Molecular Basis of Calcium-Induced Acidic Shift in Antimicrobial Zinc Sequestration by S100A12

Antimicrobial protein S100A12 sequesters Zn(II) via a His3Asp motif to inhibit pathogens during infection. Here, UV-vis and NMR spectroscopies and molecular dynamics (MD) simulations are used to gain molecular insight into the Zn(II) chelation properties of S100A12 under pH conditions relevant to infection and inflammation. UV-vis measurements show that binding of Zn(II) to apo S100A12 exhibits a sigmoidal dependence with pH, beginning its decline at pH 7.0 and vanishing at pH 4.0. In the Ca(II)-bound protein, a similar sigmoidal curve is found to exhibit an acidic shift, an effect not attributed to a Ca(II)-induced pKa suppression of the His3Asp scaffold. NMR measurements show that upon lowering the pH, resonances exhibit nonlinear migration trends with pH, suggesting the occurrence of several proton binding events, consistent with the sigmoidal pH dependence of Zn(II) binding. Analysis of the NMR chemical shifts versus pH shows that both apo- and Ca(II)-bound protein undergo conformational changes exhibiting spatial correlations, which are dispersed across the polypeptide in the apo protein and confined to discrete regions in Ca(II)-S100A12. MD simulations show the formation of a strong salt bridge within the His3Asp scaffold of the apo protein upon protonation of Zn(II)-ligating histidines. Geometric constraints imposed by Ca(II) in the Ca(II)-bound protein hinder the formation of similar salt bridges until protonation of a histidine residue external to the His3Asp and near Ca(II) takes place. Overall, our experimental and computational results support a scenario where protonation of the His3Asp motif triggers the loss of Zn(II) binding and yields protonated histidine residues prone to form stable salt bridges. In Ca(II)-S100A12, formation of stable salt bridges at pH 7 is hindered compared to the apo form, thus extending Zn(II)-binding affinity to lower pH.