Abstract
Leptospira interrogans is a pathogenic spirochete that causes disease in both humans and animals. LigB (Leptospiral immunoglobulin-like protein B) contributes to the binding of Leptospira to extracellular matrix proteins such as fibronectin (Fn), fibrinogen, laminin, and collagen. A high affinity Fn-binding region of LigB has been recently localized to LigBCen2, which contains the partial eleventh and full twelfth immunoglobulin-like repeats (LigBCen2R) and 47 amino acids of the non-repeat region (LigBCen2NR) of LigB. In this study, LigBCen2NR was shown to bind to the N-terminal domain (NTD) of Fn (K(D) = 379 nm) by an enzyme-linked immunosorbent assay and isothermal titration calorimetry. Interestingly, this sequence was not observed to adopt secondary structure by far UV circular dichroism or by differential scanning calorimetry, in agreement with computer-based secondary structure predictions. A low partition coefficient (K(av)) measured with gel permeation chromatography, a high hydrodynamic radius (R(h)) measured with dynamic light scattering, and the insensitivity of the intrinsic viscosity to guanidine hydrochloride treatment all suggest that LigBCen2NR possesses an extended and disordered structure. Two-dimensional (15)N-(1)H HSQC NMR spectra of intact LigBCen2 in the absence and presence of NTD are consistent with these observations, suggesting the presence of both a beta-rich region and an unstructured region in LigBCen2 and that the latter of these selectively interacts with NTD. Upon binding to NTD, LigBCen2NR was observed by CD to adopt a beta-strand-rich structure, suggestive of the known beta-zipper mode of NTD binding.
Highlights
Tris buffer (25 mM Tris and 150 mM sodium chloride at pH 7.0) containing 100 M calcium chloride was used in all experiments, since we have previously shown that calcium enhances the binding of LigBCen2 to N-terminal domain (NTD) (15) and that both LigBCen2R and LigBCen2NR bind calcium
Identification of the Binding Sites of NTD on LigBCen2—In order to investigate the general structural properties of LigBCen2 and its interactions with Fn, uniformly 15N-labeled LigBCen2 was prepared, and two-dimensional 15N-1H chemical shift correlation NMR experiments were performed in the absence and presence of an equimolar amount of unlabeled NTD (Fig. 2, A–D)
The number of well dispersed peaks and their positions are consistent with the -sheet-rich immunoglobulin-like fold predicted for repeat regions 11 and 12, which would place backbone N-H groups in unique chemical environments and would cause the corresponding 15N-1H correlation peaks to appear at widely varying positions in the NMR spectrum
Summary
Ious components of the extracellular matrix (16). Known interaction modes between Fn and bacterial Fn-binding proteins include the -zipper (17, 18) and the cationic cradle (19). The addition of NTD promotes the folding of LigBCen2NR from a disordered and extended structure to a folded structure. This finding is notable, since LigBCen2NR is located in the non-immunoglobulin-like region of LigB, as compared with other Fn-binding proteins, such as Staphylococcus aureus FnbpA and FnbpB (23), Streptococcus dysgalactiae FnBB (17), and Streptococcus pyogenes SfbI and SfbII (24). The binding mode appears to be similar to the known -zipper mechanism but unique in sequence-specific interactions. This finding provides the fundamental groundwork for the development of a therapeutic agent to target this interaction in order to prevent or treat Leptospira infection
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
