Structure and allosteric regulation of the alpha X beta 2 integrin I domain

Abstract

The integrin alpha X beta 2 (CD11c/CD18, p150,95) binds ligands through the I domain of the alpha X subunit. Ligands include the complement factor fragment iC3b, a key component in the innate immune defense, which, together with the expression of alpha X beta 2 on dendritic cells and on other leukocytes, suggests a role in the immune response. We now report the structure of the alpha X I domain resolved at 1.65 A by x-ray crystallography. To analyze structural requirements for ligand binding we made a mutation in the alpha X I domain C-terminal helix, which increased the affinity for iC3b approximately 200-fold to 2.4 microM compared with the wild-type domain affinity of approximately 400 microM. Gel permeation chromatography supported a conformational change between the wild-type and mutated domains. Conservation of allosteric regulation in the alpha X I domain points to the functional importance of this phenomenon.

Figure 1

The αX I domain structure and comparison to the αM I domain. (A–D) Comparison of αX (cyan) and the αM (magenta) I domains. The MIDAS metal ion present only in αM is shown as a magenta sphere, and the water molecule oxygen present only in the αX MIDAS is shown as a cyan sphere. (A) Backbones of αX and αM. (B) MIDAS region of the αX and αM I domains. Residue numbers refer to the αX sequence. (C) Residues in proximity of the αX and αM MIDAS, which form part of a putative ligand-binding interface and differ in structure or polarity between the two I domains (αX and αM residues are labeled in roman and italics, respectively). (D) Detail of the region forming the hydrophobic socket for Ile-314 (αX) or Ile-316 (αM). Residue numbers refer to the αX sequence, and Leu-164 of αM is labeled in italics. All figures were made with ribbons software (41). (E and F) Electrostatic surfaces of the αM and αX I domains. The molecular surfaces of the domains were constructed with grasp (42). The electrostatic potentials were calculated with the Delphi algorithm (43) and mapped onto the molecular surfaces on a scale from −10 kT/e⁻ (red) to +10 kT/e⁻ (blue). A Mg²⁺ ion was placed at the αX I domain MIDAS to make the electrostatic surfaces comparable. Positions of the metal ions in the αX and αM I domains are indicated with arrows.

Figure 2

Comparisons among closed I domain structures of the C-terminal β-strand and α-helix and overall secondary structure. (A) The C-terminal β6-strand and α7-helix. Superposition is based on the entire domain. The backbone segments shown are αX, residues 288–317; αM, residues 290–318 of 1JLM (15); α2, residues 306–334 of 1AOX (14); and αL, residues 280–308 of 1LFA (16). The side chains of Ile-332 in α2, Ile-316 in αM, Ile-314 in αX, and Ile-306 in αL are shown. (B) Structure-based sequence alignment of the αX, αM, αL, and α2 I domains. The same closed structures as above were superimposed. α-Helices are shown in gold and β-strands are shown in cyan. Secondary structure assignment was by the dssp algorithm (44) from the structural coordinates.

Figure 3

Binding of the αX I domain to iC3b measured in real time with surface plasmon resonance. (A) Overlay of representative sensorgrams recording the binding of the indicated concentration of the Ile-314 → Gly (I314G) αX domain to iC3b in the presence of 1 mM Mg²⁺. (B) Representative sensorgram showing the binding of the wild-type αX domain at 25.6 μM in the presence of Mg²⁺ to iC3b. (C) Binding of the wild-type (25.6 μM) and Ile-314 → Gly (12.8 μM) domains in the presence of 1 mM EDTA. In all sensorgrams, the signal from the control surface was subtracted from the signal obtained on the iC3b-coated surface.

Figure 4

Molecular size estimation under nondenaturing and denaturing conditions. (A) Representative elution profiles from analytical gel permeation chromatography of either the wild-type (dotted line, peak elution volumes are indicated in italics) or the Ile-314 → Gly mutant (solid line, peak elution volumes are indicated in bold) αX I domains. The samples were spiked with blue dextran (BD) eluting at the excluded volume of the column (8.34 ml) and bovine aprotinin (BAp), which eluted at 16.38 ml (the peak elution volumes of both internal markers are indicated with long arrows). The bed volume of the column was 18 ml. The column was calibrated with bovine serum albumin (BSA; 67.0 kDa, R_S = 35.5 Å), ovalbumin (Ova; 43.0 kDa, R_S = 30.5 Å), chymotrypsinogen A (Chy; 25.0 kDa, R_S = 20.9 Å), and ribonuclease A (Rib; 13.7 kDa, R_S = 16.4 Å). The peak elution volumes of the size markers are indicated with short arrows. (B) Nonreducing SDS/PAGE of the wild-type (wt) and Ile-314 → Gly mutant αX I domain.