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

NW12/2004G374

Crystal structure of the ankyrin repeat domain of human ribonuclease L
Nobutada TANAKA*, Yoshio KUSAKABE, Yasumitsu SAKAMOTO, Ken-ichi AOKI,
Masaru TSUNODA, Yasuyuki KITAGAWA, and Kazuo T. NAKAMURA
Laboratory of Physical Chemistry, School of Pharmaceutical Sciences, Showa University,
1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan

Introduction
In mammals, viral infections initiate an innate
immune response predominantly mediated by type I IFNs.
Type I IFNs regulate the transcription of a number ofgenes that inhibit or block viral replication throughdiverse mechanisms. There are three well-establishedantiviral pathways for interferon action: these involve thedouble-stranded RNA (dsRNA)-dependent protein kinase,
PKR, the Mx proteins, and the 2-5A system, respectively.
In the 2-5A system, treatment of cells with IFN
activates
genes
encoding
several2’,5’-linked
oligoadenylate synthetases (OASs) and a single geneencoding ribonuclease L (RNase L). The OASs areactivated by binding to dsRNA, a frequent by-product ofvirus infection. The activated OASs generate 5’triphosphorylated,2’,5’-phosphodiester-linked
oligoadenylates (2-5A) from ATP. The 2-5A moleculefunctions by binding to RNase L, changing from aninactive monomer to a catalytically active homodimer.
The activated RNase L cleaves RNA containing dyads of
UU, UA, AU, AA, and UG. The RNA degradation
inhibits protein synthesis and thus inhibits viral
replication.
RNase L is an unusual ribonuclease in that it
requires the activator molecule 2-5A to catalyze thehydrolysis of single-stranded RNA. 2-5A is itself very
unusual, comprising a type of oligoadenylates with 2’,5’internucleotide linkages, in contrast to the typical 3’,5’linkages found in RNA and DNA. The human form ofRNase L is a 741-amino acid protein with a molecularmass of 83,543 Da. RNase L consists of three domains,
namely the N-terminal ankyrin repeat, the protein kinasehomology, and the C-terminal ribonuclease domains. TheN-terminal ankyrin repeat domain (ANK), a region
containing nine ankyrin-like macromolecular recognition
repeats (the ninth ankyrin repeat is incomplete), isresponsible for 2-5A binding, and the C-terminal domain
is responsible for catalytic activity. RNase L was thought
at first to act to prevent virus replication, and recently theenzyme was proposed as a candidate risk factor for
hereditary prostate cancer. Among the naturally occurringmutants of RNase L examined, only the Arg462Gln
variant showed less RNase activity, and this variant also
showed a significant association with prostate cancer.
Therefore, elucidation of all the amino acid residues that
could influence RNase L activities, i.e. 2-5A binding,homodimerization and catalysis, is urgently required.

Experimental
Crystallization of ANK was carried out in a way
similar to the method described for non-tag ANK [1]. The
data collection was performed by rotation method at 100
K using an ADSC Q210 CCD detector with synchrotron
radiation (λ = 0.978 Å at beam line NW12 of the PF-AR,Japan). The crystals belong to an orthorhombic spacegroup P212121 with cell dimensions of a = 63.20 Å, b =72.83 Å, and c = 82.63 Å (α = β = γ = 90°). An
assumption of one molecule per asymmetric unit leads toan empirically acceptable VM value of 2.38 Å3/Da,
corresponding to a solvent content of 48 %. The best
diffraction data from a native crystal were collected up to1.8 Å resolution
Results and Discussion
We determined the crystal structure of the ANK/ 2-5A complex by the molecular replacement method
using the coordinate of “consensus ankyrin repeat
protein” (PDB code: 1MJ0) as the template forconstructing a search model and refined the resulting
model to an R-factor of 0.202 (Rfree of 0.230) at 1.8 Åresolution. The final model consists of residues 21-305,
220 water molecules, and one 2-5A molecule.
ANK folds into eight ankyrin repeat elements
and forms an extended curved structure with a groove
running across the long concave surface. Previous
primary structure analysis suggested that RNase L hadnine ankyrin repeats, but the ninth ankyrin repeat isincomplete. However, this prediction for RNase L differsfrom the present crystal structure of ANK, which consistsof eight ankyrin repeats. Residues 306 to 333,
corresponding to the incomplete ninth repeat, aredisordered. As in other ankyrin repeat proteins, each
repeat is formed by ~33 amino acid residues and consistsof pairs of antiparallel α-helices stacked side by side andconnected by a series of intervening β-hairpin motifs. The
2-5A molecule is accommodated in the concavity anddirectly interacts with ankyrin repeats 2 to 4. The
structural detail for the recognition of 2-5A by ANK will
be described elsewhere [2].
References[1] N. Tanaka et al., Protein Peptide Lett. 12, 387 (2005).
[2] N. Tanaka et al., EMBO J. 23, 3929 (2004).
* ntanaka@pharm.showa-u.ac.jp

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