NMR structure of the C-terminal domain of a tyrosyl-tRNA synthetase that functions in group I intron splicing †

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mt TyrRS; CYT-18 protein) and those of other fungi of the subphylum Pezizomycotina are bifunctional proteins that both aminoacylate mt tRNATyr and promote the splicing of mt group I introns (1, 2). Previous studies showed that CYT-18 recognizes conserved structural features of the group I intron catalytic core and promotes splicing by stabilizing the catalytically active RNA structure (3–5). The group I intron catalytic core consists of two extended helical domains: P4–P6 consisting of stacked helices P5, P4, P6, and P6a/b, and P3–P9 consisting of helices P9, P7, P3, and P8 (6). The two domains interact via a series of tertiary contacts, with the P3–P9 domain wrapping around the P4–P6 domain forming a cleft that contains the intron RNA’s active site. This active site binds the splice sites and guanosine cofactor and uses specifically bound Mg2+ ions to catalyze splicing via guanosine-initiated transesterification reactions. Biochemical and genetic experiments suggested that CYT-18 binds first to the P4–P6 domain to promote its assembly and then makes additional contacts with the P3–P9 domain that stabilize the active RNA structure relative to alternative non-native structures (3–5, 7). The structural stabilization of the group I intron core afforded by CYT-18 compensates for RNA structural defects that impair self-splicing (3). Bacterial TyrRSs are comprised of an N-terminal nucleotide-binding fold or catalytic domain, an intermediate α-helical domain, and a C-terminal tRNA-binding domain. The latter has a fold similar to that of ribosomal protein S4 and is attached to the remainder of the protein via a flexible linker (8, 9). The functional TyrRS is a homodimer, which binds tRNATyr asymmetrically across the two subunits (10, 11). The catalytic domain of one subunit (subunit A) binds the tRNA’s acceptor stem and catalyzes aminoacylation, while the intermediate α-helical and C-terminal domains of the other subunit (subunit B) bind the tRNA’s anticodon and variable arms. Although TyrRS homodimers contain two active sites, only a single tRNATyr is bound and charged, a phenomenon known as “half-sites reactivity” (12). Mt TyrRSs are structurally homologous to the bacterial enzymes, and CYT-18 uses both its N-terminal catalytic and C-terminal tRNA-binding domains to bind group I intron RNAs (13–15). However, group I intron splicing activity has been found only for the mt TyrRSs of Pezizomycotina, filamentous fungi that includes the model organisms Neurospora crassa, Aspergillus nidulans, and Podospora anserina, as well as important human pathogens, such as Histoplasma capsulatum, Coccidioides posadasii, and Aspergillus fumigatus (2). The acquisition of group I intron splicing activity by the mt TyrRSs of these fungi can be traced to a series of structural adaptations in different regions of the protein, including a number of small “insertions”, which occurred during or after the divergence of Pezizomycotina from yeast (2, 16). As illustrated in Figure 1 for several representative examples, these Pezizomycotina-specific insertions include an α-helical N-terminal extension (H0), two small insertions in the catalytic domain (Ins1 and Ins2), three additional insertions in the C-terminal domain (Ins3, 4, and 5), and a variable length C-terminal extension (CTE). Studies with CYT-18 showed that the N-terminal domain insertions H0, Ins1 and Ins2 are required for group I intron splicing but not TyrRS activity and form part of a new group I intron-binding site distinct from that which binds tRNATyr (14, 16, 17). A co-crystal structure of a splicing active C-terminally truncated CYT-18 protein (CYT-18/Δ424–669) with a group I intron RNA (the bacteriophage Twort orf142-I2 ribozyme) revealed key features of the RNA-protein interface and showed that H0, Ins1, and Ins2 bind directly to the group I intron catalytic core in position to stabilize key tertiary interactions (18). Figure 1 Comparison of splicing-active Pezizomycotina mt TyrRSs with non-splicing bacterial and mt TyrRSs. The Pezizomycotina mt TyrRSs are distinguished by a series of insertions, including an α-helical N-terminal extension H0, Ins1 and Ins2 in the catalytic ... Thus far, there has been relatively little information about how CYT-18’s C-terminal domain contributes to group I intron splicing or about the structure and function of the C-terminal domain insertions. Although a C-terminally truncated CYT-18 protein consisting of the N-terminal catalytic and intermediate α-helical domains can splice many group I introns, the C-terminal domain contributes to group I intron binding and is essential for splicing some introns, e.g., the N. crassa mt large ribosomal subunit (Nc mt LSU) intron (14). Genetic studies showed that CYT-18’s C-terminal domain is needed to compensate for certain structural mutations that impair self-splicing of group I intron RNAs, including mutations that weaken two key long-range tertiary interactions: L9-P5, which helps establish the correct relative orientation of the two catalytic core domains, and L2-P8, which helps position the P1 helix containing the 5’-splice site at the RNA’s active site (15). Additionally, small deletions within the catalytic domain’s Ins2, which binds near the L9-P5 tetraloop-tetraloop receptor interaction, have more severe effects in the C-terminally truncated CYT-18/Δ424–669 protein than in the full-length protein, suggesting that C-terminal domain binding can compensate for loss of some N-terminal domain interactions (17). Together, these findings suggest that CYT-18’s N- and C-terminal domains both contribute to group I intron binding, with greater or lesser dependence on the C-terminal domain reflecting different structural defects that must be compensated for in each intron. Site-directed hydroxyl-radical cleavage experiments showed that CYT-18’s C-terminal domain binds near P6–P6a, P3–P8 and P5 in the Nc ND1 intron and near P6–P6a, P2, P4 and P5 in the Nc mt LSU intron (19). While CYT-18’s C-terminal domain contributes to the splicing of a number of group I introns, it inhibits the second step of splicing in some heterologous group I introns, suggesting that it co-evolved to function optimally with N. crassa mt group I introns (20). The C-terminal domain of CYT-18, like those of most bacterial TyrRSs, has been intractable to X-ray crystallography in the full-length protein, presumably due to its attachment via a flexible linker that impedes crystallization in a specific orientation (8). Here, we used heteronuclear multidimensional NMR to determine the solution structure of the closely related but smaller C-terminal domain of the splicing-active Aspergillus nidulans (An) mt TyrRS. The structure confirmed the S4-like fold, but with Pezizomycotina-specific features, including the C-terminal domain insertions, which potentially contribute to novel functions. Modeling indicated that flexible attachment of the C-terminal domain is critical for its ability to interact with mt tRNATyr and group I intron RNAs on opposite sides of the catalytic domain. Surprisingly, however, NMR experiments showed that the C-terminal domains of the full-length An mt TyrRS and GeoBacillus stearothermophilus (Gs) TyrRSs do not tumble independently, implying that their attachment to the remainder of the protein is less flexible than was believed previously.