Fibronectin Binds to and Induces Conformational Change in a Disordered Region of Leptospiral Immunoglobulin-like Protein B

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 ␤-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 ␤-strand-rich structure, suggestive of the known ␤-zipper mode of NTD binding.Leptospira interrogans is a pathogenic spirochete that causes leptospirosis throughout the world, especially in developing countries but also in regions of the United States where it has reemerged (1). Weil's syndrome, a severe form of this disease, is an acute febrile illness associated with multiorgan damage, including liver failure (jaundice), renal failure (nephritis), pulmonary hemorrhage, and meningitis (1), and has a 15% mortality rate if not treated (2). The molecular pathogenesis of leptospirosis is poorly understood, and the bacterial virulence factors involved are largely unknown. Recently, several potential Leptospira virulence factors have been described, including sphingomyelinases, serine proteases, zinc-dependent proteases, and collagenase (3); LipL32 (4); lipopolysaccharide (5); a novel factor H, laminin, and Fn-binding protein (Lsa24 or Len) (6 -8); Loa 22 (9); and Lig (Leptospiral immunoglobulin-like) proteins (10 -12).Lig proteins, including LigA, LigB, and LigC, contain multiple immunoglobulin-like repeat domains (13 in LigA, 12 in LigB and LigC) (10 -12). Interestingly, the first 630 residues, from the N terminus to the first half of the seventh immunoglobulinlike domain, are conserved between LigA and LigB, but the rest of the immunoglobulin-like domains are variable (10 -12) between the two proteins. Also, a non-immunoglobulin-like repeat region found on the C-terminal...

Novel Mycobacteria Antigen 85 Complex Binding Motif on Fibronectin

“…Protein Binding Assays by ELISA-To measure the binding affinity of MAP Ag85 (Ag85A, Ag85B, and Ag85C) to Fn, human plasma Fn (0.5 g) was coated on microtiter plate wells using 0.1 M NaHCO 3 (pH 9.3) coating buffer at 4°C for overnight and blocked by PBS plus 3% BSA at 37°C for 1 h. Subsequently, various concentrations (0.1, 0.2, 0.4, 0.8, and 1.6 M) of His-tagged MAP Ag85 were added to each well and incubated at 37°C for 1 h. After three washes with PBST (PBS plus 0.05% Tween 20), bound Ag85 was detected by mouse anti-histidine (1:1000) and HRP-conjugated goat anti-mouse IgG (1:1000), serving as primary and secondary antibodies, respectively (27,28). To investigate the MAP Ag85B-binding domains of Fn, 0.5 g of full-length Fn, four Fn proteolytic domains (including NTD, GBD, CBD, and 40-kDa domain) or BSA (negative control) was coated on microtiter plate wells.…”

“…The cell-free extract was loaded onto a 10-ml nickel-nitrilotriacetic acid or GST column that was equilibrated with PBS. The protein purification procedures were the same as described previously (27,28).…”

Manganese Binds to Clostridium difficile Fbp68 and Is Essential for Fibronectin Binding

“…To detect the binding of histidine-tagged Fbp68, LigBCen2, LigBCon, mouse anti-histidine tagged (1:200), and HRP-con- jugated goat anti-mouse IgG (1:1000) were used as primary and secondary antibodies, respectively (34,35). To measure the binding of GST-fused LigBCon, Fbp68, Fbp68N, Fbp68C, rabbit anti-GST (1:200), and HRP-conjugated goat anti-rabbit IgG (1:1,00) were used as primary and secondary antibodies, respectively.…”

“…PCR products were sequentially digested with BamHI and SalI and then ligated into pQE30 or pGEX4T2 cut with BamHI and SalI, respectively. The soluble forms of all of recombinant proteins were purified from E. coli as described previously (34,35).…”

Calcium Binding to Leptospira Outer Membrane Antigen LipL32 Is Not Necessary for Its Interaction with Plasma Fibronectin, Collagen Type IV, and Plasminogen

“…Accordingly, leptospires employ surface-exposed proteins to bind to a wide range of host ligands, including laminin, collagen, cellular and plasma fibronectin, elastin, proteoglycans, fibrinogen, and plasminogen (13,30,31,36,37). LipL32 binds to plasma fibronectin, collagen types I, IV, and V, laminin, and plasminogen (11)(12)(13).…”