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. 2011;6(7):e21921.
doi: 10.1371/journal.pone.0021921. Epub 2011 Jul 8.

Crenarchaeal CdvA forms double-helical filaments containing DNA and interacts with ESCRT-III-like CdvB

Christine Moriscot  1 , Simonetta Gribaldo, Jean-Michel Jault, Mart Krupovic, Julie Arnaud, Marc Jamin, Guy Schoehn, Patrick Forterre, Winfried Weissenhorn, Patricia Renesto
Affiliations

Crenarchaeal CdvA forms double-helical filaments containing DNA and interacts with ESCRT-III-like CdvB

Christine Moriscot et al. PLoS One. 2011.

Abstract

Background: The phylum Crenarchaeota lacks the FtsZ cell division hallmark of bacteria and employs instead Cdv proteins. While CdvB and CdvC are homologues of the eukaryotic ESCRT-III and Vps4 proteins, implicated in membrane fission processes during multivesicular body biogenesis, cytokinesis and budding of some enveloped viruses, little is known about the structure and function of CdvA. Here, we report the biochemical and biophysical characterization of the three Cdv proteins from the hyperthermophilic archaeon Metallospherae sedula.

Methodology/principal findings: Using sucrose density gradient ultracentrifugation and negative staining electron microscopy, we evidenced for the first time that CdvA forms polymers in association with DNA, similar to known bacterial DNA partitioning proteins. We also observed that, in contrast to full-lengh CdvB that was purified as a monodisperse protein, the C-terminally deleted CdvB construct forms filamentous polymers, a phenomenon previously observed with eukaryotic ESCRT-III proteins. Based on size exclusion chromatography data combined with detection by multi-angle laser light scattering analysis, we demonstrated that CdvC assembles, in a nucleotide-independent way, as homopolymers resembling dodecamers and endowed with ATPase activity in vitro. The interactions between these putative cell division partners were further explored. Thus, besides confirming the previous observations that CdvB interacts with both CdvA and CdvC, our data demonstrate that CdvA/CdvB and CdvC/CdvB interactions are not mutually exclusive.

Conclusions/significance: Our data reinforce the concept that Cdv proteins are closely related to the eukaryotic ESCRT-III counterparts and suggest that the organization of the ESCRT-III machinery at the Crenarchaeal cell division septum is organized by CdvA an ancient cytoskeleton protein that might help to coordinate genome segregation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Purification of the Cdv proteins from M. sedula.
(A) Schematic representation of the proteins encoded by the cdv gene cluster from M. sedula. The black boxes correspond to protein-protein interaction motifs, i.e the CdvB-binding site of CdvA which interacts with the WH domain of CdvB and the MIM2 of CdvB that binds the MIT domain of CdvC , . (B) Recombinant full length CdvA from M. sedula was separated by SDS-PAGE (12%) and stained with Coomassie blue. Values on the left indicate molecular weights of the marker proteins. (C) Elution profile of CdvB from a Superdex 200 SEC column. The inset shows the SDS page band of purified CdvB. (D) The SEC profile (Superose 6) of CdvC (50 µM) and MALLS analysis; the inset shows the SDS-page band of CdvC. Protein elution from the column was monitored by refractometry (solid line) and the molar mass was determined by static light scattering (dots). Three different populations were observed. Pic 1: 530±50 kDa; Pic 2: 130±20 kDa; Pic 3: 40±5 kDa. (E) SEC elution profile (Superose 6) of CdvC (180 µM). Inset: Fractions corresponding to the peak separated by SDS-PAGE. The columns were standardized with the following molecular weights markers : blue dextran (2.106 kDa); tyroglobuline (670 kDa); ferritine (440 kDa); catalase (232 kDa); bovine serum albumin (67 kDa) and ribonuclease A (13.7 kDa).
Figure 2
Figure 2. CdvA forms helical filaments stabilized by DNA.
(A) Negative staining EM image of CdvA filaments (scale bar, 50 nm). (B, C) Magnifications of the areas marked by black squares in A (scale bars, 20 nm). (D) Negative staining EM micrograph of CdvA pretreated with DNase (scale bar, 50 nm). (E) SDS-PAGE of CdvA incubated for 30 min at 37°C with DNase (20 µg/ml), RNase (20 µg/ml), ATP-MgCl2 (1 mM) or ADP-MgCl2 (1 mM). (F) Protein distribution of the same samples after discontinuous sucrose density gradient centrifugation.
Figure 3
Figure 3. Interactions of M. sedula CdvA with CdvB or CdvC.
(A) CdvA and CdvB (10 µM each) were incubated overnight at 4°C and the protein distribution of the mixed sample was analyzed by sucrose density gradient centrifugation (three lower panels) in comparison to single proteins (two upper panels). The sucrose gradient contained either 50 mM or 1 M NaCl, as specified on the graph. In the lower panel, CdvA was pretreated with 20 µg/ml DNase before incubation with CdvB in presence of 50 mM NaCl. Proteins were separated on a 12% SDS-PAGE and stained with Commassie blue. Values on the left correspond to the molecular weights of the marker proteins. (B) Similar experiment as in A with CdvA or CdvC alone (upper and middle panels) or the preincubated protein mixture (lower panel) resolved on a sucrose gradient containing 50 mM NaCl. The images are representative of at least 3 different experiments carried out with different batches of purified proteins.
Figure 4
Figure 4. Structural analysis of M. sedula CdvB and CdvC homo and hetero-polymers.
(A) SEC elution profile (Superose 6) of CdvB and CdvC mixture (20 µM each). Aliquots of the column fractions were separated on 12% SDS-PAGE showing that the 7 ml void volume peak contained both proteins while the excess of CdvC was found in the 12 ml peak. (B) Negative staining EM image of the CdvB-CdvC polymers. (C) Negative staining EM image of the CdvB deletion mutant (CdvΔC). (D) Negative staining EM image of CdvC before incubation with CdvB. The insets in B and C correspond to higher magnification (×5).
Figure 5
Figure 5. Concomitant interaction of M. sedula CdvB with both CdvA and CdvC.
(A) CdvA, CdvB and CdvC (10 µM final concentration each) were incubated overnight at 4°C and the protein distribution of the mixed sample was analyzed by sucrose density gradient centrifugation. (B) The CdvA/CdvB complex was purified from the 60% sucrose fraction as shown in Figure 3 (panel A). This complex was then incubated with CdvC (10 µM final concentration) and analyzed by sucrose gradient centrifugation thus confirming results obtained when the 3 proteins were incubated together.
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