Decoding the molecular design principles underlying Ca²⁺ binding to βγ-crystallin motifs

Abstract

Numerous proteins belonging to the recently expanded βγ-crystallin superfamily bind Ca²⁺ at the double-clamp N/D-N/D-X_1-X_2-S/T-S motif. However, there have been no attempts to understand the intricacies involving Ca²⁺ binding, such as the determinants of Ca²⁺-binding affinity and their contributions to gain in stability. This work is an in-depth analysis of understanding the modes and determinants of Ca²⁺ binding to βγ-crystallin motifs. We have performed extensive naturally occurring substitutions from related proteins on the βγ-crystallin domains of flavollin, a low-affinity Ca²⁺-binding protein, and clostrillin, a moderate-affinity protein. We monitored the consequences of these modifications on Ca²⁺ binding by isothermal titration calorimetry, thermal stability and conformational and crystal structure analyses. We demonstrate that Ca²⁺ binding to the two sites of a βγ-domain is interdependent and that the presence of Arg at the fifth position disables a site. A change from Thr to Ser, or vice versa, influences Ca²⁺-binding affinity, highlighting the basis of diversity found in these domains. A subtle change in the first site has a greater influence on Ca²⁺ binding than a similar alteration in the second site. Thus, the second site is more variable in nature. Replacing an acidic or hydrophobic residue in a binding site alters the Ca²⁺-binding properties drastically. While it appears from their binding site sequence that these domains have evolved randomly, our examination illustrates the subtlety in the design of these modules. Decoding such design schemes would aid in our understanding of the functional themes underlying differential Ca²⁺ binding and in predicting these in emerging sequence information.