Trimeric structure of a C-type mannose-binding protein William I Weis, Kurt Drickamer  Structure  Volume 2, Issue 12, Pages 1227-1240 (December 1994) DOI: 10.1016/S0969-2126(94)00124-3

Figure 1 Schematic structure of MBPs and generation of carboxy-terminal fragments of MBP–A. (a) Overall structure of MBP–A and proteolytically derived fragments. The linker at the end of the bacterially expressed fragment represents the seven extra amino-terminal amino acids introduced by the expression construct. (b) Higher oligomers of collectins are found in bouquet-like (left) or cruciform (right) structures. (c) Modifications of Arg68 and Lys106 in the expression vector are shown along with sites of proteolysis by substilisin and clostripain. Structure 1994 2, 1227-1240DOI: (10.1016/S0969-2126(94)00124-3)

Figure 1 Schematic structure of MBPs and generation of carboxy-terminal fragments of MBP–A. (a) Overall structure of MBP–A and proteolytically derived fragments. The linker at the end of the bacterially expressed fragment represents the seven extra amino-terminal amino acids introduced by the expression construct. (b) Higher oligomers of collectins are found in bouquet-like (left) or cruciform (right) structures. (c) Modifications of Arg68 and Lys106 in the expression vector are shown along with sites of proteolysis by substilisin and clostripain. Structure 1994 2, 1227-1240DOI: (10.1016/S0969-2126(94)00124-3)

Figure 1 Schematic structure of MBPs and generation of carboxy-terminal fragments of MBP–A. (a) Overall structure of MBP–A and proteolytically derived fragments. The linker at the end of the bacterially expressed fragment represents the seven extra amino-terminal amino acids introduced by the expression construct. (b) Higher oligomers of collectins are found in bouquet-like (left) or cruciform (right) structures. (c) Modifications of Arg68 and Lys106 in the expression vector are shown along with sites of proteolysis by substilisin and clostripain. Structure 1994 2, 1227-1240DOI: (10.1016/S0969-2126(94)00124-3)

Figure 2 Isolation of clostripain fragment of MBP-A containing neck and CRD. Left, test digestions with clostripain. Aliquots (25 μ l) of affinity-purified protein containing the single amino acid change Arg68→Lys, at a concentration of approximately 2.5 μ g μ l–1, were digested with increasing concentrations of pre-activated clostripain for 1 h at 37°C. Lane 1, no clostripain; 2, 0.5 μ g clostripain; 3, 1 μ g clostripain; 4, 2 μ g clostripain; 5, 4 μ g clostripain; 6, 8 μ g clostripain. Reactions were halted by addition of 250 μ l sample buffer, followed immediately by heating to 100°C for 5 min. Approximately 10% of each sample was loaded onto the gel. Right, purified clostripain fragment following reverse phase HPLC. SDS/17.5% polyacrylamide gels were stained with Coomassie blue. Structure 1994 2, 1227-1240DOI: (10.1016/S0969-2126(94)00124-3)

Figure 3 Cross-linking of cl-MBP-A. Fragment purifiedby reverse phase chromatography was dissolved at a concentration of approximately 0.25 mg ml–1 in 50 mM sodium-HEPES, pH 7.5,and reacted for 1 h at room temperature with increasing concentrations of bis(sulfosuccinimidyl)-suberate. Reagent concentrations were: lane 1, 0 mM;2, 0.2 mM; 3, 0.4 mM; 4, 0.8 mM; 5, 1.6 mM; 6, 3.3 mM; 7, 6.6 mM. Reactions werestopped by addition of double strength sample buffer, followed by heating to 100°C for 5 min, and electrophoresis on an SDS/15% polyacrylamide gel which was stained with Coomassie blue. Structure 1994 2, 1227-1240DOI: (10.1016/S0969-2126(94)00124-3)

Figure 4 Circular dichroism spectra of fragments from MBP-A. Spectra were obtained at nearly the same molar concentrations of polypeptide (11–18 μ M). (a) Spectra for dimeric CRD fragment (CRD) and trimeric fragment containing neck and CRD (CRD+NECK) in 2.5 mM Tris-HCl, pH7.8 and 2.5 mM CaCl2. (b) Difference spectrum calculated from the results in (a), representing the net contribution of the neck residues. (c) Spectrum of isolated neck peptide in 5 mM Na3 PO4 , pH 7.8. (d) Spectrum of isolated neck peptide in 5 mM Na3 PO4, pH 7.8 containing 50% 2,2,2-trifluoroethanol. The mean residue ellipticity at 222 nm for the 33 amino acid neck region in the difference spectrum (b) and for the isolated neck region in 2,2,2-trifluoroethanol (d) is approximately 30x103 deg-cm2 dmol–1. Structure 1994 2, 1227-1240DOI: (10.1016/S0969-2126(94)00124-3)

Figure 5 Molscript diagrams [60] of the cl-MBP-A protomer and trimer. Calcium ions 1 and 2 are shown as green or dark spheres; number 2 is the carbohydrate-binding site. Calcium 3, which may be adventitious, is depicted as a white sphere (a) Protomer structure. The locations of residues 73 and 106, which demark the neck, are shown. Residue 221 is the carboxyl terminus of the molecule (b) Stereo Cα trace of cl-MBP-A is shown with every 10th residue marked with a circle. For clarity, residue numbers are shown for one protomer only (c) View of the trimer down the three-fold axis, looking from the CRD towards the amino terminus (d) View of the trimer perpendicular to the three-fold axis. The tight turn between the neck and CRD can be seen in the front, on the yellow protomer. Structure 1994 2, 1227-1240DOI: (10.1016/S0969-2126(94)00124-3)

Figure 5 Molscript diagrams [60] of the cl-MBP-A protomer and trimer. Calcium ions 1 and 2 are shown as green or dark spheres; number 2 is the carbohydrate-binding site. Calcium 3, which may be adventitious, is depicted as a white sphere (a) Protomer structure. The locations of residues 73 and 106, which demark the neck, are shown. Residue 221 is the carboxyl terminus of the molecule (b) Stereo Cα trace of cl-MBP-A is shown with every 10th residue marked with a circle. For clarity, residue numbers are shown for one protomer only (c) View of the trimer down the three-fold axis, looking from the CRD towards the amino terminus (d) View of the trimer perpendicular to the three-fold axis. The tight turn between the neck and CRD can be seen in the front, on the yellow protomer. Structure 1994 2, 1227-1240DOI: (10.1016/S0969-2126(94)00124-3)

Figure 5 Molscript diagrams [60] of the cl-MBP-A protomer and trimer. Calcium ions 1 and 2 are shown as green or dark spheres; number 2 is the carbohydrate-binding site. Calcium 3, which may be adventitious, is depicted as a white sphere (a) Protomer structure. The locations of residues 73 and 106, which demark the neck, are shown. Residue 221 is the carboxyl terminus of the molecule (b) Stereo Cα trace of cl-MBP-A is shown with every 10th residue marked with a circle. For clarity, residue numbers are shown for one protomer only (c) View of the trimer down the three-fold axis, looking from the CRD towards the amino terminus (d) View of the trimer perpendicular to the three-fold axis. The tight turn between the neck and CRD can be seen in the front, on the yellow protomer. Structure 1994 2, 1227-1240DOI: (10.1016/S0969-2126(94)00124-3)

Figure 5 Molscript diagrams [60] of the cl-MBP-A protomer and trimer. Calcium ions 1 and 2 are shown as green or dark spheres; number 2 is the carbohydrate-binding site. Calcium 3, which may be adventitious, is depicted as a white sphere (a) Protomer structure. The locations of residues 73 and 106, which demark the neck, are shown. Residue 221 is the carboxyl terminus of the molecule (b) Stereo Cα trace of cl-MBP-A is shown with every 10th residue marked with a circle. For clarity, residue numbers are shown for one protomer only (c) View of the trimer down the three-fold axis, looking from the CRD towards the amino terminus (d) View of the trimer perpendicular to the three-fold axis. The tight turn between the neck and CRD can be seen in the front, on the yellow protomer. Structure 1994 2, 1227-1240DOI: (10.1016/S0969-2126(94)00124-3)

Figure 6 Stereoview of packing in the triple-stranded coiled coil of the neck. Side chains of the a and d heptad positions are shown on a Cα backbone. The orientation and color coding of protomers is the same as in Figure 5d. Carbon, nitrogen and oxygen atoms are shown as white, blue and red spheres, respectively. Hydrogen bonds between His99 and Ser102 of an adjacent protomer are indicated by dashed lines. The alternative conformer of His99 in one protomer is shown in green. (Figure drawn with MOLSCRIPT [60].) Structure 1994 2, 1227-1240DOI: (10.1016/S0969-2126(94)00124-3)

Figure 7 A portion of the neck–CRD interface, shown with the 1.25σ contour of the final 2Fo–Fc map. Conserved hydrophobic residues are labeled; the residues come from the protomer whose number precedes the amino acid name. Structure 1994 2, 1227-1240DOI: (10.1016/S0969-2126(94)00124-3)

Figure 8 Representative sequences of the neck region and portions of the CRDs following the last Gly-X-Y repeat taken from various subgroups of collectins. RA, rat; HU, human. The a and d positions of the heptadrepeats are indicated. Sites of introns and the sites of subtilisin digestion that releases the carboxy-terminal CRDs of MBPs are underlined. Residues inthe neck–CRD interface (see Table 2) are denoted with asterisks. Sequences and intron positions are derived from [19,44,61–65]. Structure 1994 2, 1227-1240DOI: (10.1016/S0969-2126(94)00124-3)

Figure 9 Hypothetical model of the trimer binding to oligosaccharides. The oligosaccharide model was made by superposition of the MBP–A–Man6–GlcNAc2 Asn complex structure [7] on each protomer in the trimer. The picture is meant to emphasize the large spacing between carbohydrate-binding sites, which implies that separate oligosaccharides projecting down from a cell surface would be bound independently by the three CRDs in the trimer. Structure 1994 2, 1227-1240DOI: (10.1016/S0969-2126(94)00124-3)