Detailed architecture of a DNA translocating machine: the high-resolution structure of the bacteriophage phi29 connector particle.

The three-dimensional crystal structure of the bacteriophage phi29 connector has been solved and refined to 2.1A resolution. This 422 kDa oligomeric protein connects the head of the phage to its tail and translocates the DNA into the prohead during packaging. Each monomer has an elongated shape and is composed of a central, mainly alpha-helical domain that includes a three-helix bundle, a distal alpha/beta domain and a proximal six-stranded SH3-like domain. The protomers assemble into a 12-mer, propeller-like, super-structure with a 35 A wide central channel. The surface of the channel is mainly electronegative, but it includes two lysine rings 20 A apart. On the external surface of the particle a hydrophobic belt extends to the concave area below the SH3-like domain, which forms a crown that retains the particle in the head. The lipophilic belt contacts the non-matching symmetry vertex of the capsid and forms a bearing for the connector rotation. The structure suggests a translocation mechanism in which the longitudinal displacement of the DNA along its axis is coupled to connector spinning.

[1]  J M Carazo,et al.  The three-dimensional structure of a DNA translocating machine at 10 A resolution. , 1999, Structure.

[2]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[3]  E. Beckmann,et al.  Structure of the 13-fold symmetric portal protein of bacteriophage SPP1 , 1999, Nature Structural Biology.

[4]  J M Berger,et al.  Crystal structure of the N-terminal domain of the DnaB hexameric helicase. , 1999, Structure.

[5]  F. Major,et al.  Function of hexameric RNA in packaging of bacteriophage phi 29 DNA in vitro. , 1998, Molecular cell.

[6]  L. E. Donate,et al.  Role of the Amino-terminal Domain of Bacteriophage # 29 Connector in DNA Binding and Packaging , 2001 .

[7]  R. Hendrix,et al.  Symmetry mismatch and DNA packaging in large bacteriophages. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[8]  L. Black.  DNA packaging in dsDNA bacteriophages. , 1989, Annual review of microbiology.

[9]  M. Heel,et al.  The portal protein of bacteriophage SPP1: a DNA pump with 13‐fold symmetry. , 1993, The EMBO journal.

[10]  H. Fujisawa,et al.  Characterization of the bacteriophage T3 DNA packaging reaction in vitro in a defined system. , 1987, Journal of molecular biology.

[11]  J M Carazo,et al.  Three-dimensional reconstruction of the connector of bacteriophage phi 29 at 1.8 nm resolution. , 1986, Journal of molecular biology.

[12]  J. Carrascosa,et al.  An intrinsic-tryptophan-fluorescence study of phage phi 29 connector/nucleic acid interactions. , 1994, European journal of biochemistry.

[13]  Antonio Párraga,et al.  Purification, crystallization and preliminary X‐ray diffraction studies of the bacteriophage φ29 connector particle , 1998 .

[14]  Timothy S Baker,et al.  Assembly of a Tailed Bacterial Virus and Its Genome Release Studied in Three Dimensions , 1998, Cell.

[15]  A. Engel,et al.  The bacteriophage φ29 head–tail connector imaged at high resolution with the atomic force microscope in buffer solution , 1997, The EMBO journal.

[16]  John Kuriyan,et al.  Three-dimensional structure of the β subunit of E. coli DNA polymerase III holoenzyme: A sliding DNA clamp , 1992, Cell.

[17]  Anastassis Perrakis,et al.  Automated protein model building combined with iterative structure refinement , 1999, Nature Structural Biology.

[18]  S V Evans,et al.  SETOR: hardware-lighted three-dimensional solid model representations of macromolecules. , 1993, Journal of molecular graphics.

[19]  R Henderson,et al.  Analysis of electron microscope images and electron diffraction patterns of thin crystals of phi 29 connectors in ice. , 1994, Journal of molecular biology.

[20]  B. Matthews.  Solvent content of protein crystals. , 1968, Journal of molecular biology.

[21]  Roger W. Hendrix,et al.  Control Mechanisms in dsDNA Bacteriophage Assembly , 1988 .

[22]  P. Guo,et al.  A small viral RNA is required for in vitro packaging of bacteriophage phi 29 DNA. , 1987, Science.

[23]  K Schulten,et al.  VMD: visual molecular dynamics. , 1996, Journal of molecular graphics.

[24]  Fernando de la Cruz,et al.  The bacterial conjugation protein TrwB resembles ring helicases and F1-ATPase , 2001, Nature.

[25]  J. Carrascosa,et al.  Structure of viral connectors and their function in bacteriophage assembly and DNA packaging , 1994, Quarterly Reviews of Biophysics.

[26]  J. Navaza,et al.  AMoRe: an automated package for molecular replacement , 1994 .

[27]  M. Tasaka,et al.  DNA packaging ATPase of bacteriophage T3. , 1993, Virology.

[28]  J. Carazo,et al.  Three-dimensional reconstruction of bacteriophage φ29 neck particles at 2.2 nm resolution☆ , 1985 .

[29]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[30]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[31]  Peixuan Guo,et al.  Prohead and DNA-gp3-dependent ATPase activity of the DNA packaging protein gp16 of bacteriophage phi 29. , 1987, Journal of molecular biology.

[32]  J. Carazo,et al.  Three-dimensional reconstruction of bacteriophage phi 29 neck particles at 2 X 2 nm resolution. , 1985, Journal of molecular biology.

[33]  J. Caldentey,et al.  Gene 20 product of bacteriophage T4. II. Its structural organization in prehead and bacteriophage. , 1983, Journal of molecular biology.

[34]  A. Engelman,et al.  Crystal structure of the catalytic domain of HIV-1 integrase: similarity to other polynucleotidyl transferases. , 1994, Science.

[35]  C. Sander,et al.  Protein structure comparison by alignment of distance matrices. , 1993, Journal of molecular biology.

[36]  A. Santisteban,et al.  Structure of the head-tail connector of bacteriophage φ29 , 1982 .

[37]  J. Carrascosa,et al.  Bacteriophage lambda preconnectors. Purification and structure. , 1984, Journal of molecular biology.

[38]  W. Kabsch,et al.  Dictionary of protein secondary structure: Pattern recognition of hydrogen‐bonded and geometrical features , 1983, Biopolymers.

[39]  Charles C. Richardson,et al.  Crystal Structure of the Helicase Domain from the Replicative Helicase-Primase of Bacteriophage T7 , 1999, Cell.

[40]  G J Kleywegt,et al.  Software for handling macromolecular envelopes. , 1999, Acta crystallographica. Section D, Biological crystallography.

[41]  J. Carazo,et al.  Structure of phage φ29 connector protein assembled in Vivo , 1985 .

[42]  Marc C. Morais,et al.  Structure of the bacteriophage φ29 DNA packaging motor , 2000, Nature.

[43]  E A Merritt,et al.  Raster3D Version 2.0. A program for photorealistic molecular graphics. , 1994, Acta crystallographica. Section D, Biological crystallography.

[44]  L. E. Donate,et al.  Bacteriophage T3 connector: three-dimensional structure and comparison with other viral head-tail connecting regions. , 1988, Journal of molecular biology.

[45]  M. Feiss,et al.  Virus DNA packaging: the strategy used by phage λ , 1995, Molecular microbiology.

[46]  M. O’Donnell,et al.  Toroidal proteins: Running rings around DNA , 1998, Current Biology.

[47]  A. Engel,et al.  Gene 20 product of bacteriophage T4 its purification and structure. , 1981, Journal of molecular biology.

[48]  J. Pous,et al.  Crystallographic analysis reveals the 12-fold symmetry of the bacteriophage phi29 connector particle. , 1998, Journal of molecular biology.

[49]  C. Zhang,et al.  Inter-RNA interaction of phage phi29 pRNA to form a hexameric complex for viral DNA transportation. , 1998, Molecular cell.

[50]  J. Carrascosa,et al.  Structure and assembly of phage phi29. , 1976, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[51]  Liang Tong,et al.  REPLACE, a suite of computer programs for molecular-replacement calculations , 1993 .

[52]  J. King,et al.  Purification and organization of the gene 1 portal protein required for phage P22 DNA packaging. , 1988, Biochemistry.

[53]  M. Valle,et al.  Topology of the components of the DNA packaging machinery in the phage phi29 prohead. , 2000, Journal of molecular biology.

[54]  J. J. Fernández,et al.  Structural analysis of the bacteriophage T3 head-to-tail connector. , 2000, Journal of structural biology.

[55]  R M Esnouf,et al.  An extensively modified version of MolScript that includes greatly enhanced coloring capabilities. , 1997, Journal of molecular graphics & modelling.

[56]  M. Bjornsti,et al.  Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: oriented and quantized in vitro packaging of DNA protein gp3 , 1983, Journal of virology.

[57]  S. Casjens,et al.  Homology throughout the multiple 32-kilobase circular plasmids present in Lyme disease spirochetes , 1997, Journal of bacteriology.

[58]  John E. Johnson,et al.  Virus assembly: Imaging a molecular machine , 1999, Current Biology.

[59]  R. Hendrix.  Bacteriophage DNA Packaging RNA Gears in a DNA Transport Machine , 1998, Cell.

[60]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.