Salt-bridge modulates differential calcium-mediated ligand binding to integrin α1- and α2-I domains

Area of Science:

• Biochemistry
• Structural Biology
• Cell Biology

Background:

• Integrins are crucial transmembrane receptors mediating cell-extracellular matrix adhesion.
• A subset of integrins features an inserted (I) domain within α-subunits (αI) for ligand recognition.
• Ligand binding in I-domains is dependent on divalent cations at the metal ion-dependent adhesion site (MIDAS).

Purpose of the Study:

• To elucidate the structural basis for differential calcium ion (Ca²⁺) effects on α1I and α2I integrin I-domain ligand binding.
• To compare Ca²⁺ binding in α1I and α2I I-domains and its impact on conformation and affinity.

Main Methods:

• Co-crystallization of individual I-domains (α1I and α2I) with Ca²⁺ bound to the MIDAS.
• X-ray crystallography to determine structures at 1.4 Å (α1I) and 2.15 Å (α2I) resolution.
• Comparison of Ca²⁺-bound structures with existing Mg²⁺-bound structures.
• Site-directed mutagenesis (α2I E152A construct) to investigate the role of the E152-R192 salt bridge.

Main Results:

• Both Ca²⁺-bound α1I and α2I I-domains adopt a 'closed' ligand-binding conformation with minimal global structural changes.
• Ca²⁺ binds similarly to Mg²⁺ in α1I, promoting ligand binding.
• In α2I, Ca²⁺ is displaced from the MIDAS compared to Mg²⁺, hindering direct coordination of MIDAS residues.
• An E152-R192 salt bridge in α2I restricts MIDAS flexibility and Ca²⁺ binding.
• The α2I E152A mutation significantly increased Mg²⁺ and Ca²⁺ binding affinity and enhanced collagen ligand binding by 20%.

Conclusions:

• The E152-R192 salt bridge in α2I I-domains is a key determinant of differential Ca²⁺ binding compared to α1I I-domains.
• This structural difference explains the distinct effects of Ca²⁺ on ligand binding in α1I and α2I integrins.
• Modulating this salt bridge offers a potential mechanism to control integrin-mediated adhesion.