit of the crystal. They are there- fore ideally suited to study which waters form an integral part of the protein and whether and how the solvent structure in the crystal is uniquely determined by the amino acid sequence and three-dimensional structure of the protein.
MATERIALS AND METHODS Alignments and Superpositions-All crystallographic coordinates
were taken from the latest version of the Protein Data Bank (Bernstein et al., 1977) or were provided to us directly by the authors. The lectin structures considered in this paper, together with the relevant details of their crystallographic refinement, are described in Table I. Secondary structure assignments were performed with the program DSSP (Kabsch and Sander, 1983). The 35 available legume lectin sequences were ex- tracted from the SWISSPROT data base of amino acid sequences. Se- quence alignments were performed manually, taking into account the secondary structure elements of those lectins from which the three- dimensional coordinates are available: lentil lectin, pea lectin, Lathyrus ochrus lectin (LOL), concanavalin A, GS-IV, and E. corallodendron lec- tin. Structure superposition and characterization of protein-protein contacts were performed with X-PLOR (Briinger, 1990).
Identification of Conserved Hydration Sites-Conserved hydration sites were identified with the program FIXWAT written by A. Hem- mings (Lisgarten et al., 1993). For two structures that are compared,
The abbreviations used are: GS-IV, lectin IV from G. simplicifolia; LOL, L. ochrus lectin; r.m.s., root mean square; LOLOCT, L. ochrus lectin structure complexed with an octasaccharide.
26722
Water Structure of Legume Lectins 26723
TABLE I Legume lectin crystal structures used in this work
TABLE I1 Comparison of representative legume lectins
study. Below the diagonal is the amino acid sequence identity. Above the diagonal are backbone r.m.8. deviations after least-square superposition of the residues in variant in the proteins considered in this
Orthorhombic lentil lectin
monomer 1
monomer 2
Pea lectin Lathyrus lectin
Con- canavalin E thrina Griffonia %din lectin N (monomer '' (monomer 1) A
Lentil lectin (monomer 1) 0.18 A 0.34 A 0.35 A 0.67 A 0.48 A 0.80 A Lentil lectin (monomer 21 100 0.38 A 0.36 A 0.67 A 0.48 A 0.80 A Pea lectin (monomer 1) 89.8 89.8 0.33 A 0.69 A 0.50 A 0.79 A Lathyrus lectin (monomer 1) 89.4 89.4 92.1 0.66 A 0.51 s, 0.82 A Concanavalin A 39.7 39.7 38.8 38.8 0.56 A 1.09 A Efythrina lectin 47.1 47.1 46.7 47.1 38.4 0.79 A Griffonia isolectin IV 42.3 42.3 42.7 41.4 37.1 37.5
FIG. 1. Histognun showing the numbers of water molecules
of the same crystal as a function of the differences in their common in two lectin monomers present in the asymmetric unit
positions. Shown are orthorhombic lentil lectin (upper), pea lectin (middle), and native Lathyrus lectin (lower).
water molecules are identified that are closer than 5.0 A to the nearest protein atom, taking into account the symmetry elements of the crystal. After superposition of the relevant portions of two crystal structures, a hydration site was considered conserved between the two structures if both water molecules were found to be closer to each other than a predefined distance of 1 A and had an identical or equivalent hydrogen bonding scheme. For multiple superpositions, a sphere with radius 1 A was used.
Intersubunit Contacts and Crystal Contacts-Intersubunit contacts and crystal contacts were analyzed using the program X-PLOR (Briinger, 1990). Solvent-accessible surface areas were calculated with the BRUGEL program package (Delhaize et al., 1984). The calculated accessible surface areas for the conserved water molecules takes into
account the surfaces of only those waters that are strictly conserved. Graphics inspection was performed on an Evans and Sutherland PS390 using the FRODO program (Jones, 1978). All calculations were per- formed on a VAX-3300 computer and on a Silicon Graphics Indigo.
RESULTS Cut-off Distances for Conserued Water Sites-The crystal
structures used in this study are summarized in Table I. In total, the first hydration shell of 21 crystallographic independ- ent monomers, belonging to six different legume lectins, were compared in pairs. These lectin structures have amino acid identities ranging from 100 to 40% and superimpose with r.m.s. coordinate differences between 0.16 and 1.09 A for their equiv- alent main chain atoms, as listed in Table 11.
The three native Viciae lectin crystal structures (pea, lentil, and Lathyrus) each contain a complete functional dimer in the asymmetric unit of the crystal and are determined at a resolu- tion higher than 2.0 A. We checked for water oxygens lying between 0.0 and 2.6 A apart after least-squares superposition of the main chain nitrogen and oxygen atoms of both subunits in the same crystal. These results are preseated in Fig. 1. A clear peak is observed for water sites 0.2-0.6 A apart. With an r.m.s. coordinate difference for the backbone atoms of 0.2-0.3 A for two monomers belonging to the same protein, it can be safely concluded that these waters are identical if their coordi- nate errors are taken into account. From this we can conclude that in order to locate structurally conserved hydration sites, their presence within a 1 a sphere together with an identical or analogue hydrogen bonding pattern should be an adequate criterion.
Hydration Sites Conserved in the Lentil Lectin Crystal Structures-There were three high resolution crystal forms of lentil lectin available, each containing two monomers in the asymmetric unit. Alist of the hydration sites conserved in these six independent structures is provided in Table 111. Fig. 2 shows the distribution of these solvent sites on the lentil lectin dimer.
26724
n Water Structure of Legume Lectins
n
FIG. 2. View of the 33 conserved hydration sites in the lentil lectin crystal structures superimposed on the backbone of the lentil lectin dimer. In order to emphasize the 2-fold symmetry, the waters at the dimer interface are shown for both lectin monomers.
1
r.m.s. Backbone Atoms
I
J FIG. 3. Relationship between the number of apparently con-
served water sites of two structures, their backbone r.m.8. co- ordinate distances, and their amino acid homology. 0, compari- son of two lentil lectin monomers (100% sequence identity); A, comparison of two Lathyrus lectin monomers (100% sequence identity); 0, comparison of a lentil lectin monomer with a Lathyrw lectin or pea lectin monomer (-85% sequence identity); 0, comparisons between monomer A of orthorhombic lentil lectin, concanavalin A, GS-IV, and E. corullodendron lectin (-40% sequence identity).
These sites are not evenly distributed over the protein surface. There are two regions with a high density of conserved hydra- tion sites: around the metal and monosaccharide binding re- gions near the poles of the dimer and on the dimer interface. This polarization is not completely unexpected, as the back of the protein consists entirely of a 12-strand dimer-wide P-pleated sheet. Very few water atoms hydrating this sheet were identified in the different lentil lectin crystal structures.
This is in agreement with the results of a survey of the solva- tion characteristics of main chain atoms involved in p-sheet formation (Thanki et al., 1991). However, the relative absence of conserved waters at the front and sides of the dimer, which consist of a number of irregular loop structures and reverse turns, cannot be explained by the known general hydration preferences for secondary structure elements alone.
The packing of the lentil lectin dimers in the three crystal structures are related. In each crystal form, the two crystallo- graphically independent monomers in the asymmetric unit are positioned such that they have a highly similar crystal lattice environment. Most of the direct intermolecular hydrogen bonds are similar for each of the six lectin monomers. It was therefore also expected that a number of water molecules involved in packing interactions would be conserved among the different structures. Surprisingly, none of the conserved water molecules mediate crystal contacts. Changes in the solvent network around lattice contacts may thus mediate changes in space group or unit cell without significantly altering the global pack- ing of the protein molecules.
Hydration Sites Conserved in the Lathyrus Lectin Crystal Structures-LOL is the only legume lectin for which crystal structures of the uncomplexed lectin as well as a relatively large number of complexes with oligosaccharides have been reported. These include complexes with mannose, glucose, the trisaccharide a-Man(1+3)p-Man(l-,4)GlcNac and a bianten- nary octasaccharide (LOLOCT). In our comparison, the com- plexes with glucose were discarded as they are isomorphous with the mannose complexes, and no changes were made to the solvent during the refinement (Bourne et al., 1990b). The re- maining four crystal structures contain 10 independently re- fined lectin monomers in a wide range of crystal environments. For these 10 molecules, the solvent structure was compared in pairs. A total of 13 solvent atoms were found in common in all
Water Structure of Legume Lectins
J & 26725
J.***.... : 0 . . "... : e : : . .
. . . . . . . . . . : .* . .
.. -. : : : . " ' I I ' ..
Ala8OP .. Ala80P ..
-. . . '".e" ....
;.
. . . . . . . . . . . . . . . . ..
2.
FIG. 4. Conservation of a water site between lentil lectin (boldface lines) and pea lectin (thin lines) despite a conservative amino acid substitution. Shown are Alaasa, ThrP122, and Gln(Hi~)"~' hydrogen bonded to this water molecule.
FIG. 5. Schematic diagram of the water network on the subunit inter- face of the Viciae lectins. Upper panel, lentil lectin, middle panel, Lathyrus lec- tin; lower panel, pea lectin. Possible hy- drogen bonds involving the conserved wa- ters are shown as dotted lines.
structures, while another 22 were found in at least 8 out of 10 to otherwise conserved waters that are replaced by the bound molecules. If the two LOLOCT monomers are not considered, octasaccharide in the LOLOCT structure, and neither is it 25 water sites were found to be completely conserved, and likely to be a crystal packing effect, as all the other Lathyrus another 10 appeared in 7 of 8 molecules. This effect is not due lectin monomers considered appear in a wide variety of packing
26726 Water Structure of Legume Lectins
VTYR 4 6 c
L
VTYR 4 6 c
' Y R 4 b A
4
4 A
4 4 8
' d
4 6A
FIG. 6. Stereo representations of part of the water network on the subunit interface of the viciae lectins. Upper panel, lentil lectin, middle panel, Lathyrus lectin; lower panel, pea lectin. Waters are shown as black spheres.
modes. It is thus most likely that the aberrant behavior of the LOLOCT structure is due to its lower resolution. Indeed, the LOLOCT model was only refined at 2.3 A, the lowest resolution included in our survey. This lower resolution may have made the interpretation of the solvent density more difficult.
Effect of Side Chain Substitutions-The lectins from pea, lentil, and Lathyrus are structurally very similar. Their se- quence identities are as high as 85%, they do not contain any insertions or deletions, and the backbone atoms of their man!- mers superimpose with r.m.s. deviations between 0.3 and 0.4 A. Nevertheless, since all amino acid substitutions occur on the surface of the molecule, it was of interest to see how far this affected the water structure.
In the case of pea lectin, the only high resolution structure available is an orthor$ombic crystal form of the uncomplexed lectin refined at 1.7-A resolution and containing a complete lectin dimer in the asymmetric unit. Both monomers have 70 solvent sites in common, a number comparable with those found when comparing two high resolution ( 4 . 9 A) lentil lectin or Lathyrus lectin monomers.
Most of the hydration sites conserved in the lentil lectin structures are also found to be present in most of the Lathyrus
lectin structures and in the two pea lectin monomers, thus confirming their structural significance. These conserved wa- ters are, in general, characterized by low solvent accessibilities, a significant fraction having zero or near zero accessibility, and lower than average B-values. In Table I, it is shown that the average B-values of the conserved waters are similar to those of the protein side chain atoms. Nevertheless, some of the indi- vidual conserved water molecules can have quite high B-values in some of the structures, the highest one being 85.79 in LOLOCT. Also the correlation between the B-values of equiva- lent waters in two monomers of the same structure is low, ranging from 0.80 in the high resolution lentil lectin and pea lectin structures to as low as 0.50 in the lower resolution La- thyrus lectin structures.
Fig. 3 shows the relationship between the number of appar- ently conserved water sites of two structures, their backbone r.m.s. coordinate distances, and their amino acid homology. It is immediately clear that two structures with a limited sequence homology and relatively large r.m.s. values have only a small number of mutually conserved waters. In contrast, a large frac- tion of solvent sites can be conserved between two structures if they are highly homologous (>85% identity) and have low back-
Water Structure of Legume Lectins 26727
FIG. I . Schematic diagram of the
binding sites of lentil lectin. Possible conserved water sites near the metal
hydrogen bonds involving the conserved waters are shown as dotted lines. Interac- tions with the calcium and manganese ions are indicated with arrows.
bone r.m.s. coordinate distances. A high similarity between two structures is however a necessary but not the only condition for conservation of the solvent sites observed by crystallography. Resolution seems to be an important factor as well. The number of conserved solvent sites among pairs of lentil lectin monomers (1.9-1.75-A resolution) is significantly larger than the corre- sponding number of conserved sites among the different L. ochrus lectin monomers (2.3-1.9 A). This is especially true for the two monomers of the LOLOCT, which has the lowest reso- lution (2.3 A). The number of conserved water sites between the two lectin monomers in this structure and between each of these monomers and the other Lathyrus lectin structures is small. Within the groups of Lathyrus or lentil lectin crystal structures, there is, however, no clear correlation between reso- lution and the magnitude of the backbone r.m.s. differences.
As one intuitively expects, the waters that are completely conserved in the Lathyrus lectin, lentil lectin, and pea lectin structures always form hydrogen bond to either main chain atoms or atoms from side chains that are conserved in all three lectins. If one considers the number of hydration sites con- served between one particular lentil lectin monomer and one particular Lathyrus or pea lectin monomer, this number (30- 70) can be significantly larger than the number of completeiy conserved water sites in either the Lathyrus lectin or lentil lectin group. When considering these partly conserved sites, it became apparent that the above rule of “conserved water means conserved sequence” remains largely correct. In a lim- ited number of cases, however, it is seen that the potential water site is not destroyed if the amino acid substitution is sufficiently conservative. One such example, a water site con- served between lentil lectin and one of the pea lectin mono- mers, despite a His + Gln mutation, is shown in Fig. 4. The reason why this and numerous other partially conserved water sites are not completely conserved becomes obvious if those structures where the site in question is not conserved are su- perimposed. In contrast to the completely conserved water sites, the partly conserved sites are highly solvent exposed, and some of the side chains are free to rotate into another confor- mation that stabilizes an alternative solvent site. The reason why these side chains are found in different conformations in different structures but not observed in multiple conformations within the same structure may be either local packing con-
straints or differences in crystallization conditions such as pH or ionic strength.
Dimer Interface-The conserved water sites at the dimer interface of lentil lectin form a network. Except for interwater hydrogen bonds, all other hydrogen bonding interactions of these waters are with side chain oxygens and nitrogens. The conserved and almost conserved solvent sites found in the La- thyrus structures largely agree with those found in the lentil lectin case (Table 111). A striking difference, however, is found at the dimer interface. Although the waters at the Lathyrus lectin dimer interface are also conserved among the Lathyrus lectin structures, this conserved water network was found to be dif- ferent from the one found at the lentil lectin dimer interface. The hydrogen bonding scheme of buried waters on the interface of the lectin dimer and the Lathyrus lectin dimer are compared schematically in Figs. 5 and 6. The striking difference between the local solvent networks is somewhat unexpected in view of the extremely high local amino acid sequence conservation be- tween both lectins and the buried nature of these waters. It was shown that the introduction of an extra methyl group on the dimer interface, by the substitution of S e P 8 in the pea and Lathyrus lectins to Thr@48 in lentil lectin, causes small, but significant changes in the surrounding P-strands, especially around Thrw (Loris et al., 1993, 1994). Moreover, for sterical reasons, the side chain of Thrp48 in lentil lectin is oriented such that its methyl group is superimposed on the side chain hy- droxyls of S e P 8 in the pea and Lathyrus lectins. Since these side chains in both lentil lectin and Lathyrus lectin help to stabilize the water network on the dimer interface, it is tempt- ing to conclude that this substitution, in order to optimize the hydrogen bonding potential of the amino acid side chains and water molecules in a restricted space, is the direct cause for the establishment of an alternative water network.
If the above reasoning is true, the water network on the dimer interface of pea lectin should be identical to, or resemble strongly the one on the Lathyrus lectin dimer interface. Sur- prisingly, it was found that the corresponding water network for pea lectin is asymmetric around the dimer 2-fold axis (Fig. 5, lower panel 1. While one part of the network is identical to the one found in the Lathyrus lectin crystal structures, a second part resembles neither the network of Lathyrus lectin nor the one of lentil lectin. The explanation for this asymmetry is most
26728 Water Structure of Legume Lectins TABLE I11
Conserved hydration sites among the lentil lectin crystal structures
sign means that the corresponding atom belongs to the second monomer of the lectin dimer. Listed in this table are all water molecules that can be found simultaneously in all crystallographic independent monomers of the lectin. The #
Site Residue Atom Distance B-value Accessibility ?::Ed '?iZd Remarks lectin
A A= A' 1 Cal Ca2+ 2.37 17.13 0.000 Yes Yes Metal ligand
ASPS81 OD2 Asp@'
2.86 0
Asp@'" OD2 2.80 2.68
PhePlw 0 hns125 OD1
3.19 3.06
Wat2 0 3.31 2 Cal Ca2+ 2.38 16.98 0.000
GlyPW 0 Asp@1z1 OD1
2.74
OD1 2.84
TrpP'28 NE1 3.07 3.04
Wat' 0 3.31 Wat3 0 3.16
3 Mng Mn2+ 2.22 10.50 0.004 ~ 1 ~ 0 1 1 9 OEI G l ~ f i " ~ OE2
Packing of an irregular loop structure upon the inner p-sheet
Hydration outer sheet 2.217 No No
No No Connecting two loops 3.265
No Connecting two loops
Yes Connecting two loops
23.537
0.418
No
No
No Dimer formation
No Dimer formation
0.000
1.312
No
No
No No Dimer formation 1.564
0.780
0.000
No
Yes
No Dimer formation
No Dimer formation
0.288 Yes
No
Yes Dimer formation
No Dimer formation 1.580
0.792 No No Dimer formation
a This water is located on the local 2-fold axis of the lentil lectin dimer. The six lentil lectin monomers therefore only contain three such waters. ' Because of the local 2-fold axis relating both monomers, each of these waters will be present twice. This water is only present in five out of six lentil lectin monomers. In the sixth monomer, difference density is present suggesting the presence
of this water, but after refinement this water systematically comes too close to the OH atom of Tyrm6.
likely to be found in the very tight crystal packing of pea lectin. Evidence for packing-mediated changes in a number of loops is apparent in pea lectin structure, while no such packing effects can be seen when the two lentil lectin or Lathyrus lectin mono- mers are superimposed onto one another. The noncrystallo- graphic pseudo-2-fold axis relating both pea lectin monomers corresponds to a rotation of 178.8", while for both lentil lectin and Lathyrus lectin, it is almost identical to a real 2-fold axis
(179.8" in both cases). It is thus likely that the crystal packing forces that disturb the overall 2-fold symmetry of the pea lectin dimer also disturb part of the solvent network on the dimer interface.
Metal and Monosaccharide Binding Region-A chain of six water molecules (waters W1, W2, W3, W4, W7, and W8 in Fig. 7 and Table 111) is found near the calcium and manganese ions in all structures of lentil lectin and pea lectin and in most of the
26730 Water Structure of Legume Lectins
Lathyrus lectin structures. Of these, two are liganded to the manganese ion (W3 and W4), and two others are liganded to the calcium ion (WI and W2), W l being essential for stabilizing the unusual Ala-Asp cis-peptide bond that is found in all crystal structures of legume lectins determined to date. Several other conserved waters further stabilize the loop structures around the metal binding sites by forming a bridge with other nearby parts of the polypeptide chain. The four water molecules that directly ligate the manganese and calcium ions are also con- served in the structures of concanavalin A, GS-IV, and E. cor- allodendron lectin (Table IV). In view of the known metal re- quirements of the legume lectin family and their very high sequence homologies in this part of the structure, it is most likely that these four water molecules will be essential in all other legume lectins as well, with the exception of the Phaseo- lus a-amylase inhibitor (because this protein lacks some of the amino acids that make up the metal binding sites).
Furthermore, in those structures with a free monosaccharide binding site, a water bridgingAsp@' OD1, GlyPg9 N and AsnP"' ND2, is also observed. In all the lectin-sugar complexes studied to date, this water position is occupied by a sugar hydroxyl. In the lentil lectin structures, an oxygen atom from a phosphate anion occupying the monosaccharide binding site is found at the same place (Loris et al., 1994). As Asp@' and AsnP125 are almost completely conserved in all sequences and are key resi- dues for lectin sugar recognition, it is most likely that this solvent site will be conserved in the majority of legume lectins.
Conserved Water Molecules Associated with Secondary Struc- tural Elements-& is shown in Table 111, most of the hydration sites conserved in the structures of lentil lectin, Lathyrus lectin and pea lectin play a role in secondary structure packing (mainly bridging loops with p-strands) and the stabilization of hairpin structures. All of them have at least two, but mostly three or four hydrogen bonding partners, These are mostly main chain nitrogens and oxygens. Other conserved water mol- ecules occur as frequently as hydrogen bonding partners to a conserved water as side chain atoms.
Two of these water positions are present in all available legume lectin structures. As is shown in Fig. 8 and Table IV, the first one of these is found to stabilize a P-hairpin, while the other one interacts with a bulge-like structure in the front sheet. The P-hairpin loop is found near the metal binding site, comprising residues Valfl'" to Lysfl14' and is strongly conserved in length as well as in amino acid sequence within the whole legume lectin family. Notable exceptions are the Phaseolus a-amylase inhibitor, arcelin, and lima bean lectin. This strongly suggests that in most of the legume lectin structures, the conformation of this loop will be identical and therefore that this water site may be found in other structures as well. This is also true for the bulge-associated water site, shown in Fig. 8B. This bulge induces twist in the back sheet that may be an essential feature of all legume lectin structures, given also the high degree of sequence homology around this position. The conserved water associated with this structural feature does not only stabilizes this bulge, but also mediates sheet to sheet packing.
A survey of the Brookhaven Data Base (Bernstein et al., 1977) showed that this hydrated bulge structure is quite rare. Except for the legume lectin family, it is only found in human carbonic anhydrase (residues Le~ '~ ' -Glu~ '~ and Phe147 of entry 1CA2) and in an insertion mutant (llGG12) of staphylococcal nuclease (residues Glu'', Gly'" and VaV4 of entry 1STA) and has not been described as a motif occurring naturally in pro- teins. Despite the low number of examples found, we neverthe- less consider this type of structure of a new type of P-bulge, the hydrated p-bulge. Its topology is shown in Fig. 9. Because 2
residues are inserted into the bulged strand instead of one, as occurs in the classic and G1 types of P-bulges (Richardson et al., 19781, it is most closely related to the special type of P-bulge, and more in particular to the SW, S3, and SP3 subtypes as they occur in the classification of Chan and co-workers (Chan et al., 1993). The reason for its rarity may be related to the fact that the inserted water, although having no solvent-accessible sur- face in all of the encountered structures, has only three hydro- gen bonding partners. This is compensated in part by a bifur- cated hydrogen bond between the peptide NH group of residue X and the two lone pairs of electrons of the water oxygen atom. If one considers the bulged-out strand outside a P-sheet con- text, this structure becomes more common. The peptide NH group of the X-residue or the water molecule are then replaced by a side chain amino group from an Asn, Gln, or Arg residue. This occurrence outside a P-sheet context is not surprising, as other p-bulge structures also occur commonly as parts of turns (Milner-White, 1987).
Waters inserted in a-helices or at the ends of 6-strands have been postulated being trapped folding intermediates (Sundaralingam and Sekharudu, 1989; Finer-Moore et al., 1992). In analogy with this, it is tempting to speculate that the hydrated P-bulge is an intermediate in the formation of at least some of the types of P-bulges found commonly in proteins.
DISCUSSION
When crystal structures of homologous proteins are deter- mined, detailed comparisons are usually limited to the protein atoms themselves. The hydration spheres are in most cases only compared superficially. Most comparisons have been be- tween two particular crystal structures, e.g. the same protein in a liganded and an unliganded state, and only concerned some particular local features such as the hydration in an active site or did only take into account completely buried waters. This has led to an incomplete and inconsistent picture about the degree at which a particular solvent site is reproduced within a family of related crystal structures. This knowledge is, never- theless, essential in protein chemistry, where structure and function of a protein are adapted to an aqueous environment. Bulk and bound solvent play a major role in catalysis and molecular recognition. Although there have been a large num- ber of studies on the positioning of water on protein surfaces, these studies only indicated preferred hydration sites in func- tion of secondary structure or amino acid side chain and did not answer the question of whether a particular hydration site is occupied because it allows for the presence of a water molecule or because it needs the presence of this water (e.g. whether it is essential for the structure or stability of the protein).
The present study, at least in part, provides us with an an- swer to this question. For two identical proteins that are found in a different crystal environment, a large number of water molecules on the surface of the macromolecule are found on essentially identical places. This picture changes when a larger number of such structures are mutually compared. The number of completely conserved hydration sites diminishes, and a dominance of hydrogen bonding with main chain oxygens and nitrogens becomes clear. These hydration sites have a high probability for being conserved in close homologues of this pro- tein as well and are thus most probably an essential part of the protein. The water molecules in macromolecular crystal struc- tures may therefore be divided into two categories: ( a ) a series of structurally or functionally important waters that are inde- pendent of the crystal environment and ( b ) a large number of fortuitously identified water molecules with positions that are strongly affected by the crystal lattice and amino acid substi- tutions at the surface of the protein.
a In the case of lent; 1eZinandErythrina lectin, this otherwise conserved water is replaced by a phosphate oxygen and by 0-3 of a Gal unit, respectively. In these two cases, the distances toward these non-water oxygens are given.
Only a small number of water sites remain conserved in more distantly related homologues. These can be classified into two distinct categories; they may be conserved for structural rea- sons or for functional reasons. When amino acid sequences diverge leaving only three-dimensional homology, evolutionary pressure will demand its toll even here. Functional waters may only be conserved as far as the global function or substrate of the protein remains the same. Equally, the structural waters may also come under pressure. For example, in an extensive study on the location of buried waters in serine proteases, Sreenivasan and Axelsen (1992) found that in some cases a histidine ring could replace some of the otherwise conserved buried water molecules in serine proteases,
Also the method used for the crystallographic refinement of the macromolecular structure should be considered. In a recent publication, Hahn and Heinemann (1993) dcy-ibed the inde- pendent refinement of a DNAdecamer at 1.7-A resolution using three different refinement packages on the same x-ray data. An unexpected conclusion from this work was that although the coordinates of the DNA molecule itself seemed to be very reli- able and unaffected by the refinement protocol, the solvent structure in the three independent refinements differed mark- edly: of the -80 waters that were identified in each of the
refined structures, only 15 were present in each structure, de- spite high resolution and low R-values. These observations clearly exemplify the difficulties associated with the crystallo- graphic determination of the hydration sphere of macromolecu- lar structures. The three lentil lectin structures compared in this paper were all refined using the same program (RE- STRAIN, Haneef et al., 1985) and using an identical restraints library and similar weighting schemes and refinement proto- cols. Similarly, all of the LOL structures were refined using X-PLOR in a different laboratory (Bourne et al., 1990a, 1990b, 199Oc, 1992). It is therefore comforting to see that the con- served water molecules that were identified for both groups of structures lentil lectin and LOL) are largely identical and are, in most cases, also present in the structure of pea lectin. Fur- thermore, within the group of Viciae lectins, differences in con- served waters between lentil lectin, LOL, and pea lectin can, in the majority of the cases, be rationalized on the basis of side chain substitutions that have occurred between these lectins. One can thus safely conclude that the results of this compara- tive analysis are meaningful and do not result from artifacts resulting from the refinement strategies used in the crystal structure determination.
26732
A
Water Structure of Legume Lectins
\ t
B
FIG. 8. Hydration sites conserved in all legume lectin crystal structures. Shown are the coordinates of monomer A of orthorhombic lentil lectin, with relevant parts of the backbone atoms of concanavalin A, GS-N, and E. corallodendron lectin superimposed. A, structurally conserved water stabilizing a 6-hairpin structure. B, water molecule that stabilizes a hydrated p-bulge structure in the back sheet and that forms a bridge between the back sheet and the front sheet.
Water Structure of Legume Lectins 26733
-+ .. , IS++
+?=2? + +
-
+-+
t I FIG. 9. Topology of the hydrated p-bulge. Residues involved in the
bulge are labeled X, 1 , 2, and 3. Other residues forming part of a p-strand are labeled p. The water molecule is represented by the letter W. The arrows represent the directionality of the hydrogen bonds.
Finally, in the present study only one protein family, the legume lectins, was considered. It is clear that more cases need to be studied before a general picture can emerge. For example, the importance of water for the packing of secondary structure elements (helix-sheet and helix-helix) could not be studied here. From the studies on ribonuclease T1 (Malin et al., 1991, Pletinckx et al., 1994) and bovine pancreatic ribonuclease (Lis- garten et al., 19931, the importance of water in tertiary struc- ture formation with secondary structural elements was sug- gested. It is equally true that any study will be biased by the subset of x-ray structures that are available for a given protein family.
for help with the Protein Data Bank searches. We also thank the fol- Acknowledgments-We thank Dr. I. Lasters (CORVAS International)
lowing colleagues that made available crystallographic coordinates prior to publication: Dr. Y. Bourne and Dr. C. Cambillau for the L. ochrus lectin structures, Dr. L. Delbaere for the coordinates of lectin IV
from Griffonia simplicifolia, and Dr. J. Naismith and Dr. J. Helliwell for the refined structure of cadmium-substituted concanavalin A.
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