Metal binding is critical for the folding and function of laminin binding protein, Lmb of Streptococcus agalactiae

Abstract

Lmb is a 34 kDa laminin binding surface adhesin of Streptococcus agalactiae. The structure of Lmb reported by us recently has shown that it consists of a metal binding crevice, in which a zinc ion is coordinated to three highly conserved histidines. To elucidate the structural and functional significance of the metal ion in Lmb, these histidines have been mutated to alanine and single, double and triple mutants were generated. These mutations resulted in insolubility of the protein and revealed altered secondary and tertiary structures, as evidenced by circular dichroism and fluorescence spectroscopy studies. The mutations also significantly decreased the binding affinity of Lmb to laminin, implicating the role played by the metal binding residues in maintaining the correct conformation of the protein for its binding to laminin. A highly disordered loop, proposed to be crucial for metal acquisition in homologous structures, was deleted in Lmb by mutation (ΔLmb) and its crystal structure was solved at 2.6 Å. The ΔLmb structure was identical to the native Lmb structure with a bound zinc ion and exhibited laminin binding activity similar to wild type protein, suggesting that the loop might not have an important role in metal acquisition or adhesion in Lmb. Targeted mutations of histidine residues confirmed the importance of the zinc binding crevice for the structure and function of the Lmb adhesin.

Figure 1. Crystal structure of Lmb of Streptococcus agalactiae.

(A) Ribbon diagram of the structure of Lmb showing two domains. The metal binding site is at the interface of the two domains and the bound zinc ion is shown as cyan sphere. Near the metal binding site a long disordered loop between residues G123 and L138 is indicated by dotted lines. (B) Close up view of the metal binding site of Lmb. The zinc is coordinated by three histidines and a glutamate (shown as stick models).

Figure 2. Analysis of interaction of wt Lmb with human placental (HP) and EHS (Engelbroth-Holm-Swarm) tumor laminin.

(A) Dot blot analysis showing the interaction of wt Lmb with HP and EHS laminin. Equal concentrations (10 µg/ml) of HP and EHS laminin were spotted on a nitrocellulose membrane and probed with (10 µg/ml) wt Lmb protein. The dot blot shows that Lmb binds to both types of laminin. However, the intensity of the HP laminin spot is higher when compared with EHS Laminin spot suggesting the former has high affinity towards Lmb. (B) ELISA analysis showing the binding of wt Lmb to HP and EHS laminin. Different concentrations of wt Lmb was added to microtiter plates coated with 10 µg/ml of HP and EHS laminin and binding quantified at 405 nm. The values in the graph represent the mean+ standard deviation for the experiment in triplicates. This experiment clearly suggests that Lmb has higher affinity to HP laminin than EHS Laminin and thus supports the dot blot experiment.

Figure 3. Structure of ΔLmb.

Superposition diagram of wt Lmb (violet) and ΔLmb (yellow). In wt Lmb, the disordered “metal binding loop” between G123 and L138 is indicated by dotted lines. The close up view of the metal binding center is shown. The residues His66, His142, His206, Pro279, His264 and the segment DPH(140–142) were mutated in wt Lmb. The location of His264 (negative control-located away from the metal binding site) and Pro279 (negative control-located near the metal binding site) is also shown as a close up view.

Figure 4. CD and fluorescence spectra of Lmb and its mutants.

(A) The Far UV CD spectrum was recorded between 190–240 nm. ΔLmb, H264A and P279A showed same ellipticity as the wt Lmb. Significant changes in the ellipticity were observed for the mutants involving metal binding residues His66, His142 and His206 indicating these proteins have an altered secondary structure. (B) ANS fluorescence spectroscopy of Lmb and its mutants. Excitation wavelength was 285 nm and emission was recorded between 400 to 700 nm. Mutants of the metal binding residues show decreased fluorescence compared to wt Lmb, H264A and P279A mutant indicating they are not well folded. (C) Intrinsic tryptophan fluorescence of Lmb and its mutants. An excitation wavelength of 295 nm was used and emission was recorded between 310–400 nm. The spectra shows that the emission of the well folded wt Lmb, ΔLmb, H264A and P279A is higher compared to the partially folded or misfolded mutants.

Figure 5. Interaction of Lmb and its mutants with laminin.

(A) Dot blot analysis of HP laminin with (a) wt Lmb (b) DPH(140–142)-ARD (c) H66N (d) ΔLmb (e) H142-206A (f) H142-206-66A proteins (g) CbpA of Arcanobacterium pyogenes (negative control) (h) Jack bean urease (negative control). 10 µg/ml of laminin was spotted on the nitrocellulose membrane for binding of wt Lmb and the mutants at a concentration of 10 µg/ml. (B) Quantification of Lmb-laminin binding using ELISA. Lmb and its mutants were added to microtiter plate coated with HP & EHS laminin (10 µg/ml) and binding quantified at 405 nm. Data is expressed as a percentage of the binding observed for 1 µg/ml of the protein to laminin. Shown are the mean + standard deviations for the experiment in triplicates. (C) Dot blot analysis of (1) wt Lmb (2) H264A and (3) H142-206-66A with (a) laminin (b) fibronectin and (c) collagen. 1 µl of 0.5 mg/ml of HP laminin, fibronectin and collagen were spotted on a membrane and probed with Lmb and its mutants. Lmb shows high specificity to laminin and does not bind to fibronectin or collagen.