The Role of Calcium in Regulating the Conformational Dynamics of d-Galactose/d-Glucose-Binding Protein Revealed by Markov State Model Analysis.

The d-glucose/d-galactose-binding protein (GGBP) from is a substrate-binding protein (SBP) associated with sugar transport and chemotaxis. It is also a calcium-binding protein, which makes it unique in the SBP family. However, the functional importance of Ca binding is not fully understood. Here, the calcium-dependent properties of GGBP were explored by all-atom molecular dynamics simulations and Markov state model (MSM) analysis as well as single-molecule Förster resonance energy transfer (smFRET) measurements. In agreement with previous experimental studies, we observed the structure stabilization effect of Ca binding on the C-terminal domain of GGBP, especially the Ca-binding site. Interestingly, the MSMs of calcium-depleted GGBP and calcium-bound GGBP (GGBP/Ca) demonstrate that Ca greatly stabilizes the open conformation, and smFRET measurements confirmed this result. Further analysis reveals that Ca binding disturbs the local hydrogen bonding interactions and the conformational dynamics of the hinge region, thereby weakening the long-range interdomain correlations to favor the open conformation. These results suggest an active regulatory role of Ca binding in GGBP, which finely tunes the conformational distribution. The work sheds new light on the study of calcium-binding proteins in prokaryotes.