Calcium-Mediated Control of S100 Proteins: Allosteric Communication via an Agitator/Signal Blocking Mechanism

Allosteric proteins possess dynamically coupled residues for the propagation of input signals to distant target binding sites. The input signals usually correspond to "effector is present" or "effector is not present". Many aspects of allosteric regulation remain incompletely understood. This work focused on S100A11, a dimeric EF-hand protein with two hydrophobic target binding sites. An annexin peptide (Ax) served as the target. Target binding is allosterically controlled by Ca 2+ over a distance of ∼26 Å. Ca 2+ promotes formation of a [Ca 4 S100 Ax 2 ] complex, where the Ax peptides are accommodated between helices III/IV and III'/IV'. Without Ca 2+ hese binding sites are closed, precluding interactions with Ax. The allosteric mechanism was probed by microsecond MD simulations in explicit water, complemented by hydrogen exchange mass spectrometry (HDX/MS). Consistent with experimental data, MD runs in the absence of Ca 2+ and Ax culminated in target binding site closure. In simulations on [Ca 4 S100] the target binding sites remained open. These results capture the essence of allosteric control, revealing how Ca 2+ prevents binding site closure. Both HDX/MS and MD data showed that the metalation sites become more dynamic after Ca 2+ loss. However, these enhanced dynamics do not represent the primary trigger of the allosteric cascade. Instead, a labile salt bridge acts as an incessantly active "agitator" that destabilizes the packing of adjacent residues, causing a domino chain of events that culminates in target binding site closure. This agitator represents the starting point of the allosteric signal propagation pathway. Ca 2+ binding rigidifies elements along this pathway, thereby blocking signal transmission. This blocking mechanism does not conform to the commonly held view that allosteric communication pathways generally originate at the sites where effectors interact with the protein.