STRUCTURE NOTE

Crystal Structure of the YciO Protein From Escherichia coliJia Jia,1 Vladimir V. Lunin,1 Veronique Sauve,1 Li-Wei Huang,2 Allan Matte,1 and Miroslaw Cygler1*1Biotechnology Research Institute, National Research Council of Canada, and Montreal Joint Centre for Structural Biology,Montreal, Quebec, Canada2Biophysics Group, Physics Division, Los Alamos National Laboratory, Los Alamos, New Mexico

Introduction. The YciO protein of Escherichia coli is amember of a family of proteins that also includes YrdC,HypF in E. coli, YwlC in Bacillus subtilis, and Sua5 inSaccharomyces cerevisiae (PF01300 family in PFAM data-base1). Sequences similar to YciO are found either as (a)independent proteins, (b) with C-terminal extensions, or(c) as domains in larger proteins (PFAM). The proteinsYwlC from B. subtilis and Sua5 from S. cerevisiae areexamples of the second category. Sua5 has been identifiedas having an essential role for normal growth on lactate orglycerol medium, although the precise function of the Sua5protein remains unclear.2 HypF represents the third cat-egory where this domain is located in the middle of thelinear sequence. The hypF and hydN genes form the hydAlocus in E. coli, which encodes functions necessary for theformation of hydrogenase activity. HypF, the hydrogenasematuration protein, most likely participates in the matura-tion of all three E. coli hydrogenase isozymes 1, 2, and 3from their inactive precursor forms.3

Both crystallographic and nucleic acid binding studieswith E. coli YrdC revealed that this protein might exert itsfunction through binding to double-stranded RNA.4 Thiswould imply a role for YciO/YrdC and their homologousdomains in larger proteins as functioning in the regulationof either transcription or translation. To gain furtherinsight into this family of proteins, we have determinedthe crystal structure of E. coli YciO at 2.1 Å resolution.

Materials and Methods. The gene encoding E. coliyciO was cloned, and the protein was expressed andpurified as an N-terminal His6-tag fusion with a thrombincleavage site as described previously for E. coli MoeA.5

Crystals of the fusion protein were obtained by the hang-ing drop vapor diffusion method by equilibrating dropscontaining 2 �L of protein (15 mg/mL) in buffer (20 mMTris, pH 7.5, 40 mM NH4SO4, 60 mM NH4Ac, 5 mM2-mercaptoethanol) and 2 �L of reservoir solution (10%[w/v] PEG3350, 0.1 M MES buffer, pH 6.5, 0.1 M MgAc2,5% [v/v] ethylene glycol) suspended over 0.5 mL of reser-voir solution. The crystals belong to the orthorhombicsystem, space group P212121 with unit cell dimensions a �48.40, b � 68.77, c � 94.77 Å and one molecule in theasymmetric unit. Before data collection, the crystal wassoaked for �15 s in a cryoprotecting solution of 23% (w/v)PEG3350, 0.1 M MES, pH 6.5, 0.1 M MgAc2, 5% (v/v)

ethylene glycol, and 19% (w/v) glycerol, and was flashcooled in a cold stream of N2 gas to 100 K.

The structure of YciO was determined by MAD phasingfrom a SeMet-labeled protein crystal (Table I). Data wereprocessed and scaled with HKL2000.6 All expected Se siteswere found (SOLVE7), and the phases were calculated to2.1 Å (figure of merit of 0.52). The electron density mapafter applying density modification (RESOLVE8) allowedautomatic tracing of �80% of the main-chain and built theside-chains with the program ARP/WARP.9 The model wasthen adjusted manually by using the program O10 andrefined (CNS version 1.011) against data collected at thehard remote wavelength. During refinement, 4.8% of thereflections were set aside for the calculation of Rfree. Watermolecules were initially added automatically with CNSand verified by visual inspection of the difference map. Thefinal model contains all residues, 1–206, 127 water mol-ecules, and one sulfate ion with an R-factor of 0.211 andRfree of 0.229. The model has good geometry with noresidues in the disallowed regions of the Ramanchandranplot (Procheck12). The coordinates of YciO are deposited inthe Protein Data Bank with the accession code 1KK9.

Results and Discussion. The YciO molecule has anoverall �/� architecture and consists of a single domaincontaining a central 10-stranded �-sheet flanked by six�-helices (A–F) (Fig. 1). The �-sheet can be subdivided intotwo sheets (�1-�8-�2-�3-�7-�4, �4-�6-�5-�9-�10) with acommon middle strand �4. There are two helices on eachside of the N-terminal part of the �-sheet (A, E and B, F),whereas the remaining two helices (C, D) are on the sameside of the C-terminal part of this sheet. Helices arepositioned asymmetrically about the �-sheet, with four ofthem (A, E, C, and D) packing against one face and two (Band F) against the other (Fig. 1).

Analysis of sequences of proteins homologous to YciOidentified residues that are highly conserved within thisfamily.4 We analyzed the residue conservation by using a

*Correspondence to: Dr. Mirek Cygler, Biotechnology ResearchInstitute, NRCC, 6100 Royalmount Avenue, Montreal, Quebec H4P2R2, Canada. E-mail: mirek@bri.nrc.ca

Received 19 March 2002; Accepted 21 March 2002

Published online 00 Month 2001 in Wiley InterScience(www.interscience.wiley.com). DOI: 10.1002/prot.10178

PROTEINS: Structure, Function, and Genetics 49:139–141 (2002)

© 2002 WILEY-LISS, INC.

larger set of sequences assembled in the recent version ofPFAM.1 There is a strong concentration of the conservedresidues in two areas: within a narrow depression in theprotein surface between the C-termini of strands �7 and�10, and a much smaller area on the opposite side of themolecule defined by the side-chains of Asp35 and Ser36. Asulfate ion, originating from the crystallization motherliquor, is clearly visible in the electron density map withinthe depression mentioned above. This anion forms severalhydrogen bonds with the protein through its oxygen atoms.Its O1 atom is H-bonded to the NH group of Ser144 (strand�7) and NE of Arg57, the O2 atom makes H-bonds to NE2 ofHis63, Wat383, and through a bridging Wat316 to atomsOG1 of Thr66 and carbonyl oxygen of Asn64, the O3 atom isH-bonded to NH1 of Arg196 (�10), and NH2 of Arg57 (loopbetween helix B and �3), the O4 atom is H-bonded to NH2 ofArg196, OG of Ser144, and through the water moleculeWat313 to side-chains of Thr66, Thr98, and Arg122. Theelectrostatic potential surface shows a bimodal distributionwith opposite ends of the �-sheet having positive or negativepotentials. The depression with highly conserved residues isnear the boundary between these two areas and is character-ized by a positive potential.

Sequence comparison of YciO and YrdC shows 27%amino acid identity between the two proteins. Their three-dimensional structures are quite similar to each other.Superposition of YciO with E. coli YrdC4 gives a root-mean-squares deviation (RMSD) of 1.52 Å for 131 C� pairs. TheYciO is seven amino acids longer than YrdC and has twoadditional �-strands at the N-terminus (�1, �8). The �8strand corresponds in YrdC to a stretch with an extendedconformation but which does not form proper H-bondswith the next strand. Although the folds of YciO and YrdCare similar, molecular surfaces in the regions of hypotheti-cal active sites are somewhat different. YrdC has a widercleft then YciO. There is also a significant difference in thedistribution of their electrostatic potential. The bimodal

Fig. 1. a: The ribbon drawing of the YciO molecule. �-strands are inlight shades, �-helices in dark shades. Highly conserved residues areshown in full (black); b: molecular surface of YciO in the same orientationas above showing the depression with conserved residues (light shades).

TABLE I. Data Collection and Refinement Statistics

Peak Inflection Remote

Wavelength (Å) 0.97921 0.97942 0.95369Resolution range (last shell) 50–2.1 (2.18–2.1) 50–2.1 (2.18–2.1) 50–2.1 (2.18–2.1)Rsym (last shell) 0.067 (0.451) 0.070 (0.498) 0.068 (0.422)Completeness (last shell) 99.9 (99.0) 99.9 (99.0) 99.9 (99.9)No. of observations 202,648 203,018 205,748Unique reflections 35,651 35,705 19,133R-factor (Rfree) 0.211 (0.229)No. of nonhydrogen atoms 1628No. of water molecules 127Average B-factor (Å2)

Main-chain atoms 35.3Side-chain atoms 40.7Water molecules 48.2SO4 33.3

RMSD bond length (Å) 0.005RMSD bond angle (°) 1.3Ramachandran plot

In most favorable region (%) 92.4In disallowed regions 0

140 J. JIA ET AL.

distribution observed in YciO is much less pronounced inYrdC.4 The structural features of YciO are consistent withbinding to an extended RNA molecule.

Acknowledgments. We thank Robert Larocque fortechnical help in molecular biology, Dr. Stephane Ray-mond for help with the structural genomics database, andDr. Joseph D. Schrag for helpful discussions.

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