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).
Table 1.
Primers used for deletion and site directed mutagenesis of Lmb and the strain designations.
Table 2.
Statistics of diffraction data collection and structure refinement of ΔLmb.
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.
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.
Table 3.
Secondary structure composition of Lmb and its mutants obtained from CD spectroscopy.
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.