The α-Subunit Regulates Stability of the Metal Ion at the Ligand-associated Metal Ion-binding Site in β3 Integrins

Xianliang Rui,Mehrdad Mehrbod,Johannes F Van Agthoven,Saurabh Anand,Jian-Ping Xiong,Mohammad R.K Mofrad,M Amin Arnaout

doi:10.1074/jbc.m114.581470

Xianliang Rui, Mehrdad Mehrbod + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m114.581470

Copy DOI

Abstract

The aspartate in the prototypical integrin-binding motif Arg-Gly-Asp binds the integrin βA domain of the β-subunit through a divalent cation at the metal ion-dependent adhesion site (MIDAS). An auxiliary metal ion at a ligand-associated metal ion-binding site (LIMBS) stabilizes the metal ion at MIDAS. LIMBS contacts distinct residues in the α-subunits of the two β3 integrins αIIbβ3 and αVβ3, but a potential role of this interaction on stability of the metal ion at LIMBS in β3 integrins has not been explored. Equilibrium molecular dynamics simulations of fully hydrated β3 integrin ectodomains revealed strikingly different conformations of LIMBS in unliganded αIIbβ3 versus αVβ3, the result of stronger interactions of LIMBS with αV, which reduce stability of the LIMBS metal ion in αVβ3. Replacing the αIIb-LIMBS interface residue Phe(191) in αIIb (equivalent to Trp(179) in αV) with Trp strengthened this interface and destabilized the metal ion at LIMBS in αIIbβ3; a Trp(179) to Phe mutation in αV produced the opposite but weaker effect. Consistently, an F191/W substitution in cellular αIIbβ3 and a W179/F substitution in αVβ3 reduced and increased, respectively, the apparent affinity of Mn(2+) to the integrin. These findings offer an explanation for the variable occupancy of the metal ion at LIMBS in αVβ3 structures in the absence of ligand and provide new insights into the mechanisms of integrin regulation.

Highlights

Metal ions at ligand-associated metal ion-binding site (LIMBS) and metal ion-dependent adhesion site (MIDAS) are essential for integrin-ligand interactions
We provide computational and functional evidence that the ␣-subunit plays an essential role in stability of the metal ion coordination at LIMBS in ␤3 integrins
By combining these two approaches, we demonstrated the following. 1) interaction of the LIMBS loop with ␣V is more extensive than with ␣IIb; 2) metal ion coordination at LIMBS after 20 ns of equilibration becomes planar in ␣V␤3; 3) changing the ␣IIbLIMBS loop interface residue Phe191 to Trp destabilized the metal ion at LIMBS, whereas a Trp179 to Phe mutation in ␣V produced opposite but weaker effects; and 4) introducing F191/W in cellular ␣IIb␤3 reduced the apparent affinity of Mn2ϩ to this integrin; the reverse was observed upon introducing the W179/F mutation in cellular ␣V␤3

Summary

Background

Metal ions at LIMBS and MIDAS are essential for integrin-ligand interactions. Results: MD simulations showed strikingly different and functionally relevant conformations of LIMBS in ␣V␤3 and ␣IIb␤3. LIMBS contacts distinct residues in the ␣-subunits of the two ␤3 integrins ␣IIb␤3 and ␣V␤3, but a potential role of this interaction on stability of the metal ion at LIMBS in ␤3 integrins has not been explored. An F191/W substitution in cellular ␣IIb␤3 and a W179/F substitution in ␣V␤3 reduced and increased, respectively, the apparent affinity of Mn2؉ to the integrin These findings offer an explanation for the variable occupancy of the metal ion at LIMBS in ␣V␤3 structures in the absence of ligand and provide new insights into the mechanisms of integrin regulation. At the ligand-binding face of ␤A domain, the LIMBS loop residues Arg216, Asp217, and Ala218 contact residues in the ␣-subunit propeller domain (Fig. 1), but a potential role of the ␣-subunit in regulating metal ion occupancy in the ␤A domain has not been explored.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION