The Crystal Structure of the Plexin-Semaphorin-Integrin Domain/Hybrid Domain/I-EGF1 Segment from the Human Integrin β2 Subunit at 1.8-Å Resolution

Minlong Shi,Kumar Sundramurthy,Bin Liu,Suet-Mien Tan,S.K Alex Law,Julien Lescar

doi:10.1074/jbc.m502525200

Minlong Shi, Kumar Sundramurthy + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m502525200

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Abstract

Integrins are modular (alphabeta) heterodimeric proteins that mediate cell adhesion and convey signals across the plasma membrane. Interdomain motions play a key role in signal transduction by propagating structural changes through the molecule, thus controlling the activation state and adhesive properties of the integrin. We expressed a soluble fragment of the human integrin beta2 subunit comprising the plexin-semaphorin-integrin domain (PSI)/hybrid domain/I-EGF1 fragment and present its crystal structure at 1.8-A resolution. The structure reveals an elongated molecule with a rigid architecture stabilized by nine disulfide bridges. The PSI domain is located centrally and participates in the formation of extended interfaces with the hybrid domain and I-EGF1 domains, respectively. The hybrid domain/PSI interface involves the burial of an Arg residue, and contacts between PSI and I-EGF1 are mainly mediated by well conserved Arg and Trp residues. Conservation of key interacting residues across the various integrin beta subunits sequences suggests that our structure represents a good model for the entire integrin family. Superposition with the integrin beta3 receptor in its bent conformation suggests that an articulation point is present at the linkage between its I-EGF1 and I-EGF2 modules and underlines the importance of this region for the control of integrin-mediated cell adhesion.

Highlights

Integrins are modular (␣␤) heterodimeric proteins that mediate cell adhesion and convey signals across the plasma membrane
We expressed a soluble fragment of the human integrin ␤2 subunit comprising the plexin-semaphorin-integrin domain (PSI)/hybrid domain/I-EGF1 fragment and present its crystal structure at 1.8-Å resolution
Protein Expression—The integrin ␤2 subunit can be expressed in the absence of any integrin ␣ subunits on COS-7 cells but can only be detected by monoclonal antibodies whose epitopes map to regions outside the I-like domain [15]

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—The cDNA coding for PHE1 was constructed from the wild type human ␤2 cDNA by removing the region encoding residues Lys101–Asn339, corresponding to the ⍜-like domain insertion, and by adding a hexahistidine tag to facilitate purification followed by a stop codon after Glu460, which marks the C-terminal end of the I-EGF1 domain [17, 18] (Fig. 1A). 1 liter of culture supernatant was concentrated in a stirred cell (Amicon-8200, Millipore) through a polyethersulfone membrane with a 5-kDa molecular mass cutoff to a final volume of 50 ml in buffer A (20 mM Tris-HCl, pH 8.0, 0.2 M NaCl, 10% glycerol). The eluate was concentrated in buffer A to a final volume of 1 ml, and the sample was loaded onto a Sephacryl S-100 HR column (16 mm ϫ 60 cm) mounted on a fast protein liquid chromatography system (Amersham Biosciences). Two heavy atom binding sites were located using the program SOLVE [20], and an initial map was calculated after solvent flattening. This initial map allowed the tracing of most of the main chain atoms of the hybrid and PSI domains.

TABLE I Data collection and phasing statistics

Overall G factorc

RESULTS

DISCUSSION