Three-dimensional structure determined for a subunit of human tRNA splicing endonuclease (Sen15) reveals a novel dimeric fold

Abstract

Splicing of eukaryal intron-containing tRNAs requires the action of the heterotetrameric splicing endonuclease, which is composed of two catalytic subunits, Sen34 and Sen2, and two structural subunits, Sen15 and Sen54. Here we report the solution structure of the human tRNA splicing endonuclease subunit HsSen15. To facilitate the structure determination, we removed the disordered 35 N-terminal and 14 C-terminal residues of the full-length protein to produce HsSen15(36-157). The structure of HsSen15(36-157), the first for a subunit of a eukaryal splicing endonuclease, revealed that the protein possesses a novel homodimeric fold. Each monomer consists of three alpha-helices and a mixed antiparallel/parallel beta-sheet, arranged in a topology similar to that of the C-terminal domain of Methanocaldococcus jannaschii endonuclease. The dimeric interface is dominated by a beta-barrel structure, formed by face-to-face packing of two, three-stranded beta-sheets. Each of the beta-sheets results from reciprocal parallel pairing of one beta-strand from one subunit with two other beta-strands from the symmetric subunit. The structural model provides insights into the functional assembly of the human tRNA splicing endonuclease.

Figure 1

Correlation between the observed backbone NH dipolar couplings and couplings predicted from the alignment tensor obtained from the singular value decomposition (SVD) fit to the lowest-energy conformer of HsSen15(36–157) selected from the 20 conformers derived from the set of constraints that included residual dipolar couplings (RDCs).

Figure 2

NMR solution structure of the homodimeric HsSen15(36–157). (a) Stereoscopic view of the backbone trace of the final family of 20 conformers representing the structure with individual subunits colored red and green. (b) Ribbon diagram of the HsSen15(36–157) structure, with individual elements of secondary structure labeled. For clarity, the disordered nine-residue N-terminal tag is not shown. (c) Schematic of the secondary structure topology. The color schemes in (b) and (c) are the same as in (a).

Figure 3

(a) Ribbon diagram of the HsSen15(36–157) structure oriented with regard to three principal axes of alignment tensor. (b) The dimer interfaces of HsSen15(36–157): the side chains of selected residues from one monomer are colored in purple and labeled in red, and those from the symmetric subunit are colored in cyan and labeled in black. Otherwise the color schemes are the same as in Figure 2.

Figure 4

Multiple sequence alignment of selected splicing endonucleases. Identical and homologous residues are highlighted in green and cyan, respectively. The completely conserved tyrosine is indicated by red lettering highlighted in yellow. The secondary structural elements determined for HsSen15(36–157) and MJ_endo are shown above and below the alignment respectively. Swiss-Prot ID: HsSen15, Q8WW01; ScSen15, Q04675; SpSen15, Q7LKV3; ST_endo2, Q975V0; HsSen34, Q9BSV6; ST_endo1, Q975R3 ; MJ_endo, Q58819. The two red asterisks represent the start and end of the protein product HsSen15(36–157).

Figure 5

Comparison of the orientation of the two-fold symmetry axis between HsSen15(36–157) and MJ_endo. In MJ_endo, the C-terminal domains (residues 85–179) are painted red and green, while the N-terminal domains (residues 9–84) are painted grey. The two-fold axes and equivalent structural elements responsible for dimerization are labeled.

Figure 6

Representation of the surface electrostatic potential, with positive regions in blue and negative regions in red as calculated by the program MOLMOL.