Decoding the molecular design principles underlying Ca(2+) binding to βγ-crystallin motifs

Area of Science:

• Biochemistry
• Structural Biology
• Protein Science

Background:

• The βγ-crystallin superfamily contains numerous proteins that bind calcium ions (Ca2+) via a conserved N/D-N/D-X(1)-X(2)-S/T-S motif.
• The precise determinants of Ca2+ binding affinity and their impact on protein stability within this superfamily remain largely unexplored.

Purpose of the Study:

• To comprehensively analyze the modes and determinants of Ca2+ binding within βγ-crystallin motifs.
• To investigate how specific amino acid substitutions influence Ca2+ binding affinity and stability in flavollin and clostrillin.

Main Methods:

• Extensive naturally occurring substitutions were introduced into the βγ-crystallin domains of flavollin and clostrillin.
• Ca2+ binding was monitored using isothermal titration calorimetry, thermal stability assays, and conformational and crystal structure analyses.

Main Results:

• Ca2+ binding at the two sites within a βγ-domain is interdependent.
• The presence of Arginine at the fifth position of the motif abolishes Ca2+ binding at that site.
• Amino acid substitutions, particularly at the first binding site and involving Thr/Ser or acidic/hydrophobic residues, significantly alter Ca2+ binding affinity and properties.

Conclusions:

• The study elucidates the subtle yet critical role of specific residues in modulating Ca2+ binding affinity and stability in βγ-crystallins.
• The findings highlight the evolutionary design principles governing differential Ca2+ binding within this protein superfamily.
• Understanding these design schemes is crucial for predicting protein function and interpreting new sequence information.