title: Structure of Thermus thermophilus HB8 H-protein of the glycine-cleavage system, resolved by a six-dimensional molecular-replacement method.
abstract: The glycine-cleavage system is a multi-enzyme complex consisting of four different components (the P-, H-, T- and L-proteins). Recombinant H-protein corresponding to that from Thermus thermophilus HB8 has been overexpressed, purified and crystallized. Synchrotron radiation from BL44B2 at SPring-8 was used to collect a native data set to 2.5 A resolution. The crystals belonged to the hexagonal space group P6(5) and contained three molecules per asymmetric unit, with a solvent content of 39%. Because of the large number of molecules within a closely packed unit cell, this structure was solved by six-dimensional molecular replacement with the program EPMR using the pea H-protein structure as a search model and was refined to an R factor of 0.189 and a free R factor of 0.256. Comparison with the pea H-protein reveals two highly conserved regions surrounding the lipoyl-lysine arm. Both of these regions are negatively charged and each has additional properties that are conserved in H-proteins from many species, suggesting that these regions are involved in intermolecular interactions. One region has previously been proposed to constitute an interaction surface with T-protein, while the other may be involved in an interaction with P-protein. Meanwhile, the lipoyl-lysine arm of the T. thermophilus H-protein was found to be more flexible than that of the pea H-protein, supporting the hypothesis that H-protein does not form a stable complex with L-protein during the reaction.
authors.affiliation: RIKEN Harima Institute/SPring-8, 1-1-1 Kouto, Mikazuki, Sayo-gun, Hyogo 679-5148, Japan. nakaix@spring8.or.jp
authors.forename: Tadashi
authors.initials: T
authors.lastname: Nakai
authors.forename: Jun
authors.initials: J
authors.lastname: Ishijima
authors.forename: Ryoji
authors.initials: R
authors.lastname: Masui
authors.forename: Seiki
authors.initials: S
authors.lastname: Kuramitsu
authors.forename: Nobuo
authors.initials: N
authors.lastname: Kamiya
mesh_terms: D001665:Binding Sites; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018360:Crystallography, X-Ray / Q000379:methods*; D009097:Multienzyme Complexes / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D016415:Sequence Alignment; D016963:Thermus thermophilus / Q000737:chemistry*
chemical_list: D009097:Multienzyme Complexes