Photon Factory Activity Report 2004 #22 Part B (2005)
Biological Science

18B, NW12/2003G309

Crystal structure of tRNA-specific ribonuclease, colicin D
Shunsuke YAJIMA*1, Kotaro NAKANISHI2, Kazutoshi TAKAHASHI2, Tetsuhiro OGAWA2, Makoto
HIDAKA2, Yuichiro KEZUKA3, Takamasa NONAKA3, Kanju OHSAWA1, Haruhiko MASAKI2
1
Department of Bioscience, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156-8502, Japan2
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University ofTokyo, Bunkyo-ku, Tokyo 113-8657, Japan3
Department of BioEngineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188,Japan
Introduction
Colicin D (697 residues) has recently beencharacterized as a tRNase that kills sensitive Escherichia
coli cells via a specific tRNA cleavage. It targets
exclusively the four isoaccepting tRNAs for Arg, whose
anticodon sequences are ICG, CCG, U*CU, (U*; 5methylaminomethyluridine), and CCU, and cleaves thephosphodiester bond between positions 38 and 39, at the
3’ junction of the anticodon stem and the loop. Theresidue is A at position 38, and G or C at 39 in the fourisoacceptors; these sequences at positions 38 and 39 are
also shared by some other tRNAs, however. If colicin D
recognizes only the cleavage site, as with another tRNase
Argcolicin E5, its tartget would not exclusively be tRNA . It
could, therefore, recognize some higher order structuralfeatures of the targets with or without base recognition.
The molecular mechanisms for recognition of tRNAs
have been closely studied and characterized on aminoacytRNA synthetases (aaRS). There are, however, nohomologies between colicin D and aaRSs. Thus, westarted to analyze the crystal structure of colicin D toidentify its specific recognition mechanism.
Materials and Methods
Arg
The tRNA -specific ribonuclease activity of colicin D
exclusively locates its C-terminal domain (CRD; the Cterminal Ribonuclease Domain). The expression plasmids
used here have operons consisting of ORFs coding for
either CRD604 (604-697 residues of colicin D) or
CRD595 (595-697 residues), and Imm D (inhibitor
protein; 94 residues) tagged with six histidines at the Cterminus, expressed under control of the colicin promoter.
For a MAD experiment, selenomethionine (SeMet)
substituted CRD604-ImmD was prepared.
CRD604-ImmD crystal was obtained in mother liquor
condition with 0.09 M Mes at pH .5, 10% MPD, 5 mM
DTT, 18% PEG8000, and 0.18 M magnesium acetate at
4 °C. The space group is P41212 and the unit celldimensions are a = b = 58.5 Å, c = 149 Å for both nativeand SeMet crystals.
Results and Discussion
In the complex structure, CRD consists of a fourstranded antiparallel β-sheet, with two helices on one sideand a short helix on the other side. ImmD proved to be a228

Users' Reportfour-helix bundle protein, in which the first helix parallels
the second and the third parallels the fourth, and eachparallel pair is in a twisted position to each other (Fig. 1).
The complex was formed mainly by electrostaticinteraction between positive charges on CRD andnegative charges on ImmD.
All ribonucleases reported to date have at least one
histidine as a catalytic residue. There are two histidines in
CRD, and crystal structure supports that His611 isresponsible for the catalysis since it located on the surfaceof the protein whereas the other one was buried inside ofthe molecule.
Interestingly, the basic residues, Arg602, Lys603,
Lys607, Lys608, Lys610 and Arg624 line the surface
having a slight curve. The trace of these residues isreminiscent of the backbone curve of tRNA. The distance
between Nζ, Nη1 or Nη2 atoms of adjacent resi1ues are
4.8-9.7 Å. The distance between the atoms of the basic
residues can change according to the conformations of theside chains. These basic residues may therefore play animportant role in tRNA specific binding of CRD andrecognition of the higher order structure of tRNA.

Figure 1. The complex structure of CRD-ImmD.
References
S. Yajima et al., Biochem. Biophys. Res. Comm. 322, 966(2004).
* yshun@nodai.ac.jp