Abstract
Genome sequencing projects have focused attention on the problem of discovering the functions of protein domains that are widely distributed throughout living species but which are, as yet, largely uncharacterized. One such example is the PIN domain, found in eukaryotes, bacteria, and Archaea, and with suggested roles in signaling, RNase editing, and/or nucleotide binding. The first reported crystal structure of a PIN domain (open reading frame PAE2754, derived from the crenarchaeon, Pyrobaculum aerophilum) has been determined to 2.5 A resolution and is presented here. Mapping conserved residues from a multiple sequence alignment onto the structure identifies a putative active site. The discovery of distant structural homology with several exonucleases, including T4 phage RNase H and flap endonuclease (FEN1), further suggests a likely function for PIN domains as Mg2+-dependent exonucleases, a hypothesis that we have confirmed in vitro. The tetrameric structure of PAE2754, with the active sites inside a tunnel, suggests a mechanism for selective cleavage of single-stranded overhangs or flap structures. These results indicate likely DNA or RNA editing roles for prokaryotic PIN domains, which are strikingly numerous in thermophiles, and in organisms such as Mycobacterium tuberculosis. They also support previous hypotheses that eukaryotic PIN domains participate in RNAi and nonsense-mediated RNA degradation.
Highlights
The explosive growth of whole genome sequencing efforts, and the discovery that a large proportion of the assumed gene products are of unknown or poorly understood function, has focused attention on new approaches to assigning function
The tetrameric structure of PAE2754, with the active sites inside a tunnel, suggests a mechanism for selective cleavage of single-stranded overhangs or flap structures. These results indicate likely DNA or RNA editing roles for prokaryotic PIN domains, which are strikingly numerous in thermophiles, and in organisms such as Mycobacterium tuberculosis
As part of a pilot structural genomics project aimed at the discovery of biological function, we have focused on gene products from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum, an organism whose complete genome sequence was published recently [7]
Summary
Protein Expression, Purification, and Crystallization—The predicted open reading frame PAE2754 was amplified from genomic DNA by PCR, subcloned into the expression vector pPROeX (Invitrogen), transformed into E. coli BL21(DE3) cells, and expressed as an N-terminal His6-tagged protein. The structure of PAE2754_MM was determined by single anomalous diffraction using a single SeMet data set at ϭ 0.9794 Å This is not where the anomalous differences are maximized for selenium, but this was necessary because the “remote” wavelength data set proved to be of poor quality due to crystal decay (data were initially collected at two wavelengths in accordance with Ref. 15). Using SOLVE [16, 17], a total of 17 out of a possible 24 selenium sites (from the 8 monomers in the crystal asymmetric unit) were located, based on anomalous differences These gave initial phases to 2.8 Å with a figure of merit of 0.18 and a Z-score of 20.6. Samples were prepared in the same way using MnCl2 as the metal ion source
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