MULTIPLE MODES OF BINDING ENHANCE THE AFFINITY OF DC-SIGN FOR HIGH-MANNOSE N-LINKED GLYCANS FOUND ON VIRAL GLYCOPROTEINS

Abstract

The dendritic cell surface receptor DC-SIGN and the closely related endothelial cell receptor DC-SIGNR specifically recognize high-mannose N-linked carbohydrates on viral pathogens. Previous studies have shown that these receptors bind the outer trimannose branch Manα1-3[Manα1-6]Manα- present in high-mannose structures. Although the trimannoside binds to DC-SIGN or DC-SIGNR more strongly than mannose, additional affinity enhancements are observed in the presence of one or more Manα1-2Manα- moieties on the non-reducing termini of oligomannose structures. The molecular basis of this enhancement has been investigated by determining crystal structures of DC-SIGN bound to a synthetic six-mannose fragment of a high-mannose N-linked oligosaccharide, Manα1-2Manα1-3[Manα1-2Manα1-6]Manα1-6Man, and to the disaccharide Manα1-2Man. The structures reveal mixtures of two binding modes in each case. Each mode features typical C-type lectin binding at the principal Ca²⁺ binding site by one mannose residue. In addition, other sugar residues form contacts unique to each binding mode. These results suggest that the affinity enhancement displayed towards oligosaccharides decorated with the Manα1-2Manα- structure is due in part to multiple binding modes at the primary Ca²⁺ site, which provide both additional contacts and a statistical (entropic) enhancement of binding.

The dendritic cell receptor DC-SIGN functions in the initial recognition of pathogens, and also in adhesive interactions with T cells that scan the surface of dendritic cells for complementary peptide antigen-MHC complexes (1,2). Although the epitope for T cell interactions has not been defined, interactions with pathogens exploit the ability of DC-SIGN to recognize both branched fucosylated structures bearing terminal galactose residues, and high-mannose N-linked oligosaccharides (3-5). The latter specificity allows DC-SIGN to act as a receptor for several enveloped viruses that bear high-mannose structures on their surface glycoproteins, most notably human immunodeficiency virus (HIV⁴) (6-8). A related receptor found on endothelia in the liver, lymph nodes, and placenta, designated DC-SIGNR or L-SIGN, does not recognize fucosylated carbohydrates but shares with DC-SIGN the ability to bind tightly to high-mannose N-linked carbohydrates and to serve as a viral receptor (5,6,9-11).

DC-SIGN and DC-SIGNR are members of the C-type lectin family of Ca²⁺-dependent carbohydrate-binding proteins. The two receptors have similar primary structures, each of which comprises a short N-terminal cytoplasmic tail, a transmembrane anchor, a tetramerization domain, and a C-terminal carbohydrate-recognition domain (CRD) (6). Crystal structures of the DC-SIGN and DC-SIGNR CRDs reveal the typical long-form C-type lectin fold (4). There are three Ca²⁺ seen in these structures, of which one, designated the principal Ca²⁺, is common to all C-type lectins. The hallmark of sugar binding to C-type lectins is the direct coordination of the principal Ca²⁺ by vicinal hydroxyl groups of a pyranose ring, which also form hydrogen bonds with the amino acid side chains that serve as the other Ca²⁺ ligands (12). In the case of mannose-like ligands, vicinal, equatorial 3- and 4-OH groups form these coordination and hydrogen bonds. Specificity for particular oligosaccharides comes from additional contacts made to flanking regions of the C-type CRD.

Competition assays in which a test ligand is used to compete radiolabelled mannose-BSA from immobilized CRD have been used to examine the relative affinities of mannose-containing structures for DC-SIGN and DC-SIGNR (4,6). The trimannose core structure Manα1-3[Manα1-6]Man was found to bind 4-fold better than mannose to DC-SIGN and 2-fold better to DC-SIGNR, and the disaccharide Manα1-2Man shows similar preferences (4). A pentasaccharide corresponding to the inner five mannoses of a high-mannose oligosaccharide but lacking all terminal α1-2 linked mannoses binds 7- and 4-fold better than mannose to DC-SIGN and DC-SIGNR. The full N-linked high mannose oligosaccharide Man₉GlcNAc₂, however, shows much more substantial affinity enhancements (4). These data suggested that the presence of the Manα1-2Man moieties at the non-reducing termini of high-mannose oligosaccharides might provide substantial affinity enhancements, perhaps by interacting with a secondary binding site for this group. The surface glycoproteins of HIV and other enveloped viruses are relatively rich in Man₈ and Man₉ structures (13), so high affinity binding to such glycans contributes to selective interaction of DC-SIGN and DC-SIGNR with these pathogens.

Here, the mechanism by which terminal Manα1-2Man groups enhances affinity towards DC-SIGN and DC-SIGNR is investigated using synthetic fragments of the full N-linked high mannose structure in binding and structural studies. The data indicate that multiple modes of binding at the DC-SIGN carbohydrate-binding site provide a statistical enhancement of the affinity but do not account for all of the observed affinity differences. The different binding orientations feature contacts between the terminal mannose and different regions of the proteins, which likely provide the remaining component of the increased affinity for larger glycans.

Experimental Procedures

Protein expression

The DC-SIGN carbohydrate-recognition domain was expressed in Escherichia coli as described (6) and used for cocrystallization with Manα1-2Man. A similar construct lacking the C-terminal 12 residue extension was used for cocrystallization with Man₆. Both proteins were purified as described (6).

Synthesis and purification of Man₆ and Man₉ oligosaccharides

Compounds Man₉ and Man_6a were prepared analogously to those previously described in the literature (14,15). O-Me protection at the reducing end was chosen to diminish the possible interference of the linker with the binding site of the protein. Compound Man_6b was prepared following the same approach as Man₉, using Methyl 2,3,4-Tri-O-benzyl-α-_d-mannopyranoside (16,17) as the core sugar unit. After removal of all protecting groups, the compounds were dialyzed two times each for 12 hours against 2 L of Millipore water, then lyophilized. The Manα1-2Man disaccharide was purchased from Sigma.

Binding assays

Solid phase competition binding assays were performed using bacterially expressed CRDs of DC-SIGN and DC-SIGNR, with ¹²⁵I-Man-bovine serum albumin employed as the reporter ligand (6). Assays were performed at least twice in duplicate, except that the Man₉GlcNAc₂ glycan was assayed only once in duplicate because only limited quantities were available. Sugar concentrations were determined using the anthrone reaction (18).

Crystallization and data collection

Crystals of DC-SIGN CRD complexed with Man₂ or Man_6b (Fig. 1) were grown at 21°C by hanging drop vapor diffusion (1 μL protein to 1 μL reservoir in a drop for Man₂ and 0.6:0.6 for Man_6b). The protein solution that gave crystals for the DC-SIGN/Man₂ complex contained 10 mg ml^-1 protein, 5 mM CaCl₂ and 25 mM Man₂. The protein solution that gave crystals for the DC-SIGN/Man₆ complex contained 5 mg ml^-1 protein, 5 mM CaCl₂ and 50 mM Man_6b. The reservoir solution for both crystals contained 30% (w/v) polyethylene glycol 3000, 0.2 M NaCl, 0.1 M Tris pH 7.0. Crystals were transferred to a drop of reservoir solution with added sugar, were frozen in liquid nitrogen and maintained at 100 K during data collection. DC-SIGN-Man_6b complex data were measured on an ADSC Q315 CCD detector at beam line 11-1 of the Stanford Synchrotron Radiation Laboratory. DC-SIGN-Man₂ diffraction data were measured on an ADSC Q315 CCD detector at beam line 5.0.2 of the Advanced Light Source. Diffraction data were processed with MOSFLM and SCALA (19) (Table 1).

Figure 1. N-linked high-mannose structures.

The full nine-mannose structure (Man₉) is shown in the green box. The outer trimannose moiety, marked with a black box, is present in both the Man_6a (red box) and Man_6b, (blue box) fragments.

Table 1.

Crystallographic statistics for DC-SIGN CRD ligand complexes

	Man2	Man6b
Data collection
Space group	P43	P43
Unit cell parameters a,c (Å)	55.64, 53.20	55.96, 53.26
Resolution range (Å) (last shell)	100 - 1.95 (2.06-1.95)	100 - 2.4 (2.46-2.40)
Rsyma (last shell)	6.6 (20.8)	8.5 (19.8)
% complete (last shell)	99.9 (100)	99.6 (99.8)
Average multiplicity	4.7	4.6
Mean I/σ(I)	18.1 (7.2)	15.1 (7.6)
Refinement
No. reflections working set	11379	6198
No. reflections test set	572	303
No. protein atoms	1071	1071
No. ligand and solvent atoms	170	116
Rfreeb	0.241	0.252
Rb	0.196	0.198
Average B (Å2)	25.8	27.3
Bond length rmsd (Å)	0.005	0.006
Angle rmsd (°)	1.22	1.22
Ramachandran plot: (% in most favored/ allowed/ generous/ disallowed regions)	88.8/ 11.2/ 0/ 0	89.6/ 9.5/ 0.9/ 0

^aR_sym = Σ_hΣ_i (| I_i(h) | - | <I(h)> |) / Σ_hΣ_i I_i(h)where I_i(h) = observed intensity, and <I(h)> = mean intensity obtained from multiple measurements.

^bR and R_free = Σ ||F_o|-|F_c|| / Σ|F_o|, where |F_o| = observed structure factor amplitude and |F_c| = calculated structure factor amplitude for the working and test sets, respectively.

Structure determination

Crystals of both the Man_6b and Man₂ complexes were essentially isomorphous to the previously determined DC-SIGN-CRD-Man₄ complex (5), even though the latter was obtained using slightly different crystallization conditions. The asymmetric unit contains one copy of the protein-ligand complex. Rather than reindexing to allow direct rigid body refinement, the two structures were determined by molecular replacement with the DC-SIGN CRD model from the Man₄ complex (Protein Data Bank ID 1SL4). The Man₂ complex structure was determined with the program MOLREP (19), which gave a correlation coefficient of 70% and R value of 33% for data to 3Å. The Man_6b complex was solved with program COMO (20), which gave a correlation coefficient of 56% and R of value 31% for data to 3.5Å. Refinement and map calculations for both structures were performed with CNS (21). The maximum-likelihood amplitude target was used, with bulk solvent and anisotropic temperature factor corrections applied throughout the refinement. As refinement progressed it became clear that the ligand is bound to the site in two alternative, overlapping orientations in both structures. The two conformations were assigned occupancies of 75% and 25% based on the quality of the electron density and refined temperature factors. For each ligand orientation, Figure 2 shows the Fo-Fc electron density calculated from coordinates omitting the indicated orientation but including the other. Water molecules were added to peaks > σ in F_o-F_c maps that were within hydrogen bond distance to protein, sugar or other water molecules. The final model of the DC-SIGN CRD-Man₂ complex contains residues 253-384 of DC-SIGN, two alternative conformations of the carbohydrate ligand, 3 Ca²⁺, and 135 water molecules. The final model of the DC-SIGN CRD-Man₆ complex contains residues 253-384 of DC-SIGN, two alternative conformations of the ligand, 3 Ca²⁺, and 59 water molecules. Refinement statistics are presented in Table 1.

Figure 2. Electron density maps for bound ligands.

The indicated bound ligand orientation is shown superimposed on the Fo-Fc electron density map (green, 2σ contour) calculated from a model from which the indicated orientation was omitted, but which included the alternative orientation. A, Man_6b major orientation. B, Man_6b minor orientation. C, Man₂ major orientation. D, Man₂ minor orientation.

Results

Relative affinities of oligomannose structures for DC-SIGN and DC-SIGNR

To examine the contribution of the Manα1-2Man groups present on the termini of high-mannose oligosaccharide to DC-SIGN and DC-SIGNR binding, three synthetic oligomannose structures corresponding to fragments of Man₉GlcNAc₂ were tested in the competition assay. Man₉ is the full 9-mannose structure that would be linked to GlcNAc-GlcNAc-Asn in high-mannose N-linked carbohydrates (Fig. 1, green box). Man_6a lacks the three terminal mannoses of Man₉ (Fig. 1, red box). Man_6b is the substructure of Man₉ that lacks the α1-3 branch arm (Fig. 1, blue box). These compounds were assayed relative to mannose, and the full Man₉GlcNAc₂ structure purified from soybean agglutinin was also included for direct comparison. The two Man₆ structures bind similarly, with a 9- to 14-fold affinity enhancement relative to mannose, whereas the Man₉ compound binds roughly twice as strongly as the Man₆ glycans. The full Man₉GlcNAc₂ glycan consistently shows 2 to 3-fold stronger binding than Man₉ (Table 2).

Table 2.

Competition data for ligand binding to DC-SIGN and DC-SIGNR CRDs

	DCSIGN		DC-SIGNR
Ligand	KI μM	KI vs mannose fold increase	KI μM	KI vs. mannose fold increase
Man	2300 ± 100	1	2500 ± 200	1
Manα1-2Mana	-a	4.1 ± 0.1	-a	3.1 ± 0.5
Man6a	183 ± 18	12 ± 3	277 ± 22	10 ± 2
Man6b	157 ± 17	14 ± 1	251 ± 28	11 ± 2
Man9	73 ± 6	32 ± 4	128 ± 17	20 ± 5
Man9GlcNAc2	26	88	54	43

^aRelative values of K_I vs. mannose for Manα1-2Man taken from Ref. 4. Note that the absolute values of K_I in those experiments cannot be compared to the values shown in the rest of the Table as the solid-phase competition assays were done with a different batch of iodinated Man-BSA.

Structure of Man_6b bound to DC-SIGN

Crystallization trials of complexes between Man₉, Man_6a and Man_6b with the DC-SIGN CRD yielded co-crystals with the 50 mM Man_6b. The structure of this complex was determined at 2.4 Å resolution. The protein structure is identical to that previously described for complexes with Man₃GlcNAc₂ (GlcNAcβ1-2Manα1-3[GlcNAcβ1-2Manα1-6]Man) (4) and Man₄ (Manα1-3[Manα1-6]Manα1-6Man) (5). The ligand is bound in two overlapping orientations, in a mixture estimated at 75%, designated the major orientation (Fig. 3a,b) and 25%, designated the minor orientation (Fig. 3c,d). Of the six mannoses in the compound, only three are visible in the major orientation (Manα1-2Manα1-3Man) and two in the minor orientation (Manα1-2Man).

Figure 3. Binding of Man_6b to DC-SIGN.

The protein is shown in cyan and the carbohydrate in grey, with carbon, nitrogen, oxygen, and calcium represented as white, blue, red, and green spheres, respectively. Hydrogen bonds are shown as dashed gray lines, Ca²⁺ coordination bonds are dashed black lines, and key van der Waals interactions are indicated by dashed blue lines. Distance criteria for hydrogen bonds and van der Waals contacts are: 2.5-3.2 Å for hydrogen bonds, 3.8-4.1 Å for aliphatic carbon-aliphatic carbon contacts, 3.7 Å for aliphatic carbon-oxygen contact, aromatic carbon-oxygen 2.9-3.4 Å, aromatic carbon-aliphatic carbon contact 3.6-3.8 Å. A, Major orientation, showing all three visible sugar residues and their linkages. The positions of the links to the disordered sugars are indicated with arrows. B, Close-up of mannose bound to the principal Ca²⁺ in the major orientation. C, Minor orientation showing both visible sugars. D, close up of mannose bound to the principal Ca²⁺in the minor orientation. E, Superposition of the major orientation on the Man₄ structure (5). Man_6b is shown in grey, Man₄ in yellow. F, The major and minor orientations, shown in grey and yellow, respectively, were superimposed by aligning the pyranose rings at the principal Ca²⁺ site.

The penultimate α1-3-linked mannose that forms one arm of the outer branched trimannose unit (i.e., Manα1-2Manα1-3[Manα1-2Manα1-6]Manα1-6Man) binds to the primary Ca²⁺ site, and was similarly observed to bind to the Ca²⁺ in both the DC-SIGN/Man₄ (5) and DC-SIGN/Man₃GlcNAc₂ (4) structures. The major orientation corresponds to the arrangement seen in these earlier crystal structures (Fig. 3e). The α1-6 branch of the oligosaccharide is not visible, however, which is surprising given the shape complementarity and specific hydrogen bonds between the α1-6-linked mannose and Phe³¹³, Ser³⁶⁰, and other residues in DC-SIGN that were observed in the earlier structures (4,5). The reason for this difference is obscure, especially considering the fact that the crystal is essentially isomorphous to the Man₄ complex (5). In the Man₄ and Man₃GlcNAc₂ structures, the α1-6-linked mannose has higher temperature factors than the α1-3-linked mannose, suggesting that it may be more weakly bound. The α1-2-linked mannose at the non-reducing terminus directly contacts Val³⁵¹.

In the second, less populated orientation, the same mannose residue is bound to the Ca²⁺, but its orientation is reversed by a 180° rotation about a line bisecting the pyranose ring through the C3-C4 bond. This rotation exchanges the position of the 3- and 4-OH groups so that they still form the Ca²⁺ coordination and hydrogen bonds characteristic of C-type lectin-mannose interactions (Fig. 3b,d). A similar situation was observed in complexes of mannose-binding proteins with various ligands (22). In this orientation, only two sugars are visible: the mannose at the Ca²⁺ site, and the non-reducing terminal α1-2-linked mannose, which forms hydrogen bonds with Glu³⁵⁸, Ser³⁶⁰, and which also interacts with the face of the Phe³¹³ ring (Fig. 3c). Thus, it appears that the Phe³¹³ side chain has important roles in the recognition of ligand in either orientation. As only Manα1-2Man is visible, it is not possible to distinguish whether these residues correspond to the α1-3 or α1-6 arms of Man_6b, or if they represent a mixture of both (Fig. 1). An overlay of the major and minor orientations is shown in Fig. 3f.

Structure of Manα1-2Man bound to DC-SIGN

In order to assess if DC-SIGN might have additional subsites for the Manα1-2Man residues found at the non-reducing termini of high-mannose oligosaccharides, the CRD was co-crystallized with 25 mM Manα1-2Man. The structure of the disaccharide complex reveals binding only in the principal Ca²⁺ site; no other carbohydrate molecules were observed even at low electron density map contour levels. Manα1-2Man binds at the principal Ca²⁺ site in two orientations, again related by a 180° rotation about the C3-C4 bond bisector. The major orientation is virtually identical to that of the Manα1-2Man moiety in the minor Man₆ ligand orientation and forms the same contacts with DC-SIGN, including the contact with Phe³¹³ (Fig. 4a,b). In the minor orientation, only a single sugar is visible and forms the typical Ca²⁺ coordination and hydrogen bonds (Fig. 4c,d). This mannose is oriented identically to the Ca²⁺-bound mannose in the major Man₆ orientation. The electron density maps, however, do not make clear if the sugar bound at the Ca²⁺ is the reducing or non-reducing end of the disaccharide; it is possible that there is a mixture of the two. In particular, unlike the Man₆ structure, the non-reducing α1-2-linked mannose is not visible. The electron density for this sugar is not especially well defined in the Man_6b complex, so the lack of density for this residue in the Man₂ complex could be due to its low occupancy. It is also possible that the favorable interaction with Val³⁵¹ seen in the Man₆ structure does not compensate for the loss of entropy required to form this contact in the disaccharide.

Figure 4. Binding of Manα1-2Man to DC-SIGN.

The protein is shown in cyan and the carbohydrate in grey, with carbon, nitrogen, oxygen, and calcium represented as white, blue, red, and green spheres, respectively. Hydrogen bonds are shown as dashed gray lines, Ca²⁺ coordination bonds are dashed black lines, and key van der Waals interactions are indicated by dashed blue lines. Criteria for assigning hydrogen bonds and van der Waals contacts are given in the Figure 3 legend. A, Major orientation, side view. B, Close-up of mannose bound to the principal Ca²⁺ in the major orientation. C, Minor orientation, showing the single visible sugar. D, close up of mannose bound to the principal Ca²⁺in the minor orientation.

Models of Man₉ binding

In order to assess whether the two modes of binding observed in the Man_6b and Man₂ structures are relevant to a full, 9-mannose oligosaccharide, coordinates for Man₉GlcNAc₂ obtained by NMR analysis of the free glycan (23) were superimposed on the two orientations of Man_6b. As noted previously (4), superposition of the outer branched trimannose moiety reveals no significant steric clashes between the rest of the oligosaccharide and the protein (Fig. 5a,b), with only the side chain rotamers of Leu³⁷¹ needing adjustment to avoid clashes. Superposition of the second orientation, which places the terminal α1-2-linked mannose of the outer α1-3 arm near Phe³¹³, also reveals no clashes with the protein, with the possible exception of interference between Arg³⁴⁵ and the innermost GlcNAc residue (Fig. 5c). Further modeling, in which the terminal Manα1-2Man groups of the other arms were superimposed, shows no steric clashes (Fig. 5d,e). Similar results were obtained with crystallographic coordinates of Man₉GlcNAc₂ derived from the complex with a neutralizing anti-HIV antibody (24), although in this case some minor adjustments to the carbohydrate were required when the outer branched mannose units were superimposed (data not shown).

Figure 5. Models of Man₉GlcNAc₂ binding to DC-SIGN.

Comparison of an NMR-derived structure of Man₉GlcNAc₂ (23) with the major and minor Man_6b orientations. The protein is shown in cyan, Man₉GlcNAc₂ in green, and Man_6b, Man₄, or Manα1-2Man in yellow. Carbon, nitrogen, oxygen, and calcium are shown in white, blue, red, and green, respectively. A, Superposition of Man₉GlcNAc₂ on the major orientation of Man_6b. For clarity the GlcNAc₂ moiety of Man₉GlcNAc₂ is not shown. B, Superposition of Man₉GlcNAc₂ on Man₄ (5), which corresponds to the major Man_6b orientation but includes the α1-6-linked mannose (see text and Fig. 3e). The loop in DC-SIGN-CRD (residues 367-374) that is in the vicinity of the two GlcNAc residues is shown in orange. C, D, E, Superposition of Man₉GlcNAc₂ onto the major Manα1-2Man orientation (yellow), corresponding to the minor Man_6b orientation. C, α1-3 branch terminus of the outer trimannose core. D, α1-6 branch terminus of the outer trimannose core. E, terminus of the α 1-3 branch of the inner trimannose core.

Discussion

The structure of the Man_6b-DC-SIGN complex reveals two significantly populated binding modes for the ligand. The major binding mode corresponds to that observed in previous crystal structures, featuring a specific site for the outer branched trimannose unit of high-mannose N-linked carbohydrates. In this orientation, additional contacts are formed between a non-reducing α1-2-linked terminal mannose and Val³⁵¹. This region of DC-SIGN is also important in binding to fucosylated sugars, and a terminal GlcNAc in the GlcNAc₂Man₃ complex (4). The latter compound binds 17-fold more strongly to DC-SIGN relative to mannose and 4-fold more than the trimannose core, suggesting that the additional interactions contribute significantly to overall specificity. Surprisingly, a second binding orientation was observed in which the mannose at the principal Ca²⁺ site is reversed, thereby generating new interactions between the non-reducing terminal mannose and the region around Phe³¹³. Modeling indicates that this orientation would be able to bind to the protein as part of a full 9-mannose structure (Fig. 5c-e).

The relevance of the dual binding modes of the Man_6b compound was confirmed by the structure of the Manα1-2Man disaccharide, which shows the same interactions. In this case, the preferred binding mode leaves the non-reducing end near the Phe³¹³ site. This probably reflects a different energetic balance of the Man₂ and Man₆ compounds, but in any case it is clear that this binding mode can be significantly populated. The fact that no other binding sites for this ligand were observed suggests that there are no other secondary subsites that interact with terminal Manα1-2Man disaccharides in larger N-linked high mannose oligosaccharides that might account for enhanced binding to such glycans.

The observation of dual binding modes, each resulting in unique contacts with DC-SIGN, permits a semi-quantitative explanation of the affinity enhancements observed when high-mannose structures are decorated with α1-2-linked mannose resides at the non-reducing termini. Using [P] and [L] to denote the concentrations of free protein and ligand, respectively, and [PLn] for the concentration of the n^th distinct protein-ligand complex with an association constant Ka_n, the measured affinity constant is related to the affinity constants of the individual binding modes by the equation:${\mathrm{Ka}}_{\mathrm{meas}}=(\left[\mathrm{PL}1\right]+\left[\mathrm{PL}2\right]+\dots )∕\left[\mathrm{P}\right]\left[\mathrm{L}\right]={\mathrm{Ka}}_1+{\mathrm{Ka}}_2+\dots$ Using the definition x_n = Ka_n/Ka₁ = [PLn] / [PL1], this relationship can be restated as:${\mathrm{Ka}}_{\mathrm{meas}}={\mathrm{Ka}}_1(1+{\mathrm{x}}_2+\dots )$ Because $\Delta {\mathrm{G}}_{\mathrm{meas}}=-{\mathrm{RTlnKa}}_{\mathrm{meas}}=-{\mathrm{RTlnKa}}_1-\mathrm{RTln}(1+{\mathrm{x}}_2+\dots )=\Delta {\mathrm{G}}_1-\mathrm{RTln}(1+{\mathrm{x}}_2+\dots ),\Delta {\mathrm{G}}_1=-{\mathrm{RTlnKa}}_1$, and $\Delta {\mathrm{G}}_n=-{\mathrm{RTlnKa}}_n$ it follows that ${\mathrm{x}}_n=\exp ((-\Delta {\mathrm{G}}_n-\Delta {\mathrm{G}}_1)∕\mathrm{RT})$. Thus, for two binding modes of equal energy, ΔG₂ - ΔG₁ = 0, x₂ = 1, and ΔG_meas = ΔG₁ - RTln2, so the ability to bind in two equally energetic modes provides an additional RTln2 of free energy, corresponding to 0.41 kcal mol^-1 at 25°C. For n isoenergetic binding modes the observed association constant will be n times the intrinsic association constant, whereas additional weaker binding modes will increase the association by less than a factor of n. The effect of this statistical factor can be illustrated by comparing the binding of Manα1-2Man with the biding of mannose. With free 3- and 4-OH groups, mannose could bind in either of two orientations related by a 180° rotation that interchanges the 3- and 4-OH groups, as described above. In the disaccharide, each residue can in principle bind in either orientation, giving a total of four binding modes. Thus, if all of these modes were strictly equivalent, the relative Ka for the disaccharide would be twice that of mannose. This argument ignores the possible contribution of an alternative binding arrangement involving the 1- and 2-OH groups, which has only been observed in the case of galactose binding to mannose-binding C-type lectins (25). The predicted ratio is seen for DC-SIGNR, but the ratio is about 4 for DC-SIGN, which probably indicates that there are additional, favorable interactions with DC-SIGN made by the second sugar of the disaccharide. It is also possible that free mannose can bind in only one of two modes, as seen in crystal structures of mannose-binding proteins bound to monosaccharides, which generally show a single orientation rather than a mixture in the binding site (22,25).

For more complex ligands, we can consider the trisaccharide-binding mode observed in Man₃GlcNAc₂ and Man₄ as a relatively high affinity mode. The five-mannose core of the full Man₉ structure, which lacks all Manα1-2Man groups, binds to DC-SIGN 7-fold better than mannose and 4-fold better in the case of DC-SIGNR. Man₅ also possesses the inner branched trimannose unit in the core which, in the absence of the β-linked GlcNAc (4), might also bind. The ability to bind to either the inner or outer branched trimannose units likely explains the enhancement of Man₅ over Man₃ (4). Man_6b binds 14-fold better than mannose to DC-SIGN and 12-fold better than mannose to DC-SIGNR (Table 2). Man_6b lacks the inner trimannose, but the outer branched trimannose binding mode and the “reversed” mode in which the non-reducing terminal Man is bound near Phe³¹³, are present. The 2- and 4-fold enhancement of Man_6b over Man₅ binding to DC- SIGN and DC-SIGNR, can be accounted for in part by the second binding mode. In the major orientation, the α1-2-linked terminal mannose on Man_6b forms additional interactions relative to Man₅, which might make this orientation inherently stronger. In principle the Manα1-2Man group present on the termini of both the α1-3 and α1-6 branches of Man_6b can bind in this second orientation, and but they cannot be distinguished in the structure (see Results), potentially providing three distinct modes at the principal Ca²⁺ site (when the outer branched trimannose moiety plus the two termini of the two branches are considered).

Given the unequal occupancies of the two orientations observed in the Man_6b complex, it is likely that an inherently stronger interaction of the major, trimannose-binding mode, and the statistical effect of the second mode, both contribute to the observed affinity enhancements. If the major orientation is of higher affinity than the non-reducing Manα1-2Man binding mode, the affinity enhancements provided by the latter will be less than a factor of n modes. If we assume that the observed occupancies reflect the relative affinities of the two binding modes, with the estimated 3:1 ratio of occupancy in both the Man₆ and Man₂ structures, x = 1/3, so ΔΔG = 0.65 kcal mol^-1 would be the energy difference between these modes. If we further assume that the observed minor mode an equal mixture of the two different α1-2 termini, then the ratios are 3:0.5:0.5, and the energy difference is 1.06 kcal mol^-1. This result illustrates that small differences in energy due to differences in contacts combined with entropy losses due to conformational immobilization can give rise to preferred binding orientations and likely explains why not all possible modes are allowed even thought the binding site requires only vicinal, equatorial OH groups for Ca²⁺ ligation.

The affinities of Man_6a for DC-SIGN and DC-SIGNR are enhanced to a similar extent as for Man_6b. In this case, the outer branched trimannose unit is present, but no Manα1-2Man moieties are appended to these branches. However, two Manα1-2Man groups present on the α1-3 arm of the inner branched trimannose would provide two more binding modes. It is also possible that the inner branched trimannose unit could bind. Thus, this compound would appear to have a similar number and kind of binding modes as Man_6b, despite their different covalent structures. In the full Man₉ structure, the inner and outer branched trimannose units are present, as well as the Manα1-2Man groups attached to the branches. If we assume that the Manα1-2Manα1-2Man on the α1-3 branch of the inner trimannose structure can provide two more modes, we have a total of six modes, which would explain its further enhancement relative to Man₆.

Although this analysis makes several assumptions about which modes of binding might or might not occur, it is clear that the ability of high-mannose oligosaccharides to interact with DC-SIGN and DC-SIGNR in multiple orientations can give rise to statistical affinity enhancements that are consistent with the measured values. Energetic differences amongst the different binding modes also play an important role in determining the affinity of each compound. Nonetheless, the 2 to 3-fold increase in affinity displayed by the full Man₉GlcNAc₂ structure versus Man₉ is difficult to understand. Perhaps the inner GlcNAc residues restrict the conformation of nearby sugar groups such that there is a smaller entropy penalty for binding, or alternatively, novel contacts are formed between these residues and the surface of the protein.

DC-SIGN and DC-SIGNR serve as receptors for HIV and several other enveloped viruses by binding to the high-mannose oligosaccharides present on viral surface glycoproteins. The CRD of DC-SIGN specifically recognizes an internal portion of the carbohydrate, namely the outer branched trimannnose unit unique to these carbohydrates. The presence of Manα1-2Man enhances the affinity of oligomannose towards these receptors, even though by itself this disaccharide binds only slightly more strongly than mannose. The CRD has an intrinsically high affinity for oligomannose structures, and tetramerization likely provides further avidity enhancements for arrays of such structures (6,26). The ability of DC-SIGN and DC-SIGNR to bind high mannose glycans in multiple orientations may facilitate this multivalent binding of clusters of CRDs to glycans displayed in various arrangements on the surface of the virus, as proposed previously for cell surface recognition by mannose-binding proteins (22). There are some parallels with the mechanism by which a neutralizing antibody to HIV, 2G12, binds specifically to the terminal Manα1-2Man groups present on high-mannose carbohydrates (13,27). The binding site of 2G12 appears to recognize specifically a single orientation of Manα1-2Man present on the non-reducing termini of Man₉, but at least two of the three branch termini bind to this antibody, which would contribute some statistical enhancement of affinity. High avidity is provided in this case by the unusual domain-swapped dimeric antibody structure, which is proposed to display appropriately spaced binding sites that match the spacing of these structures on the viral surface (24,28).