Tubulins are essential for the reproduction of many eukaryotic viruses, but historically bacteriophage were assumed not to require a cytoskeleton. Here we identify a tubulin-like protein, PhuZ, from bacteriophage 201φ2-1 and show that it forms filaments in vivo and in vitro. The PhuZ structure has a conserved tubulin fold, with a novel, extended C-terminus that we demonstrate to be critical for polymerization in vitro and in vivo. Longitudinal packing in the crystal lattice mimics packing observed by EM of in vitro formed filaments, indicating how interactions between the C-terminus and the following monomer drive polymerization. Finally, we show that PhuZ assembles a spindle-like array required for positioning phage DNA within the bacterial cell. Correct positioning to the cell center and optimal phage reproduction only occur when the PhuZ filament is dynamic. This is the first example of a prokaryotic tubulin array that functions analogously to the microtubule-based spindles of eukaryotes.

I-TevI is a site-speci®c, sequence-tolerant intron endonuclease. The crystal structure of the DNA-binding domain of I-TevI complexed with the 20 bp primary binding region of its DNA target reveals an unusually extended structure composed of three subdomains: a Zn ®nger, an elongated segment containing a minor groove-binding a-helix, and a helix±turn±helix. The protein wraps around the DNA, mostly following the minor groove, contacting the phosphate backbone along the full length of the duplex. Surprisingly, while the minor groove-binding helix and the helix±turn± helix subdomain make hydrophobic contacts, the few base-speci®c hydrogen bonds occur in segments that lack secondary structure and¯ank the intron insertion site. The multiple base-speci®c interactions over a long segment of the substrate are consistent with the observed high site speci®city in spite of sequence tolerance, while the modular composition of the domain is pertinent to the evolution of homing endonucleases. Keywords: crystal structure/endonuclease/ helix±turn±helix/minor groove/Zn ®nger Introduction Intron-encoded endonucleases are proteins that promote the ®rst step in the mobility of the intron at the DNA level (Belfort and Roberts, 1997). They recognize and cleave an intronless allele of their cognate gene, initiating a replicative gene conversion event that results in the recipient allele also becoming intron-plus. These enzymes are therefore termed homing endonucleases and are grouped into a number of families based on the presence of conserved sequence elements. These are the LAGLIDADG, GIY-YIG, H-N-H and His-Cys box families (Belfort et al., 2001).I-TevI, the group I intron-encoded endonuclease of the td gene of bacteriophage T4, is the best studied member of the GIY-YIG family (Kowalski et al., 1999). The 28 kDa enzyme speci®cally recognizes its lengthy DNA substrate, or homing site, as a monomer ( Figure 1A; Mueller et al., 1995), exhibiting a high degree of sequence tolerance (Bryk et al., 1993). No single nucleotide in the 37 bp target is essential for binding and cleavage, and many multiple substitutions are well tolerated (Bryk et al., 1993. Consistent with this sequence tolerance, ethylation and methylation interference studies indicated that most of the protein±DNA contacts are via the minor groove and the phosphate backbone ( Figure 1B) (Bryk et al., 1993). The primary binding region of the enzyme is~20 bp in length, spanning the intron insertion site (IS), with a second region of contact close to the cleavage site (CS), 23±25 bp upstream of the IS ( Figure 1A). I-TevI demonstrates remarkable¯exibility, recognizing and cleaving homing site derivatives with large deletions (up to 16 bp) and insertions (up to 5 bp) between the IS and CS .The two-domain nature of the homing site is mirrored by the structure of the enzyme ( Figure 1A). I-TevI consists of two functionally distinct domains: an N-terminal catalytic domain and a C-terminal DNA-binding domain, separated by a long¯exible linker (Derbyshire et al., 1997). The catalytic ...

The crystal structures of a deletion mutant of human thymidylate synthase (TS) and its ternary complex with dUMP and Tomudex have been determined at 2.0 Å and 2.5 Å resolution, respectively. The mutant TS, which lacks 23 residues near the amino terminus, is as active as the wild-type enzyme. The ternary complex is observed in the open conformation, similar to that of the free enzyme and to that of the ternary complex of rat TS with the same ligands. This is in contrast to Escherichia coli TS, where the ternary complex with Tomudex and dUMP is observed in the closed conformation. While the ligands interact with each other in identical fashion regardless of the enzyme conformation, they are displaced by about 1.0 Å away from the catalytic cysteine in the open conformation. As a result, the covalent bond between the catalytic cysteine sulfhydryl and the base of dUMP, which is the first step in the reaction mechanism of TS and is observed in all ternary complexes of the E. coli enzyme, is not formed. This displacement results from differences in the interactions between Tomudex and the protein that are caused by differences in the environment of the glutamyl tail of the Tomudex molecule. Despite the absence of the closed conformation, Tomudex inhibits human TS tenfold more strongly than E. coli TS. These results suggest that formation of a covalent bond between the catalytic cysteine and the substrate dUMP is not required for effective inhibition of human TS by cofactor analogs and could have implications for drug design by eliminating this as a condition for lead compounds.

Endo-beta-N-acetylglucosaminidase F(3) cleaves the beta(1-4) link between the core GlcNAc's of asparagine-linked oligosaccharides, with specificity for biantennary and triantennary complex glycans. The crystal structures of Endo F(3) and the complex with its reaction product, the biantennary octasaccharide, Gal-beta(1-4)-GlcNAc-beta(1-2)-Man-alpha(1-3)[Gal-beta(1-4)-GlcNAc-be ta(1-2)-Man-alpha(1-6)]-Man-beta(1-4)-GlcNAc, have been determined to 1.8 and 2.1 A resolution, respectively. Comparison of the structure of Endo F(3) with that of Endo F(1), which is specific for high-mannose oligosaccharides, reveals highly distinct folds and amino acid compositions at the oligosaccharide recognition sites. Binding of the oligosaccharide to the protein does not affect the protein conformation. The conformation of the oligosaccharide is similar to that seen for other biantennary oligosaccharides, with the exception of two links: the Gal-beta(1-4)-GlcNAc link of the alpha(1-3) branch and the GlcNAc-beta(1-2)-Man link of the alpha(1-6) branch. Especially the latter link is highly distorted and energetically unfavorable. Only the reducing-end GlcNAc and two Man's of the trimannose core are in direct contact with the protein. This is in contrast with biochemical data for Endo F(1) that shows that activity depends on the presence and identity of sugar residues beyond the trimannose core. The substrate specificity of Endo F(3) is based on steric exclusion of incompatible oligosaccharides rather than on protein-carbohydrate interactions that are unique to complexes with biantennary or triantennary complex glycans.