Invariance of the zinc finger module: A comparison of the free structure with those in nucleic‐acid complexes

Introduction. Since the classical C2H2 zinc finger motif was predicted in 1985, 3% of the known and predicted genes in the human genome have been found encoding zinc finger proteins. Many structures have been reported of zinc finger–DNA interactions and show a common feature of zinc finger–DNA recognition. Zinc fingers read out the sequence information from the DNA major groove by forming specific hydrogen bonds between certain residues on the a-helix of zinc finger motif and the DNA bases. Most commonly, as in the canonical structure, each finger recognizes four bases on two overlapping three base pair subsites, but there are variants found in special cases. Owing to the zinc finger protein binding, the DNA is slightly unwound and has 11.3 bp/turn. The TFIIIA-5S RNA system has been extensively studied for zinc finger–nucleic acids recognition. TFIIIA (Transcription Factor IIIA) binds to the internal control region of 5S RNA gene (5S DNA) as the first step in the transcription carried out by RNA polymerase III. Its DNA binding domain is composed of nine tandem linked zinc finger motifs located at the N-terminal end. TFIIIA binds not only the 5S DNA but also the 5S RNA, the product of the gene. Biochemical data suggested that the central fingers 4–6 contribute most to the TFIIIA-5S RNA interaction. The crystal structure of a complex of 61 bases truncated 5S RNA, embodying the central region of 5S RNA, interacting with Fingers 4, 5, and 6, revealed two modes of recognition. In the case of Finger 5, the zinc finger module recognizes a region of regularly folded RNA double helix through its phosphate backbone. The other two fingers, Fingers 4 and 6 recognize specific bases presented accessibly by the intricately folded RNA loop structures. The general conclusion is that RNA is recognized by zinc fingers through a combination of its different kinds of structural elements. As well as having determined the crystal structure of the complex of the 61n RNA and the three finger peptide TFIIIA(4–6), we have also crystallized the peptide on its own and solved the structure at the resolution of 1.65 Å. This is reported here for the first time. Taking advantage of the high resolution, we here compare its individual finger modules with those in another high resolution structure and the complex of the Zif268 three finger peptide with its DNA binding site at 1.6 Å resolution, to examine if there is any significant conformational change in the zinc finger module. Materials and Methods. Protein Preparation and Crystallization: The protein was expressed and purified as described in Ref. 8. The purified protein was dialyzed into the buffer containing 20 mM HEPES pH 7.0, 2 mM MgCl2, 150 mM KCl, 5 mM DTT, 0.3 mM ZnSO4, and was then concentrated in a vivaspin unit. Protein concentrated to 7 mg/mL was used for hanging drop equilibrium evaporation. The crystals were grown in a buffer solution of 1.6M (NH4)2SO4, 25 mM MgSO4, 50 mM Tris HCl pH 8.5. For X-ray diffraction, crystals were frozen in the presence of 25% ethylene glycol. Data Collection and Structure Determination: The crystallographic data and analysis are summarized in Table I. The MAD data sets were collected at the ID29 ESRF at a resolution of 2.0 Å. The 1.65-Å data set was collected on the same beam line. The space group is P6122 with a unit cell of a 1⁄4 b 1⁄4 50.95 Å and c 1⁄4 173.43 Å. The heavy atom sites were found by Shelx, and refined by Sharp. The model was built using ARP/wARP followed by some manual modification with O, and then refined in Refmac5 to a Rfree 1⁄4 0.23, Rwork 1⁄4 0.22. The comparisons of structures were done with the Superpose Molecules program in the CCP4 package. Model figures were made in Ribbons. Coordinates are deposited in the Protein Data Bank, access code 2J7J. Results. Comparison of TFIIIA(4–6) Fingers With Zif268 Fingers: To examine the conformational flexibility of the overall zinc finger motif, we superimposed the fingers in our new structure onto those in an earlier high resolution structure, the Zif268–DNA complex at 1.6 Å. The fingers in the two peptides vary in their sequences (Fig. 1) and can be subdivided into three groups. (i)