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 Escherichia coli 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 Ca²⁺ 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 Ca²⁺ binding on the C-terminal domain of GGBP, especially the Ca²⁺-binding site. Interestingly, the MSMs of calcium-depleted GGBP and calcium-bound GGBP (GGBP/Ca²⁺) demonstrate that Ca²⁺ greatly stabilizes the open conformation, and smFRET measurements confirmed this result. Further analysis reveals that Ca²⁺ 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 Ca²⁺ binding in GGBP, which finely tunes the conformational distribution. The work sheds new light on the study of calcium-binding proteins in prokaryotes.