Overall structure and SDS/PAGE. (A) Ribbon diagram of the α_L I domain in complex with domain 1 of ICAM-3. I domain is in gold, and ICAM-3 D1 is in cyan. The Mg²⁺ ion is shown as a magenta sphere. I domain MIDAS and ICAM-3 Glu-37 side chains are shown as a ball-and-stick model. The interacting β strands C, D, and F of ICAM-1 are labeled. Five glycans and their linked asparagines are shown in silver and cyan, respectively. Residues in the epitope of MEM-83, Asp-182 and Glu-218 (42), are highlighted in green. The figure was prepared with ribbons (43). (B) SDS/PAGE (4–20%) and Coomassie blue staining of ICAM-3 D1 before and after Endo H_f treatment. Positions of molecular weight standards are shown on the left in kDa.

Structural comparisons among ICAM subfamily members and different mutant α_L I domain crystal forms. (A) Superposition of the ICAM-1/α_L I domain complex (PDB code 1MQ8, green) on ICAM-3/α_L I domain complex (red). Only D1s are used for superposition, with all Cα atoms within 2 Å. Also included is the structure of ICAM-2 (PDB ID code 1ZXQ; blue). Domain 2 of ICAM-1 and -2 are omitted for clarity. Note that the CD loops of the three ICAM subfamily members have a remarkably similar conformation. (B) Superposition of the pseudoliganded high-affinity I domain (PDB ID code 1MQ9; silver), the unliganded high-affinity I domain (PDB ID code 1MQA; gray), the ICAM-1-liganded intermediate-affinity I domain (PDB ID code 1MQ8; orange), and the ICAM-3-liganded high-affinity α_L I domain (red). B is rotated about 60° around the vertical axis from A. Notice the significant movement of the β5–α6 loop upon ligation. (C) Structure-based sequence alignment of ICAM subfamily members. The alignment is based on three-dimensional structure. The secondary structure elements of ICAM-3 D1 are marked above the sequence. The contact residues are marked (*, side chain interaction; #, backbone H-bonds; +, both side chain and backbone interactions). The critical MIDAS coordinating residue, Glu-37, is colored red. The conserved contact residues in ICAMs and the conserved residues important for restraining the unique conformation of the CD loop are colored magenta and yellow, respectively. The glycosylation sites in ICAM-2 and -3 are boxed. A was prepared with setor (44), and B was prepared with ribbons (43).

The ICAM-3/α_Lβ₂ binding interface. (A) The interface residues are shown in light cyan and light gold for ICAM-3 and the I domain, respectively. The metal ion, oxygens, and nitrogens are shown as magenta, red, and blue spheres, respectively. Water molecules are shown as red spheres. Metal coordination is shown by black dashed lines. Hydrogen bonds and a salt bridge between Lys-42 in ICAM-3 and Glu-241 in the I domain are represented by silver dashed lines. Residues involved in the hydrophobic patch are colored in silver. For clarity, MIDAS residues are omitted. (B) Structural elements that stabilize the conformation of the CD loop of ICAM-3. The CD and EF loops are colored in yellow and green, respectively. Only relevant side chains are shown in this figure for clarity. Hydrophobic, neutral hydrophilic, and basic residues are colored in orange, cyan, and blue, respectively. The broken lines represent hydrogen bonds, whereas the silver ball is a bound water molecule. A was prepared with ribbons (43), and B was prepared with setor (44).

Surface representation of the binding faces of ICAM-3 and -1. Negatively and positively charged residues are colored red and blue, respectively. Hydrophobic and neutral hydrophilic residues are colored white and yellow, respectively. Some important residues are labeled. The binding footprints are encircled with green dashed lines. Prepared with grasp (45).

Binding of the α_L I domains to ICAM-3 measured with SPR. (A) Inhibition assay. Endo H_f-cleaved or uncleaved ICAM-3 D1 from CHO Lec cells was mixed with high-affinity α_L I domain (50 nM) at a series of concentrations (15, 10, 6.6, 4.4, 3.0, 2.0, 1.3, 0.9, 0.6, and 0 μM) and then injected over the ICAM-1-Fc coated sensor chip at 10 μl/min in TBS/1 mM Mg²⁺. (B) Direct measurement of affinity and kinetics. Sensorgrams show the binding of the uncleaved ICAM-3 D1 (30, 20, 13, 9, 6, 4, 2.6, and 1.8 μM) or Endo H_f-cleaved (3, 2, 1.3, 0.9, 0.6, 0.4, 0.26, and 0.18 μM) to I domain immobilized on streptavidin chip in TBS/1 mM Mg²⁺. In all sensorgrams, the signals from the control surface, as described in Methods, were subtracted. Concentrations are shown to the right in the same order, top to bottom, as the corresponding curves.