Crystal structure of hypothetical protein YfiH from <i>Shigella flexneri</i> at 2 Å resolution

摘要

The crystal structure of an uncharacterized conserved protein (residues 1–243) expressed by yfiH gene of Shigella flexneri 2a str.2457T (gi 30042248),1 has been determined and refined to 2.01 Å by single wavelength anomalous dispersion (SAD) method. The YfiH protein belongs to a vast protein family of at least 201 uncharacterized proteins which contain conservative Pfam motif PF02578 (DUF152) (residues 29–243) and TIGR motif (TIGR00726)2, 3 (residues 25–243). The YfiH protein belongs to the COG1496 which consists of 39 proteins widely distributed in 37 species from bacteria to human. The yfiH gene (DNA bases 101,672-102,403)1 is located between the two genes, clpB and sfhB. The clpB gene is thought to encode an adenosine triphosphatase subunit of an intracellular adenosine 5′-triphosphate-dependent protease which belongs to the ClpA/ClpB family and is induced by heat shock.4 The sfhB gene expresses a protein which is known as suppressor of ftsH mutation and is responsible for synthesis of pseudouridine from uracil at three positions in 23S ribosomal RNA.5 The function of YfiH protein is unknown, but preliminary data obtained for Brevibacterium lactofermentum ATCC 13869 show that the gene is not essential for the cell's growth and viability.6 The seleno-methionine derivative of YfiH protein crystallized in the P1 space group with unit cell dimensions of a = 43.87, b = 50.58, c = 55.35 Å, α = 90.32°, β = 96.32°, and γ = 90.40°. There are two protein molecules in the asymmetric unit related by an approximate 21 symmetry along the crystallographic c axis. Both molecules adopt nearly identical backbone conformation (σ = 0.31 Å using all Cα atoms) with only a few differences in side-chain conformations. A PQS7 search predicted a monomeric form for this protein consistent with weak interface interaction between the two molecules in the asymmetric unit. The monomeric character was confirmed by size-exclusion chromatography (see Materials and Methods), which indicates that YfiH is a monomer in solution and this form may represent the biologically relevant species in vivo. The protein structure is an α/β/α fold: two layers of β-sheets, the smaller one with three antiparallel strands (S1, S10, and S4), and the bigger one with six antiparallel/parallel strands (S7, S5, S6, S3, and S2) are sandwiched between two α-helices (H2, H5) on one side and one α-helix (H1) on the other [Fig. 1(A, B)]. The N-terminal end of the H1 helix is connected to S1 in the smaller β-sheet via one of the long loops and the C-terminal end of this helix is linked to S2 in the larger β-sheet. The α-helix H2, located between S6 and S7 in the larger β-sheet, contains a kink at Gly-133 and connects to S7 by an α-helical turn. In addition, there is a small domain inserted between S7 in the larger β-sheet and the helix H5. This small domain contains a small helix-turn-helix motif (H3 and H4), followed by a β-hairpin (S8 and S9). The main α/β/α fold resumes with H5, a long loop containing a little more than one 3/10-helix H5, followed by the S10 located in the middle of the smaller β-sheet. Crystal structure of YfiH protein. A: Ribbon diagram of YfiH protein. Helices are shown in blue, β-sheets in red, and Zn2+ ions are indicated in magenta; orange indicates protein residues interacting with Zn2+ ions, among eight Zn2+ ions in the asymmetric unit, six Zn2+ ions are shown here, four of these are shared between the two protein molecules. The two Zn2+ ions not shown interact with the other protein molecule in the asymmetric unit. Secondary structure elements are also indicated in black. B: Diagram showing the secondary structure elements in YfiH protein superimposed on its primary sequence. Residues interacting with Zn2+ ions are marked with blue dots and residues interacting with acetate molecules are marked with red. β-Hairpins are depicted as red loops. C: The potential active site of YfiH protein. The Zn2+ ions are coordinated to an acetate molecule, a cysteine and two