Figure 3:

Ion engagement comparison of yeast and human proteomes captured evolutionary differences in Ca²⁺ signaling and ion coordination.

(A) Ion-induced thermal stability changes of human and yeast OGDH and IDH homologs, relative to EGTA. Human TCA cycle dehydrogenases are CRPs, whereas yeast homologs do not engage Ca²⁺. Orthologs are indicated by the same color. Star denotes that the protein is a CRP in our dataset. Dotted lines show log₂(FC) ± 0.2. Error bars: standard deviation of replicate measurements.

(B) Overlay of human OGDH (gray) and its yeast homolog Kgd1 (yellow) structures at the Ca²⁺ coordination site. Yeast Kgd1 lacks residues that coordinate Ca²⁺ in human OGDH.

(C) Ion-induced thermal stability changes of human and yeast EPS15 homologs, relative to EGTA. Yeast homolog Ede1 changes its abundance with Ca²⁺ addition only, human homologs EPS15, ITSN1, ITSN2 show thermal stability changes with Ca²⁺ or Mg²⁺. REPS1 and EHD3 do not show ion engagement. Dotted lines show log₂(FC) ± 0.2. Error bars: standard deviation of replicate measurements.

(D) Alignment of the second EF-hand domain of yeast and human EPS15 homologs. Yeast has a serine (S) where EPS15, ITSN1, ITSN2 have a conserved aspartic acid (D). REPS1 and EHD3 lack canonical EF-hand residues.

(E) Overlay of EPS15^wt structure with a predicted EPS15^D177S structure. D177S mutation (stick representation, brown) is predicted to form a smaller coordination domain resulting in coordination of smaller Mg²⁺ ions.

(F) Ca²⁺ or Mg²⁺ binding to purified EPS15^wt and EPS15^D177 proteins. Mammalian cells were transiently transfected with plasmids for expression and purification of EPS15-His proteins. Background refers to purification from cells that do not express tagged EPS15 proteins. Significance based on Student’s t-test: ns – not significant, ** - p<0.005, **** - p<0.0001.