Volume 2, Issue 8, Pages 755-766 (August 1994) Crystal structure of the extracellular region of the human cell adhesion molecule CD2 at 2.5å resolution  Dale L Bodian, E.Yvonne Jones, Karl Harlos, David I Stuart, Simon J Davis  Structure  Volume 2, Issue 8, Pages 755-766 (August 1994) DOI: 10.1016/S0969-2126(94)00076-X

Figure 1 Electron density maps contoured at 1σ displayed with the current model. (a) 3.5 å solvent flattened SIRAS map showing parts of strands A and G of domain 2 and the disulphide-bonded Cys115 and Cys179. (b) Representative region of a 2.5 å 2Fo– Fc map showing the same view as in (a). (c) Electron density for Asn65 and N-linked GlcNAc from a 2.5å 2Fo– Fc omit map computed with the sugar omitted from the model. (d) Portion of a 2.5å 2Fo– Fc map showing density for the BC loop of domain 1, residues 20– 29. A trace of the backbone of domain 1 is shown in orange, with the GFCC′C″ face of the domain pointing to the right. Structure 1994 2, 755-766DOI: (10.1016/S0969-2126(94)00076-X)

Figure 1 Electron density maps contoured at 1σ displayed with the current model. (a) 3.5 å solvent flattened SIRAS map showing parts of strands A and G of domain 2 and the disulphide-bonded Cys115 and Cys179. (b) Representative region of a 2.5 å 2Fo– Fc map showing the same view as in (a). (c) Electron density for Asn65 and N-linked GlcNAc from a 2.5å 2Fo– Fc omit map computed with the sugar omitted from the model. (d) Portion of a 2.5å 2Fo– Fc map showing density for the BC loop of domain 1, residues 20– 29. A trace of the backbone of domain 1 is shown in orange, with the GFCC′C″ face of the domain pointing to the right. Structure 1994 2, 755-766DOI: (10.1016/S0969-2126(94)00076-X)

Figure 1 Electron density maps contoured at 1σ displayed with the current model. (a) 3.5 å solvent flattened SIRAS map showing parts of strands A and G of domain 2 and the disulphide-bonded Cys115 and Cys179. (b) Representative region of a 2.5 å 2Fo– Fc map showing the same view as in (a). (c) Electron density for Asn65 and N-linked GlcNAc from a 2.5å 2Fo– Fc omit map computed with the sugar omitted from the model. (d) Portion of a 2.5å 2Fo– Fc map showing density for the BC loop of domain 1, residues 20– 29. A trace of the backbone of domain 1 is shown in orange, with the GFCC′C″ face of the domain pointing to the right. Structure 1994 2, 755-766DOI: (10.1016/S0969-2126(94)00076-X)

Figure 1 Electron density maps contoured at 1σ displayed with the current model. (a) 3.5 å solvent flattened SIRAS map showing parts of strands A and G of domain 2 and the disulphide-bonded Cys115 and Cys179. (b) Representative region of a 2.5 å 2Fo– Fc map showing the same view as in (a). (c) Electron density for Asn65 and N-linked GlcNAc from a 2.5å 2Fo– Fc omit map computed with the sugar omitted from the model. (d) Portion of a 2.5å 2Fo– Fc map showing density for the BC loop of domain 1, residues 20– 29. A trace of the backbone of domain 1 is shown in orange, with the GFCC′C″ face of the domain pointing to the right. Structure 1994 2, 755-766DOI: (10.1016/S0969-2126(94)00076-X)

Figure 2 Structure of human sCD2. (a) Stereo drawing of the Cα backbone with every tenth residue numbered. The figure was produced with the program XOBJECT (M. Noble, unpublished program). (b) Ribbon drawing. Strands for domains 1 and 2 are labeled following the convention for IgSF V-set and C2-set domains, respectively. The view is similar to that in (a) (Figure drawn with MOLSCRIPT [53].) Structure 1994 2, 755-766DOI: (10.1016/S0969-2126(94)00076-X)

Figure 3 Sequence comparison of human and rat sCD2. Structurally equivalent residues are aligned and sequence identities boxed. Every tenth residue of each sequence is numbered. Glycosylated asparagines are white on a black background, and the linker region between domains is shown in grey. Bars represent β-sheet residues. ‡ marks residues buried in the head-to-head interaction observed in the human sCD2 crystals.§ identifies residues comprising the inter-domain interface of human sCD2. The equivalent residues are involved in the head-to-head interaction and inter-domain interface of rat sCD2. Arg70 also contributes to the inter-domain interface in the rat structure; its human equivalent (Thr75) is too short to participate. Structure 1994 2, 755-766DOI: (10.1016/S0969-2126(94)00076-X)

Figure 4 Comparison of Cα traces of human and rat sCD2 crystal structures. (a) Superposition of domains 1 (b) Superposition of domains 2. Human sCD2 is in green and rat sCD2 is in yellow (copy 1) and red (copy 2). Side chains of disulnnphide bonded cysteines are displayed. Domains were aligned individually by least squares superposition of structurally equivalent residues as described in Materials and methods. (c) Comparison of the angles between IgSF domains of human sCD2 (pink) and the two copies of rat sCD2 (cyan and yellow). Orthogonal views are shown. Structures are superimposed on domain 2. Structure 1994 2, 755-766DOI: (10.1016/S0969-2126(94)00076-X)

Figure 4 Comparison of Cα traces of human and rat sCD2 crystal structures. (a) Superposition of domains 1 (b) Superposition of domains 2. Human sCD2 is in green and rat sCD2 is in yellow (copy 1) and red (copy 2). Side chains of disulnnphide bonded cysteines are displayed. Domains were aligned individually by least squares superposition of structurally equivalent residues as described in Materials and methods. (c) Comparison of the angles between IgSF domains of human sCD2 (pink) and the two copies of rat sCD2 (cyan and yellow). Orthogonal views are shown. Structures are superimposed on domain 2. Structure 1994 2, 755-766DOI: (10.1016/S0969-2126(94)00076-X)

Figure 4 Comparison of Cα traces of human and rat sCD2 crystal structures. (a) Superposition of domains 1 (b) Superposition of domains 2. Human sCD2 is in green and rat sCD2 is in yellow (copy 1) and red (copy 2). Side chains of disulnnphide bonded cysteines are displayed. Domains were aligned individually by least squares superposition of structurally equivalent residues as described in Materials and methods. (c) Comparison of the angles between IgSF domains of human sCD2 (pink) and the two copies of rat sCD2 (cyan and yellow). Orthogonal views are shown. Structures are superimposed on domain 2. Structure 1994 2, 755-766DOI: (10.1016/S0969-2126(94)00076-X)

Figure 5 Residues of the inter-domain contact region. The Cαbackbone of human sCD2 is displayed in white with side chains of residues buried in the interface (marked by § in Figure 3) coloured green. Thr75 is also displayed since its rat equivalent, Arg70, participates in the interface. The structure of copy 1 of rat sCD2 (cyan and yellow) is superimposed on the human structure by least squares fitting of the linker region. The view is similar to that in Figure 2. Figure produced with INSIGHT II (Biosym Technologies, Inc.). Structure 1994 2, 755-766DOI: (10.1016/S0969-2126(94)00076-X)

Figure 6 Residues implicated in the interaction between CD2 and CD58. The GFCC′C″ face of domain 1 is shown in CPK representation. (a) Residues that have been mutated and tested for their effect on CD58 binding. Projecting residues whose mutation disrupted ligand binding substantially are coloured red and those whose mutation had little or no effect on binding are coloured green. Mutations affecting glycosylation sites are omitted. Mutants represented are: D32A, K34A or D, E36R, K41A, K43A, M, N, E, Q, or T, Q46L, P, or E, R48A, K51A, K82A, Y86D or C, D87H, V, or A, G90E, K91A, N92A, V93A, L94S which reduced the capacity of human CD2 to bind CD58, and D20N, E50K, E52K, K55L, E56K, K57S, K61E, K89N which did not. (b) Residues buried in the head-to-head contact, marked by ‡ in Figure 3, are shown in pink. Structure 1994 2, 755-766DOI: (10.1016/S0969-2126(94)00076-X)

Figure 7 Electrostatic potential of human (left) and rat (right) sCD2. (a) GFCC′C″ faces of domain 1. (b) Complete extracellular regions. Regions of positive potential are coloured blue, negative regions red, and neutral regions white. The magnitude of the potential is indicated by the intensity of the colour. Structure 1994 2, 755-766DOI: (10.1016/S0969-2126(94)00076-X)

Figure 7 Electrostatic potential of human (left) and rat (right) sCD2. (a) GFCC′C″ faces of domain 1. (b) Complete extracellular regions. Regions of positive potential are coloured blue, negative regions red, and neutral regions white. The magnitude of the potential is indicated by the intensity of the colour. Structure 1994 2, 755-766DOI: (10.1016/S0969-2126(94)00076-X)

Figure 8 Head-to-head interaction observed in the sCD2 crystals. (a) Orthogonal views of the Cα backbone of human sCD2 (green) and its symmetry-related mate (blue) compared with symmetry-related molecules of rat sCD2 copy 1 (yellow) and copy 2 (red). Structures are superimposed on domain 1 of one molecule of human sCD2. (b) Close-up of the interacting GFC′C″ faces of human sCD2 and its symmetry-related mate. Main chain hydrogen bonds between Gly90 and Lys43 of human sCD2 (green lines) are superimposed on the Cα backbone of human sCD2 (purple). Side chains of Phe54 and Tyr86 of human sCD2 and its symmetry-related mate are displayed as green sticks. The equivalent residues and hydrogen bonds for both copy 1 and copy 2 of rat sCD2 (yellow and red, respectively) are superimposed (without their symmetry-related mates). The two-fold axis is perpendicular to the page. The figure was drawn using INSIGHT II (Biosym Technologies, Inc.). (c) Residues of human sCD2 buried in the head-to-head interaction observed in the crystal. Side chains of arginine and lysine residues are displayed in blue, aspartic acid and glutamine side chains in red, and others in white. The Cα positions of Phe54, Tyr86, and Gly90 are highlighted in purple on the Cα trace of human sCD2 (green). The Cα backbone of the symmetry-related mate is shown in yellow. The view is the same as that in (b). Structure 1994 2, 755-766DOI: (10.1016/S0969-2126(94)00076-X)

Figure 8 Head-to-head interaction observed in the sCD2 crystals. (a) Orthogonal views of the Cα backbone of human sCD2 (green) and its symmetry-related mate (blue) compared with symmetry-related molecules of rat sCD2 copy 1 (yellow) and copy 2 (red). Structures are superimposed on domain 1 of one molecule of human sCD2. (b) Close-up of the interacting GFC′C″ faces of human sCD2 and its symmetry-related mate. Main chain hydrogen bonds between Gly90 and Lys43 of human sCD2 (green lines) are superimposed on the Cα backbone of human sCD2 (purple). Side chains of Phe54 and Tyr86 of human sCD2 and its symmetry-related mate are displayed as green sticks. The equivalent residues and hydrogen bonds for both copy 1 and copy 2 of rat sCD2 (yellow and red, respectively) are superimposed (without their symmetry-related mates). The two-fold axis is perpendicular to the page. The figure was drawn using INSIGHT II (Biosym Technologies, Inc.). (c) Residues of human sCD2 buried in the head-to-head interaction observed in the crystal. Side chains of arginine and lysine residues are displayed in blue, aspartic acid and glutamine side chains in red, and others in white. The Cα positions of Phe54, Tyr86, and Gly90 are highlighted in purple on the Cα trace of human sCD2 (green). The Cα backbone of the symmetry-related mate is shown in yellow. The view is the same as that in (b). Structure 1994 2, 755-766DOI: (10.1016/S0969-2126(94)00076-X)

Figure 8 Head-to-head interaction observed in the sCD2 crystals. (a) Orthogonal views of the Cα backbone of human sCD2 (green) and its symmetry-related mate (blue) compared with symmetry-related molecules of rat sCD2 copy 1 (yellow) and copy 2 (red). Structures are superimposed on domain 1 of one molecule of human sCD2. (b) Close-up of the interacting GFC′C″ faces of human sCD2 and its symmetry-related mate. Main chain hydrogen bonds between Gly90 and Lys43 of human sCD2 (green lines) are superimposed on the Cα backbone of human sCD2 (purple). Side chains of Phe54 and Tyr86 of human sCD2 and its symmetry-related mate are displayed as green sticks. The equivalent residues and hydrogen bonds for both copy 1 and copy 2 of rat sCD2 (yellow and red, respectively) are superimposed (without their symmetry-related mates). The two-fold axis is perpendicular to the page. The figure was drawn using INSIGHT II (Biosym Technologies, Inc.). (c) Residues of human sCD2 buried in the head-to-head interaction observed in the crystal. Side chains of arginine and lysine residues are displayed in blue, aspartic acid and glutamine side chains in red, and others in white. The Cα positions of Phe54, Tyr86, and Gly90 are highlighted in purple on the Cα trace of human sCD2 (green). The Cα backbone of the symmetry-related mate is shown in yellow. The view is the same as that in (b). Structure 1994 2, 755-766DOI: (10.1016/S0969-2126(94)00076-X)