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 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.
Highlights
Has reemerged in the United States [1]
Based on the structural homology of immunoglobulin-like fold with lens ␥-crystallin type Greek key motif [31], we wondered if Lig proteins would bind Ca2ϩ [32]
We have recently shown that LigBCen2 binds strongly to the N-terminal domain (NTD) of Fn but very weakly to the gelatin binding domain (GBD) of fibronectin [15]
Summary
Reagents and Antibodies—Calcium GreenTM-1 was obtained from Molecular Probe (Eugene, OR). Absence of or 1 mM in the presence of CaCl2, GST-LigBCen, or GST in 100 l of Tris buffer (pH 7.0) was added onto microtiter plate wells and incubated for 1 h at 37 °C. protein was recorded by exciting the solution at 295 nm and measuring the emission in the 300 – 400 nm regions. For calcium or magnesium titration, 0.1, 0.3, 0.5, 0.8, or 1.0 mM of calcium chloride was mixed with 10 M of LigBCen in 50 mM Tris buffer, pH 7.2, containing 50 mM KCl, and spectra were recorded after 3 min of incubation. Fibronectin binding to LigBCen was assayed by measuring the change in Trp fluorescence upon the addition of aliquots of NTD of Fn in the presence of 100 M CaCl2 or in the absence of calcium chloride (in the presence of 100 M EGTA). Data were analyzed by Student’s t test, and statistically significant differences were claimed at p values of Ͻ0.05
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