Complex structure of engineered modular domains defining molecular interaction between ICAM-1 and integrin LFA-1

Abstract

Intermolecular contacts between integrin LFA-1 (α(L)β(2)) and ICAM-1 derive solely from the integrin α(L) I domain and the first domain (D1) of ICAM-1. This study presents a crystal structure of the engineered complex of the α(L) I domain and ICAM-1 D1. Previously, we engineered the I domain for high affinity by point mutations that were identified by a directed evolution approach. In order to examine α(L) I domain allostery between the C-terminal α7-helix (allosteric site) and the metal-ion dependent adhesion site (active site), we have chosen a high affinity variant without mutations directly influencing either the position of the α7-helix or the active sites. In our crystal, the α(L) I domain was found to have a high affinity conformation to D1 with its α7-helix displaced downward away from the binding interface, recapitulating a current understanding of the allostery in the I domain and its linkage to neighboring domains of integrins in signaling. To enable soluble D1 of ICAM-1 to fold on its own, we also engineered D1 to be functional by mutations, which were found to be those that would convert hydrogen bond networks in the solvent-excluded core into vdW contacts. The backbone structure of the β-sandwich fold and the epitope for I domain binding of the engineered D1 were essentially identical to those of wild-type D1. Most deviations in engineered D1 were found in the loops at the N-terminal region that interacts with human rhinovirus (HRV). Structural deviation found in engineered D1 was overall in agreement with the function of engineered D1 observed previously, i.e., full capacity binding to α(L) I domain but reduced interaction with HRV.

Figure 1. Complex structure of engineered α_L I domain and ICAM-1 D1.

(A&B) Schematic drawings of the integrin headpiece, denoting intra- and inter-domain rearrangements during the engagement of LFA-1 with ICAM-1. A structural transition from low (A) to high affinity conformation (B) involves downward displacement of the α7-helix, shape change in metal-ion coordination sites, and swing-out movement of the hybrid domain. SyMBS = synergistic metal binding site; MIDAS = metal-ion dependent adhesion site; ADMIDAS = adjacent to MIDAS. (C) Ribbon diagram of the engineered α_L I domain (pale yellow), containing a substitution of F265S, in complex with the engineered domain 1 (D1) of ICAM-1 (light purple). The residues coordinating to the metal ion in the MIDAS, Ser-139, Ser-141, and Thr-206 in I domain and Glu-34 in ICAM-1 D1 are shown in stick models. The Mg²⁺ ion is shown as a pink sphere. (D) The electron density map, drawn together with cartoon or stick models, shows an open conformation of the β6-strand and the α7-helix. The three hydrophobic residues (Leu-289, Phe-292, and Leu-295; cyan) are shown in stick models. (E) In comparison to the previous open structures of the I domains of different α subunits, α_M (1IDO; blue) and α₂ (1DZI; green) , the α7-helix in our structure (3TCX; magenta) shows a comparable extent of downward displacement, away from the closed structure seen in the wild-type α_L I domain (3F74; yellow) . (F) Ribbon diagrams of 14 complexes found in an asymmetric unit. I domains are drawn in grey, and 14 molecules of D1 are drawn in different colors for clarity. Dotted circles in cyan color indicate the interface between two C-terminal ends of D1. (G) Superimposed 14 complexes are shown as Ca-traces.

Figure 2. Comparison with the previous complex structures of the α_L I domain with ligands.

(A–C) Superimposed to the current α_L I domain and ICAM-1 D1 structure (3TCX; magenta) are the previously solved complex structures of (A) high affinity (HA) α_L I domain containingF265S/F292G with ICAM-5 D1D2 (3BN3; yellow) , (B) intermediate affinity (IA) α_L I domain containing L161C/F299C with ICAM-1 D1D2 (1MQ8; blue) , and (C) high affinity (HA) α_L I domain containing K287C/K294C with ICAM-3 D1 (1T0P; green) . The acidic residue of the ICAMs (Glu-34 in ICAM-1 and Glu-37 in ICAM-3 and ICAM-5) docking into the I domains and the Mg²⁺ ions are shown as stick and spheres, respectively. The β5-α6 and β6-α7 loops are circled with dotted lines.

Figure 3. Structural deviation in engineered ICAM-1 D1 and its implication in ICAM-1 interaction with LFA-1 and HRV.

(A) Cα-traces of engineered ICAM-1 D1 (magenta) and the wild-type D1 in D1D2 fragment (1IAM in yellow) are drawn with solvent-accessible surface plot (grey). (B) Hydrogen bond network formed by Thr-2, Thr-23, Ser-67, and Ser-74 at the N-terminal protein core is shown in grey dotted lines with distances in Å from the wild-type ICAM-1 structure (1IAM). Substitutions of T2V, S67A, and T23A in D1 are indicated. (C) A hydrogen bond between Pro-6 and Thr-78 is shown with a dotted line. Substitutions of P63V and T78A found in D1 are indicated. (D) D1 structures of the previous ICAM-1 D1D2 structures (1IAM in yellow , 1IC1 in blue , 1MQ8 in green [17]) were superimposed to engineered D1 (3TCX in magenta). The loops, B–C, D–E, and F–G, are circled with dotted lines. (E) The distances between the Cα atoms (dotted lines in magenta) of the triad Thr-2, Thr-23, and Ser-67, and the Cα distance between Pro-28 to Thr-23 in previous structures are compared with those in D1 mutant. (F) The superimposed ICAM-1 structures with the HRV were modeled based on the cryo-EM Cα coordinates of ICAM-1 D1D2 bound to HRV16 (1D3E and 1AYM [45]). (G) The superimposed D1 structures are shown with the α_L I domain shown as solvent-accessible surface.

Figure 4. Differences in Cα positions for the superimposed structures of ICAM-1 D1.

Pair-wise Cα-Cα distances between wild-type ICAM-1 (1IAM, 1MQ8, and 1IC1) and engineered D1 (3TCX) are plotted. Also, Cα- Cα distances among the wild-type ICAM-1 structures are plotted (1IAM to 1MQ8, 1IC1 to 1IAM, and 1MQ8 to 1IC1). Putative interacting residues of ICAM-1 D1 with HRV16 capsid as well as the residues in close contact with the α_L I domain are indicated with brackets and corresponding residue numbers. Arrow bars denote the residues that form the β-strands in ICAM-1 D1.