Calcium binding to leptospira outer membrane antigen LipL32 is not necessary for its interaction with plasma fibronectin, collagen type IV, and plasminogen

Abstract

LipL32 is the most abundant outer membrane protein from pathogenic Leptospira and has been shown to bind extracellular matrix (ECM) proteins as well as Ca(2+). Recent crystal structures have been obtained for the protein in the apo- and Ca(2+)-bound forms. In this work, we produced three LipL32 mutants (D163-168A, Q67A, and S247A) and evaluated their ability to interact with Ca(2+) and with ECM glycoproteins and human plasminogen. The D163-168A mutant modifies aspartate residues involved in Ca(2+) binding, whereas the other two modify residues in a cavity on the other side of the protein structure. Loss of calcium binding in the D163-D168A mutant was confirmed using intrinsic tryptophan fluorescence, circular dichroism, and thermal denaturation whereas the Q67A and S247A mutants presented the same Ca(2+) affinity as the wild-type protein. We then evaluated if Ca(2+) binding to LipL32 would be crucial for its interaction with collagen type IV and plasma proteins fibronectin and plasminogen. Surprisingly, the wild-type protein and all three mutants, including the D163-168A variant, bound to these ECM proteins with very similar affinities, both in the presence and absence of Ca(2+) ions. In conclusion, calcium binding to LipL32 may be important to stabilize the protein, but is not necessary to mediate interaction with host extracellular matrix proteins.

FIGURE 1.

Schematic representations of the Apo-LipL32 structure (A) (PDB 3FRL, Ref. 14) and calcium-bound LipL32 structure (B) (PDB 2WF, Ref. 16). Residues mutated to alanine in the three LipL32 mutants (Q67A, S247A, and D163–168A (D163, D164, D165, D167, and D168) are represented as stick models in yellow, red and blue for C, O, and N atoms, respectively). Residues 160–163, just preceding the polyaspartate loop region mutated in the D163-D168A mutant is unstructured in the absence of calcium ion (Hauk et al., 14). The calcium ion is shown as a magenta-colored sphere and is coordinated by oxygen atoms derived from residues Asp-132, Thr-133, Tyr-178 (shown as green sticks) as well as Asp-164 and Asp-165. The numbering scheme used in this figure is that of Hauk et al. (14) and differs from Tung et al. (16) for the calcium-bound LipL32 structure by 19 residues: Gln-67, Asp-132, Thr-133, Asp-164, Asp-165, Tyr-178, and Ser-247 in the figure correspond to Gln-48, Asp-113, Thr-114, Asp-145, Asp-146, Tyr-159, and Ser-228 in Tung et al. (16).

FIGURE 2.

Intrinsic fluorescence of LipL32 and its mutants. Fluorescence emission spectra of wild-type LipL32 (A), LipL32_Q67A (B), LipL32_D163–168A (C), and LipL32_S247A (D). Conditions were 2 μm protein in 10 mm Tris-HCl (pH 8.0), and 50 mm KCl in the presence or absence of 1 mm CaCl₂. Excitation wavelength was 285 nm.

FIGURE 3.

Calcium binding by LipL32. Calcium-induced reduction in intrinsic tryptophan fluorescence was used to accompany calcium binding using a Ca²⁺-EGTA buffer system in which the free Ca²⁺ concentration was varied between the nanomolar and millimolar ranges (pCa = 9 to 3). Conditions were 2 μm protein in 10 mm Tris-HCl (pH 7.0), 50 mm KCl, 0.5 mm EGTA. Excitation wavelength was 285 nm and emission wavelength was 338–342 nm. We observed clear transitions for wild-type LipL32 (A) and for mutants LipL32_Q67A (B) and LipL32_S247A (C) between pCa = 5 and 6, indicating that these mutants bind calcium ions with affinities between 1 and 10 μm. For the mutant LipL32_D163–168A, no clear Ca²⁺-dependent transition was observed (data not shown). Error bars present S.D. of at least 3 independent experiments.

FIGURE 4.

Thermal denaturation of wild-type LipL32 and mutants in the presence and absence of Ca²⁺ monitored by ANS fluorescence. Thermal denaturation in the presence or absence of Ca²⁺ in LipL32 wild-type (A) and mutants LipL32_Q67A (B), LipL32_D163–168A (C), and LipL32_S247A (D) was accompanied by monitoring changes in extrinsic ANS fluorescence. Conditions: 2 μm protein, 10 mm Tris-HCl, 50 mm KCl, 3 mm EDTA (pH 8.0), 8 μm ANS in the presence or absence of 5 mm CaCl₂. Excitation wavelength was 380 nm. Emission wavelength was 470 nm. The midpoint of the thermal denaturation transition (T_m) is indicated for each experiment. The pre-transition and post-transition baselines were used to calculate the fractional change in signal at each temperature, assuming that the fluorescence signals of the folded and unfolded states are linear functions of temperature in the transition region.

FIGURE 5.

Thermal denaturation of LipL32 and mutants monitored by circular dichroism. CD was used to detect temperature-induced structural changes in the presence or absence of Ca²⁺ in wild-type LipL32 (A) and mutants, LipL32_Q67A (B), LipL32_D163–168A (C), and LipL32_S247A (D). Conditions were 2 μm decalcified protein, 10 mm Tris-HCl (pH 8.0), 50 mm KCl in the presence or absence of 1 mm CaCl₂. Spectra were recorded by measuring [θ] at 216 nm at 2 °C temperature steps between 40 and 70 °C. The pre-transition and post-transition baselines were used to calculate the fractional change in signal at each temperature, assuming that the CD signals of the folded and unfolded states are linear functions of temperature in the transition region.

FIGURE 6.

Calcium is not required for LipL32 binding to F30 fragment of fibronectin. F30 binding to LipL32 proteins and to the unrelated rLIC11030 protein (negative control) were assayed by ELISA. LipL32 proteins or rLIC11030 (0–4 μm) were added to immobilized F30 and allowed to attach in the absence of Ca²⁺ or presence of Ca²⁺. After washing away unbound LipL32 proteins or rLIC11030 using the corresponding incubation buffers, all subsequent steps were carried out in the presence of CaCl₂ (see “Experimental Procedures” for details). Each point represents the mean absorbance value at 492 nm ± S.D. of three independent experiments, each performed in duplicate. Binding to F30 by wild-type LipL32_{His tag} (A), LipL32_{Q67_His tag} (B), LipL32_{D163–168A_His tag} (C), LipL32_{S247A_His tag} (D), LipL32_{185–272_His tag} (E), and rLIC11030_{His tag} (F).