Mechanistic study of classical translocation-dead SpoIIIE36 reveals the functional importance of the hinge within the SpoIIIE motor

Abstract

SpoIIIE/FtsK ATPases are central players in bacterial chromosome segregation. It remains unclear how these DNA translocases harness chemical energy (ATP turnover) to perform mechanical work (DNA movement). Bacillus subtilis sporulation provides a dramatic example of intercompartmental DNA transport, in which SpoIIIE moves 70% of the chromosome across the division plane. To understand the mechanistic requirements for DNA translocation, we investigated the DNA translocation defect of a classical nontranslocating allele, spoIIIE36. We found that the translocation phenotype is caused by a single substitution, a change of valine to methionine at position 429 (V429M), within the motor of SpoIIIE. This substitution is located at the base of a hinge between the RecA-like β domain and the α domain, which is a domain unique to the SpoIIIE/FtsK family and currently has no known function. V429M interferes with both protein-DNA interactions and oligomer assembly. These mechanistic defects disrupt coordination between ATP turnover and DNA interaction, effectively uncoupling ATP hydrolysis from DNA movement. Our data provide the first functional evidence for the importance of the hinge in DNA translocation.

FIG 1

The V429M substitution is located at the base of the hinge in the SpoIIIE motor. (A) SpoIIIE/FtsK DNA translocases have a stereotyped domain structure, which is displayed in the cartoon at the top. A model of the SpoIIIE motor domain, obtained by threading the SpoIIIE sequence (residues 317 to 389) onto the FtsK crystal structure (15), is shown below on the left, and a cartoon representation of the homology model, showing a single SpoIIIE subunit with the V429M substitution displayed in red at the base of the hinge region, is on the right. The motor forms a hexameric ring large enough to encompass a single dsDNA molecule (manually docked and shown in black). The N terminus consists of multiple transmembrane regions (dark gray), which anchor the protein to the membrane and localize the translocase to the trapped DNA (46). An unstructured linker of unknown function follows the transmembrane domain. Following the linker is the motor that uses ATP hydrolysis to power DNA translocation. The motor consists of two domains, the α domain (green), which is unique to this group of translocases and whose function is unknown, and the RecA-like β domain (light gray), which hydrolyzes ATP. The α and β domains are connected by two unstructured strands, which make up the flexible hinge (blue). The ATP-binding active site, Walker A (purple), is physically positioned at the subunit interface of adjacent β domains 15 Å way from V429M (red). Lastly, at the very C terminus is the DNA-interacting γ domain (dark gray), which senses directionality sequences in the chromosome and couples them to ATP hydrolysis of the motor. (B) Fluorescence microscopy demonstrates that SpoIIIE^V429M-CFP localizes to the middle of the sporulating septum just like the wild-type protein. Fluorescently labeled SpoIIIE-GFP and SpoIIIE^V429M-CFP are shown in cyan, and membranes of sporulating B. subtilis are dyed with FM4-64 live stain and shown in red.

FIG 2

A small residue at the base of the hinge is conserved. An alignment of representatives of the SpoIIIE/FtsK family of translocases around the hinge region reveals that the residue at the base of the hinge (equivalent to V429) is a highly conserved small residue. Sequences are those of 22 representative members of the SpoIIIE/FtsK family from Pfam (PF01580). Seventeen of 22 aligned sequences have a Val, Ala, Leu, Ser, or Ile residue at the base of the hinge. The residue at the base of the hinge, equivalent to V429, is marked with an arrow. Sequences were anchored at the Walker A motif.

FIG 3

Perturbing the base of the hinge with a large residue abolishes DNA translocation in vivo. (A) DNA translocation of two loci in the B. subtilis chromosome was tracked using a quantitative two-color fluorescence microscopy assay (20). Fluorescent yfp and cfp reporters were integrated into the genome under the P_spoIIQ promoter, which is turned on immediately after septation by the forespore-specific σ^F. To identify cells that have completed septation, the yfp reporter was inserted near the origin (ori), which always starts inside the forespore at septation. The cfp reporter was placed at different loci that start out in the mother cell (either 174° [on a 360° circular chromosome], which is near the terminus [ter], or 90°, which starts close to the division septum), allowing us to track when these marked loci were transported into the forespore. (B) Sample images of B. subtilis cells with different variants of SpoIIIE at 3 h after sporulation initiation. (C) Quantification of the apparent rate of DNA transport of the terminus (174°). SpoIIIE^V429A cells exhibit wild-type rates of terminal translocation. In contrast, SpoIIIE^V429L cells are severely impaired in translocation and none of the SpoIIIE^V429M cells are able to transport the terminus. (D) Quantification of the apparent rate of DNA transport of the 90° locus. SpoIIIE^V429A cells exhibit wild-type rates of translocation. Nearly all of the SpoIIIE^V429L cells are capable of transporting the 90° locus but at a lower rate than the wild type. SpoIIIE^V429M cells failed to transport the 90° locus even after 3 h. More than 1,500 cells were quantified for each cell type at each time point in the experiment whose results are shown in panels C and D. The efficiency of DNA transport is expressed as a percentage (CFP/YFP).

FIG 4

SpoIIIE^V429M is impaired in intersubunit assembly and protein-DNA interactions. (A) SEC studies of SpoIIIE and SpoIIIE^V429M in the nucleotide-free (Apo) state and nucleotide-bound (AMP-PNP) state demonstrate that SpoIIIE^V429M is impaired in intersubunit interactions. While SpoIIIE assembles into dimers in vitro, SpoIIIE^V429M remains a monomer. (B) EMSA reveals that wild-type SpoIIIE shifts 4 nM 42-bp lead-SRS (3 overlapping SRS on the leading strand) dsDNA fragments better than SpoIIIE^V429M. Additionally, proportional to increased protein concentrations, SpoIIIE accumulates a slow-migrating species, while SpoIIIE^V429M does not. (C) Quantification of the unshifted DNA in the EMSA reveals that SpoIIIE^V429M exhibits lower DNA affinity than the wild type. The apparent K_d (dissociation constant) was 61 ± 15 nM for SpoIIIE and 291 ± 41 nM for SpoIIIE^V429M. The x axis, displaying the protein concentration, is in log scale. Binding data were modeled using the modified Hill form of the Michaelis-Menten equation as follows: m₁ × m₀^m₃/(m₂ + m₀^m₃), where m₀ is the concentration of DNA substrate used, m₁ is V_max, m₂ is K_d, and m₃ is the Hill coefficient. The y axis shows the amount of unshifted DNA divided by the total amount of DNA. Data are represented as means ± standard errors (SE).

FIG 5

The ATPase activity of SpoIIIE^V429M is preferentially stimulated by directionality sequences. (A) Short dsDNA fragments (lead-SRS, lag-SRS [on the lagging strand], and nonspecific) were used to measure ATPase stimulation (assayed by the NADH⁺-coupled ATPase assay) primarily in response to sequence recognition. Arrows indicate the direction in which SpoIIIE can bind and translocate based on the orientation of SRS. (B) Lead-SRS dsDNA fragments preferentially stimulate SpoIIIE^V429M ATPase activity to high levels, but lag-SRS and nonspecific DNA do not. The concentrations of dsDNA fragments used were 20 μM base pairs. Data are represented as means ± SE.

FIG 6

The V429M mutation abolishes stimulation of ATPase activity by long DNA substrates. (A) Unlike the wild type, soluble SpoIIIE^V429M does not exhibit robust DNA-dependent ATPase activity. ATPase rates both in the absence and presence of 20 μM base pairs of B. subtilis gDNA are ∼50 ATP/s/molecule of SpoIIIE^V429M. The ATPase rate of SpoIIIE^V429M is elevated 4-fold above the basal rate of the wild type, which is 12 ATP/s/molecule SpoIIIE. y axis is in log scale. (B) pUC19, a long substrate with only one naturally occurring SRS, does not stimulate ATPase activity of SpoIIIE^V429M. Titrating in increasing concentrations of lead-SRS dsDNA fragments results in increased ATPase activity of SpoIIIE^V429M, which demonstrates that SpoIIIE^V429M preferentially interacts with lead-SRS. pUC19 stimulates the ATPase activity of wild-type SpoIIIE to the same levels as gDNA does, as shown by the results in panel A. pUC19 was added at 10 μM (+). Three concentrations of lead-SRS were used, 10 μM, 20 μM, and 40 μM. (C) Comparison of the ATPase stimulation of wild-type SpoIIIE with that of SpoIIIE^V429M reveals that, unlike SpoIIIE, SpoIIIE^V429M is not stimulated to similar ATPase levels by lead-SRS dsDNA fragments and gDNA, in which lead-SRS is only found approximately every 10 to 12 kb. All data except the results for lead-SRS stimulation of wild-type SpoIIIE are regraphed from Fig. 5B and 6A. Data are represented as means ± SE.