Calcium binds to leptospiral immunoglobulin-like protein, LigB, and modulates fibronectin binding.
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 Ca(2+), as evidenced by inductively coupled plasma optical emission spectrometry, energy dispersive spectrometry, (45)Ca overlay, and mass spectrometry, although there is no known motif for Ca(2+) binding. LigBCen2 binds four Ca(2+) as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The dissociation constant, K(D), for Ca(2+) binding is 7 mum, as measured by isothermal titration calorimetry and calcium competition experiments. The nature of the Ca(2+)-binding site in LigB is possibly similar to that seen in the betagamma-crystallin superfamily, since structurally, both families of proteins possess the Greek key type fold. The conformation of LigBCen2 was significantly influenced by Ca(2+) 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 Ca(2+) binding, the protein acquires significant beta-sheet conformation. Ca(2+) binding stabilizes the protein as monitored by thermal unfolding by CD (50.7-54.8 degrees C) and by differential scanning calorimetry (50.0-55.7 degrees C). Ca(2+) significantly assists the binding of LigBCen2 to the N-terminal domain of fibronectin and perturbs the secondary structure, suggesting the involvement of Ca(2+) in adhesion. We demonstrate that LigB is a novel bacterial Ca(2+)-binding protein and suggest that Ca(2+) binding plays a pivotal role in the pathogenesis of leptospirosis.
Lin
Yi-Pin
YP
College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 and the.
Raman
Rajeev
R
Centre for Cellular and Molecular Biology (Council of Scientific and Industrial Research), Uppal Road, Hyderabad 500 007, India.
Sharma
Yogendra
Y
Centre for Cellular and Molecular Biology (Council of Scientific and Industrial Research), Uppal Road, Hyderabad 500 007, India.
Chang
Yung-Fu
YF
College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 and the. Electronic address: yc42@cornell.edu.
D018829:Adhesins, Bacterial / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000378:metabolism / Q000502:physiology*; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry; D002417:Cattle; D002942:Circular Dichroism; D005353:Fibronectins / Q000737:chemistry*; D007700:Kinetics; D007919:Leptospira / Q000378:metabolism*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
D018829:Adhesins, Bacterial; D000942:Antigens, Bacterial; D002135:Calcium-Binding Proteins; D005353:Fibronectins; C487006:LigB-m protein, Leptospira interrogans; D002118:Calcium