Phosphorylation-Independent Regulation of the Diguanylate Cyclase WspR
Figure 2
Cyclic di-GMP Binding and Gel Filtration Profile of Wild-Type and Mutant WspR
(A) Detection of guanosine nucleotides by a reverse-phase HPLC-based assay. GTP, GDP, linear di-GMP (pGpG), c-di-GMP, and an intermediate condensation product (linear GTP-GMP; pppGpG) are well separated in this assay (grey dashed line). Products corresponding to pppGpG and pGpG were identified by mass spectrometry (unpublished data). Cyclic di-GMP was commercially available. WspR expressed in E. coli purifies with c-di-GMP bound (red trace) that is accessible for PDEs (black trace).
(B) I-site and active site mutants of WspR purify nucleotide-free. Mutant proteins with disrupted I-sites (WspRR242A or WspRR198A; blue and purple traces, respectively) or catalytic site (WspRGGAAF; orange trace) were analyzed. Nucleotide-free WspRwt is obtained by PDE treatment followed by repurification using affinity and size exclusion columns (green trace).
(C) PDE treatment triggers a conformational change in WspR. Cyclic di-GMP–bound WspRwt (0.24 mM) was incubated with PDE (0.008 mM) in gel filtration buffer (25 mM Tris-Cl [pH 7.5], 100 mM NaCl, and 1 mM DTT) supplemented with 10 mM Mn2+ for 0.5, 1, or 2 h at 25 °C. Reactions were analyzed by SEC on a Superdex200 10/300 column (GE Heathcare).
(D) SEC profiles of mutant and wild-type WspR. Nucleotide-bound and nucleotide-free WspRwt (red and green traces, respectively), WspRGGAAF (orange trace), WspRR242A (blue trace), and WspRR198A (purple trace) (0.24 mM) were analyzed by analytical gel filtration in gel filtration buffer. Peak maxima at 11.7 (peak 1), 12.1 (peak 2), and 13.3 ml (peak 3) are labeled. Peaks 1–3 (Superdex 200 10/30 column; GE Healthcare) correspond to peaks 1–3 (Shodex KW-803 column; JM Science, Inc.) in Table 1, obtained from the SEC coupled to the static multiangle light-scattering detectors.