Machine learning-based modulation of Ca2+-binding affinity in EF-hand proteins and comparative structural insights into site-specific cooperative binding

Ca2+-binding proteins are present in almost all living organisms and different types display different levels of binding affinities for the cation. Here, we report two new scoring schemes enabling the user to estimate and manipulate the calcium binding affinities in EF hand containing proteins. To validate this, we designed a unique EF-hand loop capable of binding calcium with high affinity by altering five residues. The N-terminal domain of Entamoeba histolytica calcium-binding protein1 (NtEhCaBP1) is used for site-directed mutagenesis to incorporate the designed loop sequence into the second EF-hand motif of this protein, referred as Nt-EhCaBP1-EF2 mutant. The binding isotherms calculated using ITC calorimetry showed that Nt-EhCaBP1-EF2 mutant site binds Ca2+ with higher affinity than Wt-Nt-EhCaBP1, by similar to 600 times. The crystal structure of the mutant displayed more compact Ca2+-coordination spheres in both of its EF loops than the structure of the wildtype protein. The compact coordination sphere of EF-2 causes the bend in the helix-3, which leads to the formation of unexpected hexamer of NtEhCaBP1-EF2 mutant structure. Further dynamic correlation analysis revealed that the mutation in the second EF loop changed the entire residue network of the monomer, resulting in stronger coordination of Ca2+ even in another EF-hand loop.