Calcium binding to a disordered domain of a type III-secreted protein from a coral pathogen promotes secondary structure formation and catalytic activity

Strains of the Gram-negative bacterium Vibrio coralliilyticus cause the bleaching of corals due to decomposition of symbiotic microalgae. The V. coralliilyticus strain ATCC BAA-450 (Vc450) encodes a type III secretion system (T3SS). The gene cluster also encodes a protein (locus tag VIC_001052) with sequence homology to the T3SS-secreted nodulation proteins NopE1 and NopE2 of Bradyrhizobium japonicum (USDA110). VIC_001052 has been shown to undergo auto-cleavage in the presence of Ca(2+) similar to the NopE proteins. We have studied the hitherto unknown secondary structure, Ca(2+)-binding affinity and stoichiometry of the “metal ion-inducible autocleavage” (MIIA) domain of VIC_001052 which does not possess a classical Ca(2+)-binding motif. CD and fluorescence spectroscopy revealed that the MIIA domain is largely intrinsically disordered. Binding of Ca(2+) and other di- and trivalent cations induced secondary structure and hydrophobic packing after partial neutralization of the highly negatively charged MIIA domain. Mass spectrometry and isothermal titration calorimetry showed two Ca(2+)-binding sites which promote structure formation with a total binding enthalpy of −110 kJ mol(−1) at a low micromolar K(d). Putative binding motifs were identified by sequence similarity to EF-hand domains and their structure analyzed by molecular dynamics simulations. The stoichiometric Ca(2+)-dependent induction of structure correlated with catalytic activity and may provide a “host-sensing” mechanism that is shared among pathogens that use a T3SS for efficient secretion of disordered proteins.