Selection and expression of the key components of cellulosomes
The docking protein DocA (PDB ID code: 2CCL) and the adhesion protein Coh (PDB ID code: 1OHZ), whose crystal structures had been investigated, were selected as the targets. The selected genes were initially optimized and synthesized in accordance with codons of E. coli and were then transfected into E. coli BL21(DE3) with the help of the pET-28a(+) plasmid for gene expression. When the optical density at 600 nm reached 1.0, IPTG at a final concentration of 0.2 mmol/L was added to induce protein expression, and induction was carried out at 22 °C for 10 h. After ultrasonic fragmentation and centrifugation, Coh and DocA-G were purified by salting out, ultrafiltration, and affinity chromatography using a HisTrap HP column and were then analyzed by SDS-PAGE. As shown in Fig. 2, the molecular weight of Coh was about 16.7 kDa (lanes 2 and 4), whereas the molecular weight of DocA-G was about 36.7 kDa (lanes 1 and 3). The molecular weights of the purified proteins were close to the theoretical values, which indicated that Coh and DocA-G, i.e., the key components of cellulosomes, were successfully purified.
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Analysis of binding ability of DocA-G and Coh
After the purification process, the docking mechanisms of Coh and DocA-G at different calcium ion concentrations were investigated using a Biacore T200 molecular interaction analyzer (GE Healthcare, Chicago, IL, USA). It was found that the higher was the concentration of calcium ions in the range from 1.00 × 10−7 to 1.00 × 10−4 mol/L, the higher was the binding ability of Coh and DocA-G. However, when the calcium ion concentration was lower than 1.00 × 10−4 mol/L, the binding ability of Coh and DocA-G was similar to that when the calcium ion concentration was 1.00 × 10−7 mol/L. It can be tentatively concluded that the interaction between Coh and DocA-G to form a stable structure requires the participation of calcium ions at a concentration of about 1.00 × 10−4 mol/L.
Design and selection of DocA-G mutants
Mutation sites in DocA-G were selected with the help of PyMOL software. The amino acids V40, D41, K42, N43, and S45, which were within 0.4 nm of the calcium ion-binding site of DocA-G, were selected as the key residues involved in calcium binding. Then, the key residues were altered and simulated using the Rosetta website. The two highest-scoring mutants, namely, DocA-D40 (containing T40, S41, N42, D43, and Y45) and DocA-D41 (containing T40, S41, N42, and T45), were selected for protein–protein docking and analysis.
The genes encoding DocA-D40 and DocA-D41 were constructed using a rapid site-specific mutagenesis kit (Tiangen, Beijing, China). Using the pET-28a(+)-DocA-G vector as a template, the genes docA-D40 and docA-D41 were amplified with the 40-F, 40-R primers and the 41-F, 41-R primers, respectively. The primer sequences used are shown in Table 2. The target PCR products were gel-purified, inserted into the pET-28a(+) plasmid, and transfected into E. coli BL21(DE3). The resulting recombinant vectors, namely, pET-28a(+)-DocA-D40 and pET-28a(+)-DocA-D41, were identified by DNA sequencing. The specific primer sequences used for mutagenesis are listed in Fig. 3.
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In vitro confirmation of binding of DocA-G mutants to Coh.
As shown in Fig. 4, when the calcium ion concentration was in the range from 1.00 × 10−7 to 1.00 × 10−4 mol/L the binding capacities of DocA-D41 and DocA-G were almost identical, although the binding capacity of DocA-D41 was slightly higher at a calcium ion concentration of 1.00 × 10−4 mol/L and was about 1.2 times that of DocA-G. When the calcium ion concentration was in the range from 1.00 × 10−5 to 1.00 × 10−2 mol/L the binding capacities of DocA-D40 and DocA-D41 were about 3.68 times and 4.11 times that of the original protein DocA-G, respectively. Moreover, DocA-D41 exhibited the highest binding capacity for Coh at a calcium ion concentration of 5 × 10−4 mol/L.
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Molecular dynamics simulation and structural analysis of DocA-G mutants
We constructed different DocA-D40-Coh and DocA-D41-Coh complexes using RosettaDock 3.4 and then performed an MD simulation for 5 ns using GROMACS 4.5 software. Using the g_rms tool in GROMACS 4.5, the difference parameters (i.e., RMSD) for the structures of the mutants and that of the original docking protein DocA (with/without Ca2+) were calculated. The RMSD values for the mutants DocA-D40 and DocA-D41 (0.232 and 0.228, respectively) were lower than that for DocA (0.378), which implies that the structures of DocA-D40 and DocA-D41 are more stable than that of DocA.
Please insert Fig. 5 here.