Molecular mechanism of sequence-directed DNA loading and translocation by FtsK - PubMed (original) (raw)
Molecular mechanism of sequence-directed DNA loading and translocation by FtsK
Jan Löwe et al. Mol Cell. 2008.
Abstract
Dimeric circular chromosomes, formed by recombination between monomer sisters, cannot be segregated to daughter cells at cell division. XerCD site-specific recombination at the Escherichia coli dif site converts these dimers to monomers in a reaction that requires the DNA translocase FtsK. Short DNA sequences, KOPS (GGGNAGGG), which are polarized toward dif in the chromosome, direct FtsK translocation. FtsK interacts with KOPS through a C-terminal winged helix domain gamma. The crystal structure of three FtsKgamma domains bound to 8 bp KOPS DNA demonstrates how three gamma domains recognize KOPS. Using covalently linked dimers of FtsK, we infer that three gamma domains per hexamer are sufficient to recognize KOPS and load FtsK and subsequently activate recombination at dif. During translocation, FtsK fails to recognize an inverted KOPS sequence. Therefore, we propose that KOPS act solely as a loading site for FtsK, resulting in a unidirectionally oriented hexameric motor upon DNA.
Figures
Figure 1. Crystal Structures of the FtsKγ Domain with and without DNA
(A) Cocrystallization of PaFtsKγ with the KOPS-containing DNA duplex 5′-ACCA
GGGCAGGG
CGAC-3′ (KOPS: GGGCAGGG) produced a structure containing three PaFtsKγ domains bound to double-stranded DNA. The asymmetric unit of these crystals contains two complete complexes. Protein chains A, B, and C are bound to the first duplex; chains D, E, and F are bound to the second. (B) In the complex, the winged helix domains insert with the loop between H2 and H3 into the major groove of the DNA. The wing, as is common for WHD domains, interacts with the minor groove of the DNA. (C) The three PaFtsKγ domains are arranged along the DNA to follow the major groove, leading to an arrangement in which they are ∼90 degrees apart when looking along the axis of the DNA (A to B: ∼100°, B to C: ∼80°). (D) When superimposing the PaFtsKγ-complexed DNA duplex with ideal B DNA, it becomes clear that the DNA is not, or is only very slightly, bent from straight. (E) The three PaFtsKγ subunits together recognize the GGGCAGGG KOPS motif. Chains A and C recognize a GGG triplet, whereas chain B slots in between the two and recognizes mostly the CA duplet, although very few direct contacts exist, explaining the lack of conservation of the middle bases of the KOPS canonical sequence. The binding mode for chains A and C is very similar, with a tight interaction of the wing with the minor groove. Chain B is tilted and binds in a slightly different way, as expected, because it recognizes a different DNA sequence despite being the same protein. (F) The recognition of different stretches of DNA by the same domain is possible because the three subunits also interact with themselves. They all bind in the same overall orientation to the DNA, though chain B is slightly tilted.
Figure 2. Model of the Motor and FtsKγ Domains onto DNA
(A) When modeling the KOPS:PaFtsKγ complex onto the motor domain (PaFtsK αβ)(Massey et al., 2006), it becomes clear that the γ domains do not follow the 60° rotational repeat of the motor domains. (B) Using the same model, a prediction can be made for the direction of the motor domain hexamer on KOPS-containing DNA. The model was generated by placing the N termini of the γ domains close to the C termini of the motor domains (Massey et al., 2006). The two parts of the molecules are joined by a linker of 14 amino acids in the full-length protein that are missing between the two structures. This then places the KOPS sequence in a way that the permissive direction of the KOPS sequence goes from left to right. This means that it is likely that the motor complex moves the DNA from the wider end of the cone to the narrower end where the N-terminal α domains are located, as is indicated. (C) An isothermal titration calorimetry (ITC) experiment shows that three PaFtsKγ domains bind to one KOPS, N = 0.37 (0.33 for 1:3). (D) Control ITC experiment using double-stranded DNA of the same length without KOPS. No binding is observed.
Figure 3. A Recombination Reaction Dependent upon FtsK Loading at KOPS
(A) Schematic diagram of recombination substrates. The dif site, present on both substrates, is shown as a gray box containing a triangle, and KOPS is shown as an arrow on the DNA that points in the permissive direction. Presence of a radioactive label is shown by the asterisk. Each longer substrate is named according to the order of its component parts. For example, the substrate shown in (A) is _dif_-5-KOPS-25, where 5 and 25 represent the length of duplex DNA (bp) in these positions. This nomenclature is used for all the subsequent substrates. In this substrate, the XerD binding site of dif is closest to KOPS. (B) Autoradiogram of a recombination gel. Each lane contained XerCD, FtsK50C, ATP, and the indicated substrates. The percent recombination shown below each lane is an average derived from two independent experiments using the same conditions. When the level of recombination was too low to quantify, it is indicated as ≤0.1%. (C) Recombination is dependent upon ATP. Recombination reactions carried out on the indicated substrates in the presence or absence of ATP as indicated. The smeary band seen in the _difrev_-5-KOPS-25 lane in (B) and the similar bands in the _dif_-2-KOPS-25 lanes in (C) are background bands and unrelated to recombination since they appear with these substrates in the absence of protein.
Figure 4. Three γ Domains in a Hexamer Are Sufficient for KOPS-Dependent Recombination
(A) Schematic representation of the covalent dimers of FtsK50C and their indicated nomenclature. (B) DNA binding of monomeric and covalent dimeric/trimeric FtsK50C derivatives. The substrate is shown schematically above the gel. The KOPS site is flanked by 1 bp to one side and by 35 bp on the other side, upon which FtsK could load. The complexes are putatively identified on the right; the smallest complex, present with FtsK50C but absent in the covalent dimer, is, thus, assigned to be a monomer bound to DNA. Similarly, the smallest complex with the covalent dimer, absent with the trimer, is assigned as a single dimer bound to DNA. The largest complex, common to all the proteins tested, is presumed to be a hexamer and is marked as such. There is no indication that the covalent multimers form higher-order complexes of different composition than monomeric FtsK50C. (C) Recombination activity of the covalent dimeric proteins. Each dimeric protein was active except the γ−/γ− variant. (D) Comparison of recombination efficiency on substrates of identical length, K, with KOPS, and N, without KOPS. This confirms that the dimeric proteins retain the dependence on KOPS for efficient recombination.
Figure 5. FtsK Cannot Recognize KOPS in the Reverse Direction
(A) Schematic representation of the substrates used. Nomenclature is as in Figure 3. In these substrates, a second (nonpermissive) KOPS is added between the dif site and the KOPS loading site. An “x” represents the distance in bp between dif and this additional KOPS. (B) Recombination reactions using substrates with a reversed KOPS. The control is _dif_-20-KOPS-25, used previously (Figure 3), which is a similar length to the other substrates here. The quantified percentage of recombination from each substrate is shown below the autoradiograph and is normalized to the control reaction. Multiple repeats of this experiment give the same trends. (C) Graphs showing recombination against time for the substrates shown in (B). The top panel compares the control substrate with the substrate having reversed KOPS 5 bp from dif. The lower panel shows the effect of varying this distance between dif and the reversed KOPS. Again, a representative set of data is shown, but repeats give similar trends. (D) Model for collision of FtsK hexamers following directional KOPS loading. Each hexamer is represented as a cone on the DNA, and, for clarity, only a single γ domain is shown as a connected triangle. Since γ binds KOPS in a specific orientation (Figure 1), recognition of an inverted KOPS would require the linker between the motor and γ to loop over the entire length of the γ-KOPS complex. This is disallowed (red cross). Thus, FtsK is never loaded in the “wrong” orientation, nor can it recognize an inverted KOPS during translocation. Translocating hexamers have a black arrow to show their movement relative to the DNA. Initially, both hexamers are loaded directionally at KOPS, and the yellow hexamer has begun translocation. In scheme (Di), both hexamers translocate and collide in the intervening DNA. In (Dii), one hexamer remains bound at KOPS. Both schemes could result in pausing of translocation upon collision. The various subsequent outcomes are then shown (Da)–(Dc), with (arbitrary) reference to the initially translocating hexamer: in (Da) and (Db), one motor pushes the other, whereas in (Dc), one motor dissociates and the other is free to translocate.
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