SpoIIIE protein achieves directional DNA translocation through allosteric regulation of ATPase activity by an accessory domain

Abstract

Bacterial chromosome segregation utilizes highly conserved directional translocases of the SpoIIIE/FtsK family. These proteins employ an accessory DNA-binding domain (γ) to dictate directionality of DNA transport. It remains unclear how the interaction of γ with specific recognition sequences coordinates directional DNA translocation. We demonstrate that the γ domain of SpoIIIE inhibits ATPase activity of the motor domain in the absence of DNA but stimulates ATPase activity through sequence-specific DNA recognition. Furthermore, we observe that communication between γ subunits is necessary for both regulatory roles. Consistent with these findings, the γ domain is necessary for robust DNA transport along the length of the chromosome in vivo. Together, our data reveal that directional activation involves allosteric regulation of ATP turnover through coordinated action of γ domains. Thus, we propose a coordinated stimulation model in which γ-γ communication is required to translate DNA sequence information from each γ to its respective motor domain.

Keywords: ATPases; Allosteric Regulation; Chromosomes; DNA-Protein Interaction; Molecular Motors.

FIGURE 1.

γ domain modulates ATPase activity of SpoIIIE. A, soluble SpoIIIE exhibits DNA-dependent ATPase activity. ATP turnover was measured using an NADH⁺-coupled ATPase assay. The basal rate is 15 and the maximal rate is 800 ATP/s/molecule. Fits modeled using the Hill form of Michaelis-Menten equation as follows: 15 + m1·m0^m3/(m2 + m0^m3), where m1 is V_max, m2 is K_m, and m3 is the Hill coefficient. B, deletion of the γ domain results in an elevated basal rate at 80 ATP/s/molecule, which is not stimulated by DNA. C, ATPase activity of ATP concentration titration for SpoIIIE and SpoIIIE^Δγ in the presence of 20 μm gDNA. Inset shows curve at lower ATP concentrations. Fits modeled using the substrate activation form of the Michaelis-Menten equation (34): m1/(1 + m2/m0)·((1 + m3·m0/m4)/(1 + m0/m4)), where m1 is the V_max, m2 is the K_m, m3 is the efficiency of product formation, and m4 is the activation constant.

FIGURE 2.

γ domain confers sequence-specific ATPase stimulation. A, dsDNA fragments (3×SRS, 3×revSRS, random) were used to measure ATPase activity that is primarily generated by initial DNA binding and sequence recognition. B, γ domain only stimulates ATPase activity when binding to permissive SRS. On the x axis, 0 = no DNA; 22, 42, 61, and 80 = length of dsDNA fragment in base pairs; and g = gDNA. The data are presented in semi-log format (y axis is log scale). C, SpoIIIE is insensitive to nonpermissive SRS. ATPase experiments suggested that 3×revSRS dsDNA fragments do not stimulate ATPase activity above basal rate. To confirm, we compared ATPase stimulation by dsDNA fragments that contain SRS followed by revSRS. The 64-bp 3×SRS-3×revSRS (schematic). On the x axis, 1 = no DNA: 2 = gDNA; 3 = 64-bp 3×SRS-3×revSRS; 4 = 50-bp 3×SRS-1×revSRS; 5 = 50-bp 1×SRS-3×revSRS; 6 = 61-bp 1×SRS. Fits modeled using the Hill form of Michaelis-Menten equation: 15 + m1·m0^m3/(m2 + m0^m3), where m1 is V_max; m2 is K_m, and m3 is the Hill coefficient. D, K_m values for 3×SRS fragments are lower than for gDNA. ATPase activity measured during DNA titrations of 42-, 61-, and 80-bp 3×SRS dsDNA fragments. The x axis was scaled to emphasize the differences in V_max, which somewhat obscures the fact that all dependences presented here are sigmoidal. The data are presented in linear format (y axis is linear scale). Data are represented as mean ± S.E.

FIGURE 3.

γ domain exhibits higher DNA affinity for SRS-containing DNA. A, γ domain confers DNA binding and sequence specificity. Fluorescence polarization studies of SpoIIIE and SpoIIIE^Δγ with SRS and random DNA templates. 10 nm of f-labeled 2×SRS 30-bp dsDNA was used as binding template. Binding data were modeled using the Hill form of the Michaelis-Menten equation: m1·m0^m3/(m2 + m0^m3), where m1 is V_max; m2 is K_d, and m3 is the Hill coefficient. B, SpoIIIE preferentially binds to SRS over random DNA. After incubating 152 nm SpoIIIE with 10 nm of 30-bp f-2×SRS dsDNA fragment, we titrated in increasing concentrations of unlabeled 30-bp 2×SRS or random DNA. SpoIIIE binds tighter to 2×SRS than to random by a factor of 7.6. K_i of 2×SRS is 27.8 ± 15.8 nm, and random is 211.5 ± 41.1 nm. C, SpoIIIE^Δγ does not exhibit sequence specificity in binding. After incubating 1550 nm of SpoIIIE^Δγ with 10 nm of 30-bp fluorescein-2×SRS dsDNA fragment, we titrated in increasing concentrations of unlabeled 30-bp 2×SRS or random DNA. SpoIIIE^Δγ binds SRS and random sequences with the affinity. K_i of 2×SRS is 856.3 ± 93.3 nm, and random is 876.0 ± 131.7 nm. K_i values were calculated after modeling data using the following equation: K_i = [I]₅₀/([L]₅₀/K_d + [P]₀/K_d + 1), where [I]₅₀ denotes the concentration of the free inhibitor at 50% inhibition; [L]₅₀ is the concentration of the free labeled ligand at 50% inhibition; [P]₀ is the concentration of the free protein at 0% inhibition, and K_d is the dissociation constant of the protein-ligand complex. Data are represented as mean ± S.E.

FIGURE 4.

γ domain contribution to DNA binding is separable from sequence specificity. A, both SpoIIIE and SpoIIIE^Δγ accumulate a slow-migrating species. DNA binding of SpoIIIE, SpoIIIE^Δγ, and SpoIIIE^V429M was observed using EMSA on 4 nm of 42-bp 3×SRS dsDNA fragments. Free DNA is labeled with an arrow, and slow-migrating species are labeled with asterisks. B, quantification of the free DNA in EMSA shows that SpoIIIE has a K_d of 63.2 ± 11.7 nm, and SpoIIIE^Δγ has a K_d of 204.5 ± 37.6 nm for 3×SRS fragments. EMSAs were performed with 4 nm 42-bp 3×SRS dsDNA fragments. C, SpoIIIE preferentially binds to SRS. ATPase activity was stimulated by either 2 or 1 μm base pairs of 80-bp 3×SRS dsDNA fragments while titrating in 80-bp random dsDNA fragments. K_i values were calculated after modeling data using the following equation: K_i = [I]₅₀/([L]₅₀/K_d + [P]₀/K_d + 1), where [I]₅₀ denotes the concentration of the free inhibitor at 50% inhibition; [L]₅₀ is the concentration of the free ligand at 50% inhibition; [P]₀ is the concentration of the free protein at 0% inhibition, and K_d is the dissociation constant of the protein-ligand complex. Data are represented as mean ± S.E.

FIGURE 5.

Mutations in the putative γ subunit interface uncouple DNA binding from SRS recognition. A, sequence alignment of γ domains from FtsK in E. coli, FtsK in P. aeruginosa, and SpoIIIE in B. subtilis. Arrow points to conserved Arg residues (Arg-745 and Arg-756 in SpoIIIE). SpoIIIE sequence numbering is based on a revised sequence (YP_007533637.1). B, schematic of threaded structure of trimer of SpoIIIE γ domains onto γ domain structure from FtsK in P. aeruginosa (Protein Data Bank code 2VE9). Arg-745 and Arg-756 are shown in black. C, SpoIIIE^R745A, SpoIIIE^R745E, and SpoIIIE^R756E exhibit low stimulation of ATPase activity by gDNA, but 80-bp dsDNA fragments stimulate higher ATPase activity. All DNA templates were provided at 20 μm base pair concentration. Shorter templates stimulate higher ATPase activity of SpoIIIE^R745A (D) and SpoIIIE^R745E (E) than longer templates. An ATPase assay was initiated in the presence of 10 μm base pairs of gDNA, before 80-bp dsDNA fragments (3×SRS or random) were titrated in. On the x axis for D and E: 1 = no DNA; 2 = gDNA, no addition; 3 = gDNA, 20 μm of 80-bp dsDNA fragment; 4 = gDNA, 40 μm of 80-bp dsDNA fragment; 5 = gDNA, 100 μm of 80-bp dsDNA fragment. Data are represented as mean ± S.E.

FIGURE 6.

Perturbing the γ-γ interfaces results in tighter DNA binding and loss of sequence specificity. A, ATPase activity of ATP concentration titration for SpoIIIE^R745A and SpoIIIE^R745E compared with SpoIIIE and SpoIIIE^Δγ in the presence of 20 μm gDNA. The inset curve zooms into the lower concentrations, which are included in the full graph. Fits were modeled using substrate activation form of the Michaelis-Menton equation (34): m1/(1 + m2/m0) × ((1 + m3 × m0/m4)/(1 + m0/m4)), where m1 is the V_max, m2 is the K_m, m3 is the efficiency of product formation, and m4 is the activation constant. SpoIIIE and SpoIIIE^Δγ curves are the same as shown in Fig. 1C. B, SpoIIIE^R745A and SpoIIIE^R745E maintain the prevalence of the slowest migrating species by EMSA. EMSAs were performed with 4 nm of 42-bp 3×SRS dsDNA fragments. Free DNA labeled with an arrow and slow-migrating species labeled with asterisks. C, SpoIIIE^R745A binds DNA in a sequence-unspecific manner. Fluorescence anisotropy measurements with 10 nm of 30-bp 2×SRS and random dsDNA fragments show the same binding curves. Additionally, SpoIIIE^R745A binds DNA twice as tightly as SpoIIIE. D, SpoIIIE^R745E exhibits mild sequence specificity in DNA binding. Fluorescence anisotropy measurements were performed with 10 nm of 30-bp 2×SRS and random dsDNA fragments. Additionally, SpoIIIE^R745E binds DNA twice as tightly as SpoIIIE. Binding data were modeled using Hill form of the Michaelis-Menten equation as follows: m1·m0^m3/(m2 + m0^m3), where m1 is V_max; m2 is K_d, and m3 is the Hill coefficient. Data are represented as mean ± S.E.

FIGURE 7.

Coordinated activity of γ domains is important for maintaining robust chromosome transport in vivo. A, transport of several loci on the B. subtilis chromosome were analyzed using a quantitative two-color fluorescence microscopy assay (23). The genes for two fluorescent proteins (YFP and CFP) were placed under P_spoIIQ promoter, which is turned on immediately after septation by a forespore-specific transcription factor (σ^F). To identify cells that have completed septation, the yfp reporter was placed near the origin, which is found inside the forespore prior to septation. The cfp reporter was placed at different loci that were trapped in mother cell, which allowed us to track when the marked loci were transported into the forespore. B, apparent rate of transport by SpoIIIE of all chromosomal loci is essentially indistinguishable. >1500 cells were analyzed for each strain at each time point. Inset depicts circular B. subtilis chromosome with dotted lines designating 3 megabases of the chromosome that are trapped in the mother cell, and cfp-marked loci on right arm of the chromosome are indicated. C, SpoIIIE^Δγ exhibits a severe translocation defect along the entire length of the chromosome. At 3 h after induction of sporulation, only 5% of cells are able to complete chromosome transport. D, sample images taken of strains containing cfp integrated at 174° locus. Transport of this locus by cells with SpoIIIE and SpoIIIE^Δγ was monitored over time. Images depict transport at 3 h after sporulation synchronization. E, schematic representation of the coordinated stimulation model. Three types of DNA sequence are depicted as follows: random, permissive SRS, and nonpermissive SRS. Once bound to DNA, an SpoIIIE hexamer requires coordination between the γ domains to sense the identity of the DNA sequence. If the sequence is random, the γ domains will inhibit ATP turnover by the motor domains, keeping the motor domain in an ATPase off state. In this state, SpoIIIE translocates slowly along the DNA. Upon encounters with a permissive SRS, the γ domain coordination is necessary to sense the SRS and activate robust ATPase activity of the motor domain. This ATPase on state results in rapid and processive translocation along the DNA. A nonpermissive SRS is not recognized by the γ domain and thus does not result in an effect on the ATPase state of the motor domain. Mutations in the γ-γ subunit interface abrogate the ability of the γ domains to communicate with one another and thus prevent SpoIIIE from sensing specific sequences. Thus, a γ interface variant neither inhibits nor stimulates ATPase activity, and it translocates at the same rate regardless of the DNA sequence it encounters.