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. 2015 Mar;71(Pt 3):346-53.
doi: 10.1107/S2053230X15001351. Epub 2015 Feb 21.

Improved crystallization and diffraction of caffeine-induced death suppressor protein 1 (Cid1)

Luke A Yates  1 , Benjamin P Durrant  1 , Michael Barber  1 , Karl Harlos  1 , Sophie Fleurdépine  2 , Chris J Norbury  2 , Robert J C Gilbert  1
Affiliations

Improved crystallization and diffraction of caffeine-induced death suppressor protein 1 (Cid1)

Luke A Yates et al. Acta Crystallogr F Struct Biol Commun. 2015 Mar.

Abstract

The post-transcriptional addition of uridines to the 3'-end of RNAs is an important regulatory process that is critical for coding and noncoding RNA stability. In fission yeast and metazoans this untemplated 3'-uridylylation is catalysed by a single family of terminal uridylyltransferases (TUTs) whose members are adapted to specific RNA targets. In Schizosaccharomyces pombe the TUT Cid1 is responsible for the uridylylation of polyadenylated mRNAs, targeting them for destruction. In metazoans, the Cid1 orthologues ZCCHC6 and ZCCHC11 uridylate histone mRNAs, targeting them for degradation, but also uridylate microRNAs, altering their maturation. Cid1 has been studied as a model TUT that has provided insights into the larger and more complex metazoan enzyme system. In this paper, two strategies are described that led to improvements both in the crystallogenesis of Cid1 and in the resolution of diffraction by ∼1.5 Å. These advances have allowed high-resolution crystallographic studies of this TUT system to be initiated.

Keywords: Cid1; terminal uridylyltransferases.

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Figures

Figure 1
Figure 1
(a) Heparin affinity chromatography of tCid1 (without GST fused). The co-purified nucleic acids bound to the tCid1 enzyme (and mutants) were displaced by heparin affinity chromatography of tCid1. The protein that bound to the immobilized heparin was eluted using a linear sodium chloride gradient from 50 to 2 M. A single symmetrical peak eluted as a result of the sodium chloride gradient and clearly displays a significant absorbance at 280 nm and a greatly reduced 260:280 nm absorbance ratio. (b) SDS–PAGE analysis of the protein composition of the two peaks.
Figure 2
Figure 2
(a) Size-exclusion chromatography (SEC) of tCid1 (without GST fused or contaminating nucleic acids). A single symmetrical peak with a 260:280 nm ratio of 0.5 was observed when monitoring the elution at 280 and 260 nm. (b) SDS–PAGE analysis of the protein composition from SEC, demonstrating the presence of a single ∼45 kDa protein (tCid1).
Figure 3
Figure 3
Crystals of (a) tCid1, produced to determine the structure with PDB code 4e7x (Yates et al., 2012 ▶), (b) mCid1 crystal form I (space group C2), (c) mCid1 crystal form II (space group P21), (d) tCid1 (K133A/R137A/R277A/K282A) mutant crystal form I (space group P1) and (e) tCid1 (K133A/R137A/R277A/K282A) mutant crystal form II (space group P21). See Table 2 ▶ for further information.
Figure 4
Figure 4
(a) Crystal structure of tCid1 (PDB entry 4e7x) highlighting the domain architecture, with the N-terminal domain (NTD) rendered in blue, the C-­terminal domain (CTD) rendered in green and the β-trap door (β-TD) feature of the structure which helps in substrate containment (Yates et al., 2012, 2015 ▶) rendered in red. The sites that are mutated in our tCid1 RNA-binding mutant are shown as side chains (spheres) and are rendered in orange. (b) The crystal packing of PDB entry 4e7x showing the location of Lys133, Arg137, Arg277 and Lys282 and how this relates to crystal packing. This crystal structure possesses four molecules per asymmetric unit, which are rendered in green (chain A), cyan (chain B), purple (chain C) and yellow (chain D). In all chains the positions of Lys133, Arg137, Arg277 and Lys282 are rendered in orange and are shown as side chains (spheres). (c, d) The crystal packing of the RNA-binding mutant crystal structures in space groups P1 (c) and P21 (d). The positions of K133A/R137A/R277A/K282A are highlighted in orange. Both crystal forms possess two molecules per asymmetric unit, which are rendered in green (chain A) and cyan (chain B) in both cases. This figure was generated using PyMOL (Schrödinger).
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