Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, México D.F., México 04510 and 2Departamento de Química, Universidad Autónoma Metropolitana Iztapalapa, A.P. 55534, México D.F., México 09340
Received on February 16, 2000; revised on May 9, 2000; accepted on May 17, 2000.
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Abstract |
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Key words: hydrogen bonding/hydropathy/packing/proteinprotein complex/protein-sugar complex
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Introduction |
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In an attempt to further understand the molecular origins of the affinity between lectins and carbohydrates, we have carried out a detailed statistical analysis of the interfacial structure of 32 LC complexes comprising eight different lectin families. In the examination of properties such as interfacial surface area, surface hydropathy, amino acid propensities, hydrogen bonding and packing, novel demographic trends were revealed. In addition, the overall properties of these heterologous interfaces were compared with those characterizing protein dimers, an exercise that helps contextualize LC complexes within the wide and varied spectrum of complex classes that proteins are able to form. In general, the study shows that LC interactions have been optimized in such a way that their packing is significantly tighter than the packing of proteinprotein (PP) interfaces or protein interiors. The origin of this behavior seems to be related to a much stronger prevalence of polarpolar interactions and a higher interaction cooperativity. Finally, an energetic comparison of LC and PP complexes is presented to further reinforce the emerging notion that systems differing from each other in their stereochemical metrics also rely on different energetic bases (Xu et al., 1997; García-Hernández and Hernández-Arana, 1999
).
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Results and discussion |
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Interface hydropathy
Interface hydropathy indexes (¶p) were calculated from the ratio of the change in accessibility of polar surfaces (ASAp) to
ASAt. It can be seen in Table II that ¶p values of LC interfaces vary across a broad interval, ranging from 0.342 to 0.662. Notably, marked differences in polar content can be observed also between complexes of the same lectin, as in the case of the concanavalin A complexes 5cna and 1cvn. Thus, even when common binding patterns can be distinguished within a given lectin family (Sharma and Surolia, 1997
), complexes of homologous lectins are not necessarily redundant under the interfacial hydropathy metrics.
A plot of ¶p versus ASAt (Figure 1A) reveals that these parameters are strongly correlated (p = -0.91) in the case of lectinmonosaccharide complexes. Although this correlation dissipates considerably in oligosaccharide complexes (p = -0.38), when only their primary binding subsites (see Materials and methods) are considered, the data gather into the same trend as monosaccharide complexes (Figure 1B). Saliently, this demographic behavior is delineated by lectins with unrelated phylogenetic origin, indicating that independent interface architectures have evolved through common optimization paths. In contrast, since protein interiors and PP interfaces do not present a covariance between size and hydropathy (p = 0.24 and -0.32, respectively), it seems that PP recognition events are based on different principles to those observed in LC interactions.
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Being polyhydroxy compounds, carbohydrates are highly hydrophilic molecules. Not surprisingly, binding sites in lectins were found to have a higher hydrophilicity than other protein regions, including protein exteriors. Hence, lectin binding sites could be described as hydrophilic patches on protein surfaces. This picture contrasts with that for binding sites of protein homodimers, which have been realized to be more hydrophobic than the remainder of the protein exterior (Stites, 1997).
Surface complementarity
As detailed in Materials and methods, the interface compactness was measured using two different approaches: the gap index (GI), which is a measure of the total cavity volume at an interface, normalized by ASAt, and the average distance (D) between closest interface atom pairs.
GI values for LC interfaces were found to be distributed in the range of 0.201.98 Å, with a mean of 1.00 ± 0.48 Å. This value is significantly smaller (risk level <1%) than the mean GI of 2.20 ± 0.83 found for protein homodimer interfaces (Jones and Thornton, 1996).
As a second measure of intermolecular packing, average distances for closest polar-polar (Dp), polar-apolar (Dp-ap), apolar-apolar (Dap), and any (Dt) atom pairs at LC interfaces were obtained. Furthermore, Di values were normalized by the sum of the van der Waals radii to obtain a relative measure (Ri) of the interatomic distance to the size of the two neighboring atoms. As shown in Table VI, distances of closest polarpolar neighbors at LC interfaces were found to be significantly shorter than distances of other atom pair classes. As indicated by an average Rp value less than one, they are the only pair class whose van der Waals spheres tend to overlap. Moreover, differences in interatomic distances were also realized between subsites, with much shorter Dt values (0.4 Å) at primary subsites than at other loci. Table VI also shows average distances for closest atom pairs at PP interfaces. According to these data, intermolecular distances are significantly larger for PP than for LC adducts. It is interesting to note that while Dap and Rap are similar in the two systems, Dp and Rp are significantly shorter in LC complexes.
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Hydrogen bonding
With the aim of comparing the metrics of interfacial hydrogen bonds, the structures of LC and PP complexes were analyzed by means of the program HBPLUS (McDonald and Thornton, 1994). As shown in Table VII, the average hydrogen bond density per unit of total area buried (
hb/At) was found to be almost three times larger in the case of LC complexes. This dramatic discrepancy is partially due to differences in the abundance of polar surfaces, since the ¶p magnitude of LC interfaces is only 55% higher than the mean value of PP interfaces. In this respect, it is interesting to note that the density of hydrogen bonds per unit of polar area (
hb/Ap) is significantly larger (
87%) for LC complexes. Indeed, by considering the relative magnitudes of ¶p and
hb/Ap, the global hydrogen densities of the two systems become interconvertible. Finally, an analysis of the fraction of cooperative hydrogen bonds (¶C-hb, in Table VII) reveals that hydrogen bonding is more cooperative at LC interfaces in an excess that allows to account for the difference in
hb/Ap values.
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Comparison of LC and PP heterocomplexes
LC complexes are transient complexes (i.e., both parts of the complex can exist as separate entities), whereas most of the homodimers studied here are permanent (i.e., the subunits unfold upon dissociation). In general, PP heterointerfaces have been found to be smaller in size than protein homodimeric interfaces. For instance, both protease-inhibitor and antibody-antigen interfaces amount on average to 1600 Å2 per interface (Janin and Chothia, 1990; Jones and Thornton, 1996
). However, this value is still considerably larger than the average size of LC interfaces. The mean size of a set of proteinnucleic acid complexes was found to be 3100 Å2 (Nadassy et al., 1999
), which indicates that LC interfaces are also small in comparison with other heteromolecular interfaces. Furthermore, packing at protein heterodimeric interfaces seems to be, at best, as good as those in homodimeric complexes or protein interiors (Jones and Thornton, 1996
; Conte et al., 1999
; Nadassy et al., 1999
). Thus, LC compounds stand out as the protein complexes with the highest packing density at the contact interface.
Similarly, the polar character of LC interfaces is prominent among protein complexes; their average hydropathy (0.5) is only paralleled by those of antibodyantigen (0.49 ± 0.04) and proteinnucleic acid (0.48 ± 0.08) complexes (Conte et al., 1999
; Nadassy et al., 1999
). However, even these latter types of complexes display average hydrogen bonding densities (smaller than one bond per 100 Å2) which are much smaller than the bond density for LC complexes.
Comparison of LC and PP interaction energetics
For a data set of PP complexes including 3 homo- and 18 heterodimers, Xu et al. (1997) found that the average standard free energy,
G, of binding is 13.1 ± 4.3 cal · mol1 at 25 °C. From the analysis of these authors it is seen that the average change in accessible molecular area (
AMA) is 505 Å2. Normalization of the binding free energy by
AMA gives a value of
G/
AMA equal to 26 ± 5 cal · mol1 · Å2. In the case of LC complexes, the average
G of binding at 25 °C is only 5.4 ± 1.2 calmol1 (García-Hernández and Hernández-Arana, 1999
); however, the molecular area buried is so small (90 to 190 Å2) that when the normalization by
AMA is performed the resulting
G/
AMA amounts to 41 ± 7 cal · mol1 · Å2. As expected, these magnitudes indicate that as the contact area in a recognition process decreases, the diminution in free energy per unit area is larger.
What is the origin of the larger stabilization (free energy change) per unit of area buried in LC interactions? In a previous report, which analyzed the energetics of LC interactions versus protein folding (García-Hernández and Hernández-Arana, 1999), it has been shown that the larger stabilization per unit of contact area in LC complexes has an enthalpic origin. All evidence there analyzed seems to indicate that polar-polar contacts at LC interfaces are stronger than those in the protein interior. According to the results obtained in the present work, the same conclusion may hold true for a comparison between LC and PP associations. Better packing of polarpolar contacts, especially manifested in shorter hydrogen bond distances, would likely be a source of more favorable binding enthalpy for LC complexes. Unfortunately, calorimetric data for PP associations are scarce, so that no direct comparison of binding enthalpies is possible at the present.
Concluding remarks
Undoubtedly, good surface complementarity is a requisite to achieve tight packing at the interface of two interacting molecules. In this regard, binding sites in lectins, and in particular primary subsites, seem to be well adapted for the recognition of sugar molecules. They constitute, in a broad sense, regions with the appropriate hydropathy to interact with the highly polar carbohydrate surface. At a finer level of detail, there is a precise stereochemical matching between the carbohydrate and its binding site that enhances hydrogen bonding cooperativity, in particular, and polar interatomic interactions, in general. At the same time, apolarapolar interactions do not appear to be seriously compromised, since their packing is as good as at PP interfaces. This evidence is at variance with the notion that hydrophobic stacking could be the predominant force in LC binding. Overall, the energetic consequences of such a high surface complementarity between a lectin and its ligand seem to be that stable complexes can be formed despite the reduced number of contacts across the interface.
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Materials and methods |
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Changes in accessible surface area upon binding
Structure-based determinations of water-accessible surface areas (ASA) were performed with the NACCESS program (Hubbard and Thornton, 1993), using a probe radius of 1.4 Å, and a slice width of 0.1 Å. Total changes in ASA upon binding (
ASAt) were estimated from the difference between the complex and the sum of free molecules. Polar area changes (
ASAp) were calculated from the changes in accessibility of nitrogen plus oxygen atoms.
Area buried in globular protein interiors
ASAp and
ASAt values for the folding of 42 globular monomeric proteins, estimated as the difference between the native and unfolded states, were taken from García-Hernández and Hernández-Arana (1999).
Amino acid compositions and propensities
A residue was considered to form part of the binding site when its ASA was >1 Å2. A residue was counted as exterior when having at least 5% of its total surface area accessible to the solvent. From these counts, percentage frequencies for each amino acid in the protein surface (Fls) and at the binding site (Fbs) were calculated. The propensity (P) of the residue i to form a binding site was evaluated by means of the ratio Fbs/Fls (Jones and Thornton, 1996
). P values > 1 (< 1) indicate that the residue is more (less) preferred to be incorporated at binding sites than elsewhere in the protein surface.
Surface complementary
For each carbohydrate atom whose accessibility changed upon binding by more than 0.1 Å2, the distance (D) to the nearest lectin-atom was determined. Closest atom pairs were classified into polar-polar (O··O, O··N), polar-apolar (O··C, N··C), apolar-apolar (C··C), and any (X··X) contacts, and the corresponding average distances (Dp, Dp-ap, Dap, and Dt, respectively) were obtained. D values were normalized with the sum of the van der Waals radii (Rvdw) to obtain a relative measure (R) of the inter-atomic distance to the size of the two neighboring atoms: R = D/(Rvdw1 + Rvdw2).
Surface complementary was also evaluated by determining the volume of the gaps existing between lectin and carbohydrate atoms beyond their van der Waals spheres by means of the SURFNET software (Laskowski, 1995). A gap index (GI) was then obtained by dividing the total interfacial gap volume by the
ASAt value.
Hydrogen bonding analysis
LC interfaces were analyzed by means of the software HBPLUS (McDonald and Thornton, 1994) to identify hydrogen bonds. Since hydrogen atoms are rarely observed in x-ray solved structures of proteins, the identification of potential hydrogen bonds was based only on the geometric arrangement of atoms forming suitable acceptor-donor pairs, imposing the constraints derived by Stickle et al. (1992)
from small-molecule crystal structures. To reduce the uncertainties due to the experimental determination of atomic positions, only structures with a resolution of 2.3 Å or better (i.e., 25 LC and 18 PP complexes) were considered in the statistics of hydrogen bond lengths and scalar angles. The mean resolutions for the reduced data sets of LC and homodimer structures are 2.03 and 1.95 Å, respectively, while the mean temperature factors of the interfacial atoms are 22 and 18 Å2, respectively. Thus, the structural qualities of both data sets are comparable.
Identification of the primary binding subsite
For the majority of the LC complexes here analyzed, the primary binding subsite was operationally identified as that zone of the protein surface occupied by the monosaccharide that defines the primary selectivity of the lectin. Experimentally, the primary selectivity is established by determining the monosaccharide that inhibits the activity of the lectin. Nevertheless, the cholerae toxin (1chb) and the GS4 lectin (1led) are not able to bind monosaccharides, reason for which they were not considered in the analysis of primary binding subsites. Five complexes in Table I have oligosaccharide ligands composed of only one type of monosaccharide. For two of these cases (1cvn and 1jpc), the corresponding monosaccharide complexes are included in the data set (5cna and 1msa). The primary binding subsites of the complexes 1a78 and 1npl were identified by comparison with the homologous complexes 1gan and 1jpc, respectively. Finally, the primary subsite for 2msb was established according with the structural analysis performed by Weis et al. (1992) over different kinds of monosaccharides.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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