Crystal structure of the YgfY from Escherichia coli, a protein that may be involved in transcriptional regulation

Introduction. The ygfY gene from Escherichia coli encodes an 88 amino acid protein of unknown function, and is a member of a large sequence family present in both, prokaryotes and eukaryotes. A Psi-Blast search of the nonredundant and environmental nonredundant databases revealed 249 family members. 1 Of these sequences, 152 are from environmental samples, mostly from a Sargasso Sea sample filtered to include only microbial cells. 2 The first cycles of Psi-Blast iteration yielded 78 homologous bacterial proteins, including 42 from the Sargasso Sea. Plant and fungal proteins emerged in the second cycle, and insect and mammalian sequences emerged in the third cycle. While the bacterial proteins have approximately the same size as YgfY (except for incomplete sequences from environmental samples), the eukaryotic proteins are at least double in size. None of the sequence homologues has known biochemical function. The cellular role is also unknown, except for a remote relationship (E-score 10 4 ) to a protein from Saccharomyces cerevisiae, YOL071W/EM15 (GI:6324501), which is required for sporulation and for transcriptional induction of the early meiotic-specific transcription factor IME1. 3 This 162amino-acid residue yeast protein exhibits 25% identity to YgfY over a stretch of 60 amino acids. The crystal structure of YgfY was determined at 1.2 A ˚ resolution as a part of our structural genomics project (http://s2f.umbi.umd.edu), revealing a five-helix fold similar to that of the homologous protein NMA1147 from Neisseria meningitidis (30% identity) determined recently by NMR methods. 4 Yet, there are inconsistencies in the details of these two structures that reflect the different levels of accuracy of the two methods of structure determination. We propose that the functional region is different from the one proposed based on the NMR structure, and highlight a particularly important structural difference associated with the proposed activity center. Materials and Methods. YgfY from E. coli was amplified using PfuTurbo DNA polymerase (Stratagene), genomic DNA, and 5- and 3-end primers. In addition to the native gene sequence, a sequence consistent with a thrombin cleavage site was introduced, and a NdeI restriction site was designed to convert the 6xHis-tagged construct into a construct coding for native protein. The PCR product was introduced into the pET100/D-TOPO expression vector by TOPO directional cloning procedure (Invitrogen). For wildtype protein production, the E. coli strain BL21 Star (DE3) was transformed with the recombinant plasmid. An expression screen showed that the His-tagged protein was soluble, whereas the native protein was insoluble. Wild-type protein was produced by growing the cells at 37°C in Super Broth medium supplemented by ampicillin (100 g/mL). Once the cell culture reached A600 0.6, expression was induced by the addition of 1 mM IPTG, and afte r3ht he cells were harvested by centrifugation. To prepare selenomethionine (SeMet) containing protein, the E. coli B834 (DE3) strain was transformed with the recombinant vector. The cells were grown at 30°C in minimal medium supplemented with ampicillin (50 g/mL), SeMet, and 19 amino acids other than methionine. When the cell culture reached A600 0.5, 1 mM IPTG was added, and after 3 h the cells were harvested. Cells were suspended in 20 mM Tris HCl (pH 8.0), 0.5 M NaCl, and 5 mM imidazole, and lysed by passage through a French press. The soluble fraction was loaded on Ni-NTA metal affinity column (Qiagen). Protein was eluted with 20 mM Tris HCl (pH 8.0), 0.5 M NaCl, and 250 mM imidazole. To remove the N-terminal sequence containing the 6xHis tag, human thrombin (Haemtech) was added at 1:2000 molar ratio and incubated overnight at 4°C in Tris HCl (pH 8.0), and 500 mM NaCl. Thrombin was removed by passing the protein mixture through benzamidine column (Amersham Biosciences). The cleaved and uncleaved protein and the N-terminal peptide were separated on a second Ni-NTA column. The protein was dialyzed against a buffer of 50 mM NaCl and 20 mM Tris HCl (pH 7.5), and further purified with a size-exclusion chromatography