- Email: [email protected]
The Hexameric Ring Structure of the Escherichia coli RuvB Branch Migration Protein
doi:10.1016/S0022-2836(02)00353-4 available online at http://www.idealibrary.com on
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J. Mol. Biol. (2002) 319, 587–591
COMMUNICATION
The Hexameric Ring Structure of the Escherichia coli RuvB Branch Migration Protein Yen-Ju Chen, Xiong Yu and Edward H. Egelman* Department of Biochemistry and Molecular Genetics University of Virginia Health Sciences, Box 800733 Charlottesville, VA 22908-0733 USA
The RuvB protein is part of the homologous recombination machinery in prokaryotic cells. Many studies have shown that RuvB is organized into hexameric rings functioning as DNA pumps at Holliday junctions, using ATP hydrolysis to drive branch migration. Structures now exist for two RuvB proteins, as well as for several structurally homologous proteins, including the replication factor-C small subunit (RFCS). Two models for the possible hexameric organization of RuvB subunits have been proposed, based upon the hexameric structures of NSF and HslU, two AAA-ATPases involved in vesicle fusion and proteolysis, respectively. We have used electron microscopy to generate an improved three-dimensional reconstruction of the double hexamers formed by Escherichia coli RuvB on double-stranded DNA. We find that an atomic model of the hexameric RFCS provides a significantly better fit to the RuvB hexamer than do the models for RuvB generated from NSF and HslU. This suggests that there may be a highly conserved structure for many proteins involved in different aspects of DNA replication, recombination, transcription and repair. q 2002 Elsevier Science Ltd. All rights reserved
*Corresponding author
Keywords: Holliday junction; helicase; electron microscopy; image analysis; molecular modeling
RuvB participates in homologous recombination by promoting Holliday junction branch migration.1 Cooperating with RuvA, RuvB uses the energy derived from ATP hydrolysis to pump DNA.2,3 Originally, RuvB was identified as a DNA helicase based on in vitro activity and sequence comparison.4 Subsequent sequence analysis suggested that RuvB belongs to the AAA þ family (ATPases associated with various cellular activities) rather than the helicase family.5 Electron microscopy and image analysis were used to show that RuvB forms a hexameric ring, similar in morphology to those formed by hexameric helicases.6 These rings form around doublestranded DNA, and two RuvB rings are separated by RuvA and oriented in opposite directions on a Holliday junction.7 Crystal structures of RuvB from Thermos thermophilus HB88 and Thermotoga maritima9 have been Abbreviations used: RFCS, replication factor-C small subunit; AAAþ family, ATPases associated with various cellular activities; EM, electron microscope. E-mail address of the corresponding author: [email protected]
solved. However, the packing within the RuvB hexamer is still unknown. Two different models of hexameric RuvB packing based on the ATPase domain of the NSF and HslU, two AAA-ATPases involved in vesicle fusion and proteolysis, respectively, have been proposed.9,10 We have used the constraint of an improved low-resolution reconstruction of Escherichia coli RuvB to generate a model for the hexameric packing of the existing RuvB crystal structures. Electron microscope (EM) reconstruction and model fitting In the presence of double-stranded DNA and ATPgS, RuvB forms double rings that encircle the DNA (Figure 1). From such images of negativelystained samples, 8463 RuvB double-rings were extracted manually. Three-dimensional reconstructions of E. coli rho,10 a hexameric transcription termination helicase, and bacteriophage T7 gp4,11 a hexameric replicative helicase/primase, were used separately as starting models. These initial models were used to determine the relative orientation of the 8463 projections, and this information
0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved
588
Structure of RuvB Hexamer
Figure 1. Electron micrograph of E. coli RuvB bound to dsDNA in the presence of ATPgS. The specimens were stained with 2% (w/v) uranyl acetate and imaged with minimal dose, at a magnification of 30,000£, in a JEOL 1200 EXII electron microscope. The scale bar represents ˚. 1000 A
was used to generate new three-dimensional reconstructions. By iterating this process, the reconstructions can diverge considerably from the starting models. Examples are shown in Figure 2(e) – (h) of how images may be averaged into characteristic classes after their relative orientations have been determined using the 3D reconstruction as a reference. A previous lowerresolution RuvB reconstruction6 could be used as a starting model, but it is better to use unrelated structures in order to avoid biasing the result. Using E. coli rho and bacteriophage T7 gp4 as starting models, our reconstruction converged to the same structure without the potential bias that might have resulted from the use of the earlier RuvB reconstruction. Previous EM analysis showed that the homologous T. thermophilus RuvB protein forms a heptamer in the absence of DNA and a hexamer when bound to DNA.12 To avoid any such polymorphism that might be present with the E. coli protein, only double rings bound to DNA were analyzed. D6 symmetry (two C6 rings that are oriented back-to-back) was imposed each cycle. After 40 cycles of 3D reconstruction and projection matching, the reconstructions converged to the same solution, and were therefore independent of the two starting models (Figure 3). The ˚ (as resolution of the reconstruction is , 22 A measured by the criterion of the Fourier shell ˚ correlation falling to 0.5). Each hexamer is , 115 A ˚ in diameter and extends , 70 A along the 6-fold ˚ when axis. The diameter of central hole is , 30 A
Figure 2. (a)– (d) Comparison of the projections of the reconstruction with (e) – (h) the corresponding referencefree averages24for subsets of images. The tilt angles between the 6-fold axis of the double hexamers and the normal of the plane of projection are: (a) and (e) 67.58; (b) and (f) 758; (c) and (g) 82.58; and (d) and (h) 908. The number of images used in reference-free averages are (e) 336, (f) 872, (g) 281 and (h) 59. The scale bar ˚ in (e). (i) The distribution of doublerepresents 100 A hexamer images based upon Eulerian angles u and w, where u is the tilt of the 6-fold axis from the normal to the plane of projection, and the third Eulerian angle is the rotation in the plane. Thus, u ¼ 908 would correspond to the long axis of the double-hexamer lying in the plane of projection. In this convention, w is the rotation about the 6-fold axis. The size of each circle is proportional to the number of images corresponding to each specific tilt and rotation angle. The largest circle contains 872 images at 758 tilt (u) and 08 rotation (w). Each filled circle contains less than 100 images. Because of the 6-fold symmertry, every point in this plot generates five additional points in the full angular plot.
the surface threshold is chosen to enclose the expected molecular volume. This reconstruction has been improved significantly from our earlier effort for several reasons.6 First, more than 8000 images have been used in this reconstruction, while the original reconstruction involved only 434 images. Second, the original reconstruction was generated from only two views (generating 12 views after the imposition of 6-fold symmetry), while the present reconstruction involves 50 views (and 300 views after the
589
Structure of RuvB Hexamer
Figure 3. (a) Surface of the 3D reconstruction of E. coli RuvB and (b) a cut-away view of the double-ring. The surface has been chosen so as to include 100% of the expected molecular volume, assuming a partial specific volume for protein of 0.75 cm3/g. (c) The crystal structure of Thermos thermophilus RuvB has been fit into the reconstruction, and the three domains of the subunit are labeled. Residues identified by mutagenesis that were proposed to be the in different interfaces are colored: red for DNA-binding (Arg300, Gly301, and Arg302), yellow for neighboring subunit contact (Glu115 and Arg158), green for RuvA-binding (Gly124, Gly126, Ile132, and Ile134). Three contour plots of RuvB are cut ˚ apart, perpendicular to the 6-fold axes and spaced 16 A and the positions of these sections are indicated by the three short bars in (c). The reconstruction was generated using the SPIDER software package.25
imposition of symmetry). Third, the assumption was made previously that the 6-fold axis of the double rings was always exactly in the plane of the micrographs. The present analysis (Figure 2(i)) shows that the most probable angle found for this axis is , 158 out of this plane. Nevertheless, the new reconstruction is in very good agreement with the general features observed originally. More importantly, the new reconstruction provides an excellent match with the actual averaged projections (Figure 2(a) –(h)), has higher resolution than the original reconstruction, and greatly increases
our confidence in using this reconstruction to build a pseudo-atomic model of the hexamer. In the two RuvB crystal structures, the subunit is monomeric. Superimposing the T. thermophilus HB8 RuvB structure8 on the structure of the homologous protein from T. maritima9 shows that the main difference between these two involves a small rotation of the C-terminal domain III (Figure 4(a)). We have used these two crystal structures to build two slightly different hexameric models constrained by our reconstruction (Figure 4(b), only T. thermophilus shown). In each case, the best fit was chosen by the match between the model and the density of the EM reconstruction, the extent to which the model extended outside the reconstruction surface, and the amount of steric clashes between subunits. Both models are quite different from the proposed models8,9 based on the structures of the hexameric ATPase domains of HslU13 and NSF.14 Comparing these models, both of our models show that the angle of the subunit with respect to the 6-fold axis of the ring is in between the HslU and NSF models, and provides a better fit to the 3D reconstruction. In particular, the surface of the reconstruction displays a prominent ridge that is now occupied by the subunit, but would not be in the other two models. The overall packing is also tighter than in the other two models. Our structure occupies the whole volume of the reconstruction and only a few flexible loops extend outside the surface. Since it is possible that these loops will be disordered in the hexamer, they would not generate surface features in the reconstruction. The crystal structure of another AAAþ protein, the replication factor-C small subunit (RFCS) of Pyrococcus furiosus, has been solved,15 and a hexameric model built based upon the constraint of a 3D EM reconstruction.15,16 Sequence analysis shows that RFCS is more distant from RuvB than is HslU, but the crystal structure of RFCS is more similar to the RuvB structure (z-score17 ¼ 15.6) than the structure of HslU is (z-score ¼ 13.3). The main difference between HslU and RFCS is the slight rotation of domain III (Figure 4(a)), but this difference affects the model fitting significantly. Based on the hexameric RFCS structure, we can build another RuvB packing model by superimposing the RuvB crystal structure onto the hexameric RFCS model (Figure 4(c)), and it is more similar to our model based on the EM reconstruction than it is to the models based on either the NSF or HslU hexamer. Locating the interface Using data obtained from mutational analysis,18,19 we can identify residues at the interface between RuvB and RuvA, between RuvB subunits, and between RuvB and DNA, in our RuvB hexameric models. For both the T. thermophilus and T. maritima RuvB, the RuvB – RuvA interface is at the extended loop (119 – 134) of domain I (all
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Figure 4. (a) Comparison of Thermus thermophilus RuvB (blue), Thermotoga maritima (red) and Pyrococcus furiosus RFCS (yellow). (b) Hexameric packing of T. maritima RuvB fit into the 3D recontruction of E. coli RuvB, and (c) the hexameric model of T. maritima RuvB based on the replication factor small subunit (RFCS) of P. Furiosus.
sequence numbers refer to T. thermophilus RuvB). Mutations in this loop inhibit cell growth or make the strain more sensitive to UV radiation.18 The I150T mutation in E. coli RuvB, which corresponds to Leu134 in T. thermophilus RuvB, disrupts the complex between RuvB and RuvA. Our model shows that this loop is at the bottom of the hexameric structure (Figure 3(c), green), at the interface between the rings in the double hexamer, and would be at the side of the hexamer that faces RuvA.7 This orientation has been proposed in the other models.8,9 The mutations G140D, G142D, I148T and I150T in E. coli RuvB (corresponding to Gly124, Gly126, Ile132 and Ile134 in T. thermophilus RuvB) reduce colony growth,18 suggesting that the interaction between RuvB and RuvA is a hydrophobic interaction. This suggests that the RuvB double-hexamers are held together by similar hydrophobic interactions. Glu131 and Arg174 in E. coli RuvB (corresponding to Glu115 and Arg158 in T. thermophilus RuvB) are described as interacting with the ATP molecule bound to a neighbor-
Structure of RuvB Hexamer
ing subunit.9,18 Our model supports this idea that these two residues of neighboring subunits face the nucleotide binding pocket and form the protein –ATP – protein sandwich (Figure 3(c), yellow). The RuvB subunits of T. maritima in the crystal with ADP form a helix rather than a hexamer,9 consistent with the observation that RuvB hexamer formation requires ATP binding.6 But it is not a general property of hexameric helicases, since, for example, the RepA protein can form a ring without any cofactors.20 The putative DNA-binding sites are in domain III and motif II in domain II.18 Domain III is structurally similar to so-called winged helix DNA-binding proteins.9,21 Winged helix proteins use either the H3 helix (DtxR) or W1 wing (RFX1) for DNA binding.21,22 Our model suggests that RuvB uses the same W1 wing as RFX1 for binding DNA, because this loop is closer to the central hole than is the H3 helix (Figure 3(c), red). Mutations on this wing in RuvB (R316H, G317E, R318C in E. coli RuvB, corresponding to Arg300, Gly301, Arg302 in T. thermophilus RuvB) show that it plays an important role in DNA binding.18 The L268S mutation in E. coli RuvB (corresponding to Ile252 in T. thermophilus RuvB) has an interesting phenotype: it behaves like wild type E. coli RuvB in vitro, but confers UV-sensitivity in a dominant negative manner in vivo.23 Mutating this residue to alanine (L268A) results in a moderate dominant negative phonotype.18 In the crystal structures, this residue is in domain III and close to the loop between domains III and II. It faces the center of domain III, which would prevent it from interacting with other proteins. In our model, it is not near either the subunit – subunit interface, or the RuvA or DNA interfaces. Therefore, our model does not explain the effect of these mutations and future studies will hopefully address possible allosteric effects involving this residue. The D113N mutation in E. coli RuvB (corresponding to Asp97 in T. thermophilus RuvB) in motif II affects both DNA binding and ATP hydrolysis, but not hexamer formation.19 In our model, the corresponding Asp97 is far from the central hole for DNA binding. Therefore, it implies that DNA binding in the central channel may be coupled allosterically with ATP hydrolysis near the ATP-binding pocket.
Acknowledgments We thank Margaret S. VanLoock for helpful discussions, and Steve West for the gift of RuvB protein. This research was supported by NIH GM35269 (to E.H.E.).
References 1. Parsons, C. A., Stasiak, A., Bennett, R. J. & West, S. C. (1995). Structure of a multisubunit complex that
Structure of RuvB Hexamer
2.
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9.
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promotes DNA branch migration. Nature, 374, 375–378. Shiba, T., Iwasaki, H., Nakata, A. & Shinagawa, H. (1991). SOS-inducible DNA repair proteins, RuvA and RuvB, of Escherichia coli: functional interactions between RuvA and RuvB for ATP hydrolysis and renaturation of the cruciform structure in supercoiled DNA. Proc. Natl Acad. Sci. USA, 88, 8445–8449. Tsaneva, I. R., Muller, B. & West, S. C. (1992). ATP-dependent branch migration of Holliday junctions promoted by the RuvA and RuvB proteins of E. coli. Cell, 69, 1171– 1180. Tsaneva, I. R., Muller, B. & West, S. C. (1993). RuvA and RuvB proteins of Escherichia coli exhibit DNA helicase activity in vitro. Proc. Natl Acad. Sci. USA, 90, 1315– 1319. Neuwald, A. F., Aravind, L., Spouge, J. L. & Koonin, E. V. (1999). AAAþ a class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res. 9, 27 –43. Stasiak, A., Tsaneva, I. R., West, S. C., Benson, C. J., Yu, X. & Egelman, E. H. (1994). The Escherichia coli RuvB branch migration protein forms double hexameric rings around DNA. Proc. Natl Acad. Sci. USA, 91, 7618– 7622. Yu, X., West, S. C. & Egelman, E. H. (1997). Structure and subunit composition of the RuvAB– Holliday junction complex. J. Mol. Biol. 266, 217– 222. Yamada, K., Kunishima, N., Mayanagi, K., Ohnishi, T., Nishino, T., Iwasaki, H. et al. (2001). Crystal structure of the Holliday junction migration motor protein RuvB from Thermus thermophilus HB8. Proc. Natl Acad. Sci. USA, 98, 1442– 1447. Putnam, C. D., Clancy, S. B., Tsuruta, H., Gonzalez, S., Wetmur, J. G. & Tainer, J. A. (2001). Structure and mechanism of the RuvB Holliday junction branch migration motor. J. Mol. Biol. 311, 297–310. Yu, X., Horiguchi, T., Shigesada, K. & Egelman, E. H. (2000). Three-dimensional reconstruction of transcription termination factor rho: orientation of the N-terminal domain and visualization of an RNAbinding site. J. Mol. Biol. 299, 1279– 1287. VanLoock, M. S., Chen, Y. J., Yu, X., Patel, S. S. & Egelman, E. H. (2001). The primase active site is on the outside of the hexameric bacteriophage T7 gene 4 helicase-primase ring. J. Mol. Biol. 311, 951– 956. Miyata, T., Yamada, K., Iwasaki, H., Shinagawa, H., Morikawa, K. & Mayanagi, K. (2000). Two different oligomeric states of the RuvB branch migration motor protein as revealed by electron microscopy. J. Struct. Biol. 131, 83– 89.
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13. Bochtler, M., Hartmann, C., Song, H. K., Bourenkov, G. P., Bartunik, H. D. & Huber, R. (2000). The structures of HsIU and the ATP-dependent protease HsIU-HsIV. Nature, 403, 800– 805. 14. Lenzen, C. U., Steinmann, D., Whiteheart, S. W. & Weis, W. I. (1998). Crystal structure of the hexamerization domain of N-ethylmaleimide-sensitive fusion protein. Cell, 94, 525– 536. 15. Oyama, T., Ishino, Y., Cann, I. K., Ishino, S. & Morikawa, K. (2001). Atomic structure of the clamp loader small subunit from Pyrococcus furiosus. Mol. Cell, 8, 455– 463. 16. Mayanagi, K., Miyata, T., Oyama, T., Ishino, Y. & Morikawa, K. (2001). Three-dimensional electron microscopy of the clamp loader small subunit from Pyrococcus furiosus. J. Struct. Biol. 134, 35 – 45. 17. Holm, L. & Sander, C. (1996). Mapping the protein universe. Science, 273, 595–603. 18. Iwasaki, H., Han, Y. W., Okamoto, T., Ohnishi, T., Yoshikawa, M., Yamada, K. et al. (2000). Mutational analysis of the functional motifs of RuvB, an AAAþ class helicase and motor protein for Holliday junction branch migration. Mol. Microbiol. 36, 528– 538. 19. Mezard, C., Davies, A. A., Stasiak, A. & West, S. C. (1997). Biochemical properties of RuvBD113N: a mutation in helicase motif II of the RuvB hexamer affects DNA binding and ATPase activities. J. Mol. Biol. 271, 704–717. 20. Scherzinger, E., Ziegelin, G., Barcena, M., Carazo, J. M., Lurz, R. & Lanka, E. (1997). The RepA protein of plasmid RSF1010 is a replicative DNA helicase. J. Biol. Chem. 272, 30228– 30236. 21. Gajiwala, K. S. & Burley, S. K. (2000). Winged helix proteins. Curr. Opin. Struct. Biol. 10, 110 – 116. 22. Emery, P., Strubin, M., Hofmann, K., Bucher, P., Mach, B. & Reith, W. (1996). A consensus motif in the RFX DNA binding domain and binding domain mutants with altered specificity. Mol. Cell Biol. 16, 4486 –4494. 23. Mezard, C., George, H., Davies, A. A., van Gool, A. J., Zerbib, D., Stasiak, A. & West, S. C. (1999). Escherichia coli RuvBL268S: a mutant RuvB protein that exhibits wild-type activities in vitro but confers a UV-sensitive ruv phenotype in vivo. Nucl. Acids Res. 27, 1275 –1282. 24. Penczek, P., Radermacher, M. & Frank, J. (1992). Three-dimensional reconstruction of single particles embedded in ice. Ultramicroscopy, 40, 33 – 53. 25. Frank, J., Radermacher, M., Penczek, P., Zhu, J., Li, Y., Ladjadj, M. & Leith, A. (1996). SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190 –199.
Edited by W. Baumeister (Received 24 January 2002; received in revised form 3 April 2002; accepted 4 April 2002)
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J. Mol. Biol. (2002) 319, 587–591
COMMUNICATION
The Hexameric Ring Structure of the Escherichia coli RuvB Branch Migration Protein Yen-Ju Chen, Xiong Yu and Edward H. Egelman* Department of Biochemistry and Molecular Genetics University of Virginia Health Sciences, Box 800733 Charlottesville, VA 22908-0733 USA
The RuvB protein is part of the homologous recombination machinery in prokaryotic cells. Many studies have shown that RuvB is organized into hexameric rings functioning as DNA pumps at Holliday junctions, using ATP hydrolysis to drive branch migration. Structures now exist for two RuvB proteins, as well as for several structurally homologous proteins, including the replication factor-C small subunit (RFCS). Two models for the possible hexameric organization of RuvB subunits have been proposed, based upon the hexameric structures of NSF and HslU, two AAA-ATPases involved in vesicle fusion and proteolysis, respectively. We have used electron microscopy to generate an improved three-dimensional reconstruction of the double hexamers formed by Escherichia coli RuvB on double-stranded DNA. We find that an atomic model of the hexameric RFCS provides a significantly better fit to the RuvB hexamer than do the models for RuvB generated from NSF and HslU. This suggests that there may be a highly conserved structure for many proteins involved in different aspects of DNA replication, recombination, transcription and repair. q 2002 Elsevier Science Ltd. All rights reserved
*Corresponding author
Keywords: Holliday junction; helicase; electron microscopy; image analysis; molecular modeling
RuvB participates in homologous recombination by promoting Holliday junction branch migration.1 Cooperating with RuvA, RuvB uses the energy derived from ATP hydrolysis to pump DNA.2,3 Originally, RuvB was identified as a DNA helicase based on in vitro activity and sequence comparison.4 Subsequent sequence analysis suggested that RuvB belongs to the AAA þ family (ATPases associated with various cellular activities) rather than the helicase family.5 Electron microscopy and image analysis were used to show that RuvB forms a hexameric ring, similar in morphology to those formed by hexameric helicases.6 These rings form around doublestranded DNA, and two RuvB rings are separated by RuvA and oriented in opposite directions on a Holliday junction.7 Crystal structures of RuvB from Thermos thermophilus HB88 and Thermotoga maritima9 have been Abbreviations used: RFCS, replication factor-C small subunit; AAAþ family, ATPases associated with various cellular activities; EM, electron microscope. E-mail address of the corresponding author: [email protected]
solved. However, the packing within the RuvB hexamer is still unknown. Two different models of hexameric RuvB packing based on the ATPase domain of the NSF and HslU, two AAA-ATPases involved in vesicle fusion and proteolysis, respectively, have been proposed.9,10 We have used the constraint of an improved low-resolution reconstruction of Escherichia coli RuvB to generate a model for the hexameric packing of the existing RuvB crystal structures. Electron microscope (EM) reconstruction and model fitting In the presence of double-stranded DNA and ATPgS, RuvB forms double rings that encircle the DNA (Figure 1). From such images of negativelystained samples, 8463 RuvB double-rings were extracted manually. Three-dimensional reconstructions of E. coli rho,10 a hexameric transcription termination helicase, and bacteriophage T7 gp4,11 a hexameric replicative helicase/primase, were used separately as starting models. These initial models were used to determine the relative orientation of the 8463 projections, and this information
0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved
588
Structure of RuvB Hexamer
Figure 1. Electron micrograph of E. coli RuvB bound to dsDNA in the presence of ATPgS. The specimens were stained with 2% (w/v) uranyl acetate and imaged with minimal dose, at a magnification of 30,000£, in a JEOL 1200 EXII electron microscope. The scale bar represents ˚. 1000 A
was used to generate new three-dimensional reconstructions. By iterating this process, the reconstructions can diverge considerably from the starting models. Examples are shown in Figure 2(e) – (h) of how images may be averaged into characteristic classes after their relative orientations have been determined using the 3D reconstruction as a reference. A previous lowerresolution RuvB reconstruction6 could be used as a starting model, but it is better to use unrelated structures in order to avoid biasing the result. Using E. coli rho and bacteriophage T7 gp4 as starting models, our reconstruction converged to the same structure without the potential bias that might have resulted from the use of the earlier RuvB reconstruction. Previous EM analysis showed that the homologous T. thermophilus RuvB protein forms a heptamer in the absence of DNA and a hexamer when bound to DNA.12 To avoid any such polymorphism that might be present with the E. coli protein, only double rings bound to DNA were analyzed. D6 symmetry (two C6 rings that are oriented back-to-back) was imposed each cycle. After 40 cycles of 3D reconstruction and projection matching, the reconstructions converged to the same solution, and were therefore independent of the two starting models (Figure 3). The ˚ (as resolution of the reconstruction is , 22 A measured by the criterion of the Fourier shell ˚ correlation falling to 0.5). Each hexamer is , 115 A ˚ in diameter and extends , 70 A along the 6-fold ˚ when axis. The diameter of central hole is , 30 A
Figure 2. (a)– (d) Comparison of the projections of the reconstruction with (e) – (h) the corresponding referencefree averages24for subsets of images. The tilt angles between the 6-fold axis of the double hexamers and the normal of the plane of projection are: (a) and (e) 67.58; (b) and (f) 758; (c) and (g) 82.58; and (d) and (h) 908. The number of images used in reference-free averages are (e) 336, (f) 872, (g) 281 and (h) 59. The scale bar ˚ in (e). (i) The distribution of doublerepresents 100 A hexamer images based upon Eulerian angles u and w, where u is the tilt of the 6-fold axis from the normal to the plane of projection, and the third Eulerian angle is the rotation in the plane. Thus, u ¼ 908 would correspond to the long axis of the double-hexamer lying in the plane of projection. In this convention, w is the rotation about the 6-fold axis. The size of each circle is proportional to the number of images corresponding to each specific tilt and rotation angle. The largest circle contains 872 images at 758 tilt (u) and 08 rotation (w). Each filled circle contains less than 100 images. Because of the 6-fold symmertry, every point in this plot generates five additional points in the full angular plot.
the surface threshold is chosen to enclose the expected molecular volume. This reconstruction has been improved significantly from our earlier effort for several reasons.6 First, more than 8000 images have been used in this reconstruction, while the original reconstruction involved only 434 images. Second, the original reconstruction was generated from only two views (generating 12 views after the imposition of 6-fold symmetry), while the present reconstruction involves 50 views (and 300 views after the
589
Structure of RuvB Hexamer
Figure 3. (a) Surface of the 3D reconstruction of E. coli RuvB and (b) a cut-away view of the double-ring. The surface has been chosen so as to include 100% of the expected molecular volume, assuming a partial specific volume for protein of 0.75 cm3/g. (c) The crystal structure of Thermos thermophilus RuvB has been fit into the reconstruction, and the three domains of the subunit are labeled. Residues identified by mutagenesis that were proposed to be the in different interfaces are colored: red for DNA-binding (Arg300, Gly301, and Arg302), yellow for neighboring subunit contact (Glu115 and Arg158), green for RuvA-binding (Gly124, Gly126, Ile132, and Ile134). Three contour plots of RuvB are cut ˚ apart, perpendicular to the 6-fold axes and spaced 16 A and the positions of these sections are indicated by the three short bars in (c). The reconstruction was generated using the SPIDER software package.25
imposition of symmetry). Third, the assumption was made previously that the 6-fold axis of the double rings was always exactly in the plane of the micrographs. The present analysis (Figure 2(i)) shows that the most probable angle found for this axis is , 158 out of this plane. Nevertheless, the new reconstruction is in very good agreement with the general features observed originally. More importantly, the new reconstruction provides an excellent match with the actual averaged projections (Figure 2(a) –(h)), has higher resolution than the original reconstruction, and greatly increases
our confidence in using this reconstruction to build a pseudo-atomic model of the hexamer. In the two RuvB crystal structures, the subunit is monomeric. Superimposing the T. thermophilus HB8 RuvB structure8 on the structure of the homologous protein from T. maritima9 shows that the main difference between these two involves a small rotation of the C-terminal domain III (Figure 4(a)). We have used these two crystal structures to build two slightly different hexameric models constrained by our reconstruction (Figure 4(b), only T. thermophilus shown). In each case, the best fit was chosen by the match between the model and the density of the EM reconstruction, the extent to which the model extended outside the reconstruction surface, and the amount of steric clashes between subunits. Both models are quite different from the proposed models8,9 based on the structures of the hexameric ATPase domains of HslU13 and NSF.14 Comparing these models, both of our models show that the angle of the subunit with respect to the 6-fold axis of the ring is in between the HslU and NSF models, and provides a better fit to the 3D reconstruction. In particular, the surface of the reconstruction displays a prominent ridge that is now occupied by the subunit, but would not be in the other two models. The overall packing is also tighter than in the other two models. Our structure occupies the whole volume of the reconstruction and only a few flexible loops extend outside the surface. Since it is possible that these loops will be disordered in the hexamer, they would not generate surface features in the reconstruction. The crystal structure of another AAAþ protein, the replication factor-C small subunit (RFCS) of Pyrococcus furiosus, has been solved,15 and a hexameric model built based upon the constraint of a 3D EM reconstruction.15,16 Sequence analysis shows that RFCS is more distant from RuvB than is HslU, but the crystal structure of RFCS is more similar to the RuvB structure (z-score17 ¼ 15.6) than the structure of HslU is (z-score ¼ 13.3). The main difference between HslU and RFCS is the slight rotation of domain III (Figure 4(a)), but this difference affects the model fitting significantly. Based on the hexameric RFCS structure, we can build another RuvB packing model by superimposing the RuvB crystal structure onto the hexameric RFCS model (Figure 4(c)), and it is more similar to our model based on the EM reconstruction than it is to the models based on either the NSF or HslU hexamer. Locating the interface Using data obtained from mutational analysis,18,19 we can identify residues at the interface between RuvB and RuvA, between RuvB subunits, and between RuvB and DNA, in our RuvB hexameric models. For both the T. thermophilus and T. maritima RuvB, the RuvB – RuvA interface is at the extended loop (119 – 134) of domain I (all
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Figure 4. (a) Comparison of Thermus thermophilus RuvB (blue), Thermotoga maritima (red) and Pyrococcus furiosus RFCS (yellow). (b) Hexameric packing of T. maritima RuvB fit into the 3D recontruction of E. coli RuvB, and (c) the hexameric model of T. maritima RuvB based on the replication factor small subunit (RFCS) of P. Furiosus.
sequence numbers refer to T. thermophilus RuvB). Mutations in this loop inhibit cell growth or make the strain more sensitive to UV radiation.18 The I150T mutation in E. coli RuvB, which corresponds to Leu134 in T. thermophilus RuvB, disrupts the complex between RuvB and RuvA. Our model shows that this loop is at the bottom of the hexameric structure (Figure 3(c), green), at the interface between the rings in the double hexamer, and would be at the side of the hexamer that faces RuvA.7 This orientation has been proposed in the other models.8,9 The mutations G140D, G142D, I148T and I150T in E. coli RuvB (corresponding to Gly124, Gly126, Ile132 and Ile134 in T. thermophilus RuvB) reduce colony growth,18 suggesting that the interaction between RuvB and RuvA is a hydrophobic interaction. This suggests that the RuvB double-hexamers are held together by similar hydrophobic interactions. Glu131 and Arg174 in E. coli RuvB (corresponding to Glu115 and Arg158 in T. thermophilus RuvB) are described as interacting with the ATP molecule bound to a neighbor-
Structure of RuvB Hexamer
ing subunit.9,18 Our model supports this idea that these two residues of neighboring subunits face the nucleotide binding pocket and form the protein –ATP – protein sandwich (Figure 3(c), yellow). The RuvB subunits of T. maritima in the crystal with ADP form a helix rather than a hexamer,9 consistent with the observation that RuvB hexamer formation requires ATP binding.6 But it is not a general property of hexameric helicases, since, for example, the RepA protein can form a ring without any cofactors.20 The putative DNA-binding sites are in domain III and motif II in domain II.18 Domain III is structurally similar to so-called winged helix DNA-binding proteins.9,21 Winged helix proteins use either the H3 helix (DtxR) or W1 wing (RFX1) for DNA binding.21,22 Our model suggests that RuvB uses the same W1 wing as RFX1 for binding DNA, because this loop is closer to the central hole than is the H3 helix (Figure 3(c), red). Mutations on this wing in RuvB (R316H, G317E, R318C in E. coli RuvB, corresponding to Arg300, Gly301, Arg302 in T. thermophilus RuvB) show that it plays an important role in DNA binding.18 The L268S mutation in E. coli RuvB (corresponding to Ile252 in T. thermophilus RuvB) has an interesting phenotype: it behaves like wild type E. coli RuvB in vitro, but confers UV-sensitivity in a dominant negative manner in vivo.23 Mutating this residue to alanine (L268A) results in a moderate dominant negative phonotype.18 In the crystal structures, this residue is in domain III and close to the loop between domains III and II. It faces the center of domain III, which would prevent it from interacting with other proteins. In our model, it is not near either the subunit – subunit interface, or the RuvA or DNA interfaces. Therefore, our model does not explain the effect of these mutations and future studies will hopefully address possible allosteric effects involving this residue. The D113N mutation in E. coli RuvB (corresponding to Asp97 in T. thermophilus RuvB) in motif II affects both DNA binding and ATP hydrolysis, but not hexamer formation.19 In our model, the corresponding Asp97 is far from the central hole for DNA binding. Therefore, it implies that DNA binding in the central channel may be coupled allosterically with ATP hydrolysis near the ATP-binding pocket.
Acknowledgments We thank Margaret S. VanLoock for helpful discussions, and Steve West for the gift of RuvB protein. This research was supported by NIH GM35269 (to E.H.E.).
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Structure of RuvB Hexamer
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Edited by W. Baumeister (Received 24 January 2002; received in revised form 3 April 2002; accepted 4 April 2002)