Cyclic-di-GMP and ADP bind to separate domains of PilB as mutual allosteric effectors

Abstract

PilB is the assembly ATPase for the bacterial type IV pilus (T4P), and as a consequence, it is essential for T4P-mediated bacterial motility. In some cases, PilB has been demonstrated to regulate the production of exopolysaccharide (EPS) during bacterial biofilm development independently of or in addition to its function in pilus assembly. While the ATPase activity of PilB resides at its C-terminal region, the N terminus of a subset of PilBs forms a novel cyclic-di-GMP (cdG)-binding domain. This multi-domain structure suggests that PilB binds cdG and adenine nucleotides through separate domains which may influence the functionality of PilB in both motility and biofilm development. Here, Chloracidobacterium thermophilum PilB is used to investigate ligand binding by its separate domains and by the full-length protein. Our results confirm the specificity of these individual domains for their respective ligands and demonstrate communications between these domains in the full-length protein. It is clear that when the N- and the C-terminal domains of PilB bind to cdG and ADP, respectively, they mutually influence each other in conformation and in their binding to ligands. We propose that the interactions between these domains in response to their ligands play critical roles in modulating or controlling the functions of PilB as a regulator of EPS production and as the T4P assembly ATPase.

Keywords: ATPase; PilB; biofilm; cyclic-di-GMP; motility; type IV pili.

Figure 1.. N- and C-terminal domains of CtPilB bind to their respective ligands specifically.

ITC results and their analysis are shown for the binding of (A) PilB_N with cdG, (B) PilB_ΔN with ATPγS, and (C) PilB_ΔN with ADP. In each panel, the ITC thermogram is shown on the top and the isotherm from curve fitting at the bottom. DP stands for power differential and the time interval between injections was 150 s. Shown with the isotherm is the thermodynamic parameter calculated from curve fitting. These include ΔH and −TΔS in kilocalorie per mole as well as the dissociation constant (K_D) and the number of ligand binding sites (N) per protein. Standard deviations for the relevant parameters from curve fitting are listed in Table 1.

Figure 2.. Full-length CtPilB binds cdG, ATPγS, and ADP with higher affinity.

Shown are ITC results and their analysis for the binding of CtPilB with (A) cdG, (B) ATPγS, and (C) ADP as presented in Figure 1. The time interval between injections was 150 s in (A) and 120 s in (B) and (C). In addition, panels (A′), (B′), and (C′) show graphical comparisons of the thermodynamic signatures for the binding of the same ligand to the full-length PilB and its relevant ligand-binding domains individually (either PilB_N or PilB_ΔN). The comparisons for K_D and N are on the top and those for ΔG, ΔH, and −TΔS at the bottom. The unit for K_D is nanomolar in (A′) and micromolar in (B′) and (C′).

Figure 3.. ADP, but not ATPγS, enhances the binding PilB with cdG.

Shown are the ITC results and their analysis for the titration of cdG into CtPilB pre-incubated with 50 μM of (A) ATPγS or (B) ADP presented in the same format as in Figure 1. The time interval between injections was 150 s. For the purpose of comparison, the isotherm for the binding of cdG and CtPilB without any adenine nucleotide from Figure 2 is included as the red dashed lines in both panels. Panel (C) shows the graphical comparison of thermodynamic signatures of cdG and CtPilB binding with (+) or without (−) pre-incubation with ADP and ATPγS as indicated at the top of the panel.

Figure 4.. cdG inhibits the binding of PilB to ADP, but not ATPγS.

ITC results and their analysis for the titration of CtPilB pre-incubated with 100 μM cdG with (A) ATPγS and (B) ADP. The time interval between injections was 120 s. For comparison, the isotherm for the binding of ADP with CtPilB without cdG is included in (B) as a red dashed line. Panel (B′) shows the graphical comparison of thermodynamic signatures for the titration of ADP into PilB without (−) or with (+) pre-incubation with cdG as indicated at the top of the panel.

Figure 5.. Removal of MshE_N domain increases ATPase activity of CtPilB.

ATPase activity (nmol P/mg protein/min) of PilB_ΔN and full-length CtPilB was determined by MLG-based endpoint assays using 75 nM protein and ATP at specified concentrations. Shown are the averages and the standard deviations (error bar) from three independent experiments, each of which was conducted in triplicate samples. Pairs of stars (**) indicate the activities of PilB_ΔN and full-length CtPilB at the indicated ATP concentration are significantly different with a P-value <0.05 by unpaired student t-tests.

Figure 6.. Model for the regulation of PilB signaling activity by cdG and ADP.

PilB is inactive in EPS signaling when it is associated with ADP. In this ADP-bound state, however, PilB has high affinity for cdG and it has a high probability to bind cdG. The binding of cdG, in turn, results in a decrease in the affinity of PilB for ADP and the release of ADP leads to the PilB–cdG complex to its active EPS-signaling conformation. The dissociation of cdG inactivates PilB in EPS signaling and may simultaneously facilitates the ATP hydrolysis cycle by PilB. The left side of this model is functionally analogous to the conversion of a G-protein from a GDP-bound and inactive signaling state to GTP-bound and active state which is catalyzed by GEF.