Calcium binds to Leptospiral immunoglobulin-like protein, LigB, and modulates fibronectin binding

Abstract

Pathogenic Leptospira spp. express immunoglobulin-like proteins, LigA and LigB, which serve as adhesins to bind to extracellular matrices and mediate their attachment on host cells. However, nothing is known about the mechanism by which these proteins are involved in pathogenesis. We demonstrate that LigBCen2 binds Ca²⁺, as evidenced by inductively coupled plasma optical emission spectrometry, energy dispersive spectrometry, ⁴⁵Ca overlay, and mass spectrometry, although there is no known motif for Ca²⁺ binding. LigBCen2 binds four Ca²⁺ as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The dissociation constant, K_D, for Ca²⁺ binding is 7 μm, as measured by isothermal titration calorimetry and calcium competition experiments. The nature of the Ca²⁺-binding site in LigB is possibly similar to that seen in the βγ-crystallin superfamily, since structurally, both families of proteins possess the Greek key type fold. The conformation of LigBCen2 was significantly influenced by Ca²⁺ binding as shown by far- and near-UV CD and by fluorescence spectroscopy. In the apo form, the protein appears to be partially unfolded, as seen in the far-UV CD spectrum, and upon Ca²⁺ binding, the protein acquires significant β-sheet conformation. Ca²⁺ binding stabilizes the protein as monitored by thermal unfolding by CD (50.7-54.8°C) and by differential scanning calorimetry (50.0-55.7°C). Ca²⁺ significantly assists the binding of LigBCen2 to the N-terminal domain of fibronectin and perturbs the secondary structure, suggesting the involvement of Ca²⁺ in adhesion. We demonstrate that LigB is a novel bacterial Ca²⁺-binding protein and suggest that Ca²⁺ binding plays a pivotal role in the pathogenesis of leptospirosis.