RNA Repair: Hiding in Plain Sight.

Enzymes that phosphorylate, dephosphorylate, and ligate RNA 5' and 3' ends were discovered more than half a century ago and were eventually shown to repair purposeful site-specific endonucleolytic breaks in the RNA phosphodiester backbone. The pace of discovery and characterization of new candidate RNA repair activities in taxa from all phylogenetic domains greatly exceeds our understanding of the biological pathways in which they act. The key questions anent RNA break repair in vivo are (a) identifying the triggers, agents, and targets of RNA cleavage and (b) determining whether RNA repair results in restoration of the original RNA, modification of the RNA (by loss or gain at the ends), or rearrangements of the broken RNA segments (i.e., RNA recombination). This review provides a perspective on the discovery, mechanisms, and physiology of purposeful RNA break repair, highlighting exemplary repair pathways (e.g., tRNA restriction-repair and tRNA splicing) for which genetics has figured prominently in their elucidation.

RNA circles with minimized immunogenicity as potent PKR inhibitors.

Exon back-splicing-generated circular RNAs, as a group, can suppress double-stranded RNA (dsRNA)-activated protein kinase R (PKR) in cells. We have sought to synthesize immunogenicity-free, short dsRNA-containing RNA circles as PKR inhibitors. Here, we report that RNA circles synthesized by permuted self-splicing thymidylate synthase (td) introns from T4 bacteriophage or by Anabaena pre-tRNA group I intron could induce an immune response. Autocatalytic splicing introduces ∼74 nt td or ∼186 nt Anabaena extraneous fragments that can distort the folding status of original circular RNAs or form structures themselves to provoke innate immune responses. In contrast, synthesized RNA circles produced by T4 RNA ligase without extraneous fragments exhibit minimized immunogenicity. Importantly, directly ligated circular RNAs that form short dsRNA regions efficiently suppress PKR activation 103- to 106-fold higher than reported chemical compounds C16 and 2-AP, highlighting the future use of circular RNAs as potent inhibitors for diseases related to PKR overreaction.

The oxidoreductase PYROXD1 uses NAD(P)+ as an antioxidant to sustain tRNA ligase activity in pre-tRNA splicing and unfolded protein response.

The tRNA ligase complex (tRNA-LC) splices precursor tRNAs (pre-tRNA), and Xbp1-mRNA during the unfolded protein response (UPR). In aerobic conditions, a cysteine residue bound to two metal ions in its ancient, catalytic subunit RTCB could make the tRNA-LC susceptible to oxidative inactivation. Here, we confirm this hypothesis and reveal a co-evolutionary association between the tRNA-LC and PYROXD1, a conserved and essential oxidoreductase. We reveal that PYROXD1 preserves the activity of the mammalian tRNA-LC in pre-tRNA splicing and UPR. PYROXD1 binds the tRNA-LC in the presence of NAD(P)H and converts RTCB-bound NAD(P)H into NAD(P)+, a typical oxidative co-enzyme. However, NAD(P)+ here acts as an antioxidant and protects the tRNA-LC from oxidative inactivation, which is dependent on copper ions. Genetic variants of PYROXD1 that cause human myopathies only partially support tRNA-LC activity. Thus, we establish the tRNA-LC as an oxidation-sensitive metalloenzyme, safeguarded by the flavoprotein PYROXD1 through an unexpected redox mechanism.

Evolution of the substrate specificity of an RNA ligase ribozyme from phosphorimidazole to triphosphate activation.

The acquisition of new RNA functions through evolutionary processes was essential for the diversification of RNA-based primordial biology and its subsequent transition to modern biology. However, the mechanisms by which RNAs access new functions remain unclear. Do RNA enzymes need completely new folds to support new but related functions, or is reoptimization of the active site sufficient? What are the roles of neutral and adaptive mutations in evolutionary innovation? Here, we address these questions experimentally by focusing on the evolution of substrate specificity in RNA-catalyzed RNA assembly. We use directed in vitro evolution to show that a ligase ribozyme that uses prebiotically relevant 5'-phosphorimidazole-activated substrates can be evolved to catalyze ligation with substrates that are 5'-activated with the biologically relevant triphosphate group. Interestingly, despite catalyzing a related reaction, the new ribozyme folds into a completely new structure and exhibits promiscuity by catalyzing RNA ligation with both triphosphate and phosphorimidazole-activated substrates. Although distinct in sequence and structure, the parent phosphorimidazolide ligase and the evolved triphosphate ligase ribozymes can be connected by a series of point mutations where the intermediate sequences retain at least some ligase activity. The existence of a quasi-neutral pathway between these distinct ligase ribozymes suggests that neutral drift is sufficient to enable the acquisition of new substrate specificity, thereby providing opportunities for subsequent adaptive optimization. The transition from RNA-catalyzed RNA assembly using phosphorimidazole-activated substrates to triphosphate-activated substrates may have foreshadowed the later evolution of the protein enzymes that use monomeric triphosphates (nucleoside triphosphates, NTPs) for RNA synthesis.

Biochemical and structural insights into a 5' to 3' RNA ligase reveal a potential role in tRNA ligation.

ATP-grasp superfamily enzymes contain a hand-like ATP-binding fold and catalyze a variety of reactions using a similar catalytic mechanism. More than 30 protein families are categorized in this superfamily, and they are involved in a plethora of cellular processes and human diseases. Here, we identify C12orf29 (RLIG1) as an atypical ATP-grasp enzyme that ligates RNA. Human RLIG1 and its homologs autoadenylate on an active site Lys residue as part of a reaction intermediate that specifically ligates RNA halves containing a 5'-phosphate and a 3'-hydroxyl. RLIG1 binds tRNA in cells and can ligate tRNA within the anticodon loop in vitro. Transcriptomic analyses of Rlig1 knockout mice revealed significant alterations in global tRNA levels in the brains of female mice, but not in those of male mice. Furthermore, crystal structures of a RLIG1 homolog from Yasminevirus bound to nucleotides revealed a minimal and atypical RNA ligase fold with a conserved active site architecture that participates in catalysis. Collectively, our results identify RLIG1 as an RNA ligase and suggest its involvement in tRNA biology.

Cross-chiral exponential amplification of an RNA enzyme.

An RNA ligase ribozyme that catalyzes the joining of RNA molecules of the opposite chiral handedness was optimized for the ability to synthesize its own enantiomer from two component fragments. The mirror-image D- and L-ligases operate in concert to provide a system for cross-chiral replication, whereby they catalyze each other's synthesis and undergo mutual amplification at constant temperature, with apparent exponential growth and a doubling time of about 1 h. Neither the D- nor the L-RNA components alone can achieve autocatalytic self-replication. Cross-chiral exponential amplification can be continued indefinitely through a serial-transfer process that provides an ongoing supply of the component D- and L-substrates. Unlike the familiar paradigm of semiconservative nucleic acid replication that relies on Watson-Crick pairing between complementary strands, cross-chiral replication relies on tertiary interactions between structured nucleic acids "across the mirror." There are few examples, outside of biology, of autocatalytic self-replication systems that undergo exponential amplification and there are no prior examples, in either biological or chemical systems, of cross-chiral replication enabling exponential amplification.

Characterization of tRNA splicing enzymes RNA ligase and tRNA 2'-phosphotransferase from the pathogenic fungi Mucorales.

Fungal Trl1 is an essential trifunctional tRNA splicing enzyme that heals and seals tRNA exons with 2',3'-cyclic-PO4 and 5'-OH ends. Trl1 is composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase end-healing domains that generate the 3'-OH,2'-PO4 and 5'-PO4 termini required for sealing by an N-terminal ATP-dependent ligase domain. Trl1 enzymes are present in many human fungal pathogens and are promising targets for antifungal drug discovery because their domain structures and biochemical mechanisms are unique compared to the mammalian RtcB-type tRNA splicing enzyme. Here we report that Mucorales species (deemed high-priority human pathogens by WHO) elaborate a noncanonical tRNA splicing apparatus in which a monofunctional RNA ligase enzyme is encoded separately from any end-healing enzymes. We show that Mucor circinelloides RNA ligase (MciRNL) is active in tRNA splicing in vivo in budding yeast in lieu of the Trl1 ligase domain. Biochemical and kinetic characterization of recombinant MciRNL underscores its requirement for a 2'-PO4 terminus in the end-joining reaction, whereby the 2'-PO4 enhances the rates of RNA 5'-adenylylation (step 2) and phosphodiester synthesis (step 3) by ∼125-fold and ∼6200-fold, respectively. In the canonical fungal tRNA splicing pathway, the splice junction 2'-PO4 installed by RNA ligase is removed by a dedicated NAD+-dependent RNA 2'-phosphotransferase Tpt1. Here we identify and affirm by genetic complementation in yeast the biological activity of Tpt1 orthologs from three Mucorales species. Recombinant M. circinelloides Tpt1 has vigorous NAD+-dependent RNA 2'-phosphotransferase activity in vitro.

Fungi of the order Mucorales express a "sealing-only" tRNA ligase.

Some eukaryotic pre-tRNAs contain an intron that is removed by a dedicated set of enzymes. Intron-containing pre-tRNAs are cleaved by tRNA splicing endonuclease, followed by ligation of the two exons and release of the intron. Fungi use a "heal and seal" pathway that requires three distinct catalytic domains of the tRNA ligase enzyme, Trl1. In contrast, humans use a "direct ligation" pathway carried out by RTCB, an enzyme completely unrelated to Trl1. Because of these mechanistic differences, Trl1 has been proposed as a promising drug target for fungal infections. To validate Trl1 as a broad-spectrum drug target, we show that fungi from three different phyla contain Trl1 orthologs with all three domains. This includes the major invasive human fungal pathogens, and these proteins can each functionally replace yeast Trl1. In contrast, species from the order Mucorales, including the pathogens Rhizopus arrhizus and Mucor circinelloides, have an atypical Trl1 that contains the sealing domain but lacks both healing domains. Although these species contain fewer tRNA introns than other pathogenic fungi, they still require splicing to decode three of the 61 sense codons. These sealing-only Trl1 orthologs can functionally complement defects in the corresponding domain of yeast Trl1 and use a conserved catalytic lysine residue. We conclude that Mucorales use a sealing-only enzyme together with unidentified nonorthologous healing enzymes for their heal and seal pathway. This implies that drugs that target the sealing activity are more likely to be broader-spectrum antifungals than drugs that target the healing domains.

Kinetic and structural insights into the requirement of fungal tRNA ligase for a 2'-phosphate end.

Fungal RNA ligase (LIG) is an essential tRNA splicing enzyme that joins 3'-OH,2'-PO4 and 5'-PO4 RNA ends to form a 2'-PO4,3'-5' phosphodiester splice junction. Sealing entails three divalent cation-dependent adenylate transfer steps. First, LIG reacts with ATP to form a covalent ligase-(lysyl-Nζ)-AMP intermediate and displace pyrophosphate. Second, LIG transfers AMP to the 5'-PO4 RNA terminus to form an RNA-adenylate intermediate (A5'pp5'RNA). Third, LIG directs the attack of an RNA 3'-OH on AppRNA to form the splice junction and displace AMP. A defining feature of fungal LIG vis-à-vis canonical polynucleotide ligases is the requirement for a 2'-PO4 to synthesize a 3'-5' phosphodiester bond. Fungal LIG consists of an N-terminal adenylyltransferase domain and a unique C-terminal domain. The C-domain of Chaetomium thermophilum LIG (CthLIG) engages a sulfate anion thought to be a mimetic of the terminal 2'-PO4 Here, we interrogated the contributions of the C-domain and the conserved sulfate ligands (His227, Arg334, Arg337) to ligation of a pRNA2'p substrate. We find that the C-domain is essential for end-joining but dispensable for ligase adenylylation. Mutations H227A, R334A, and R337A slowed the rate of step 2 RNA adenylation by 420-fold, 120-fold, and 60-fold, respectively, vis-à-vis wild-type CthLIG. An R334A-R337A double-mutation slowed step 2 by 580-fold. These results fortify the case for the strictly conserved His-Arg-Arg triad as the enforcer of the 2'-PO4 end-specificity of fungal tRNA ligases and as a target for small molecule interdiction of fungal tRNA splicing.

Characterisation and engineering of a thermophilic RNA ligase from Palaeococcus pacificus.

RNA ligases are important enzymes in molecular biology and are highly useful for the manipulation and analysis of nucleic acids, including adapter ligation in next-generation sequencing of microRNAs. Thermophilic RNA ligases belonging to the RNA ligase 3 family are gaining attention for their use in molecular biology, for example a thermophilic RNA ligase from Methanobacterium thermoautotrophicum is commercially available for the adenylation of nucleic acids. Here we extensively characterise a newly identified RNA ligase from the thermophilic archaeon Palaeococcus pacificus (PpaRnl). PpaRnl exhibited significant substrate adenylation activity but low ligation activity across a range of oligonucleotide substrates. Mutation of Lys92 in motif I to alanine, resulted in an enzyme that lacked adenylation activity, but demonstrated improved ligation activity with pre-adenylated substrates (ATP-independent ligation). Subsequent structural characterisation revealed that in this mutant enzyme Lys238 was found in two alternate positions for coordination of the phosphate tail of ATP. In contrast mutation of Lys238 in motif V to glycine via structure-guided engineering enhanced ATP-dependent ligation activity via an arginine residue compensating for the absence of Lys238. Ligation activity for both mutations was higher than the wild-type, with activity observed across a range of oligonucleotide substrates with varying sequence and secondary structure.

RTP801 interacts with the tRNA ligase complex and dysregulates its RNA ligase activity in Alzheimer's disease.

RTP801/REDD1 is a stress-responsive protein overexpressed in neurodegenerative diseases such as Alzheimer's disease (AD) that contributes to cognitive deficits and neuroinflammation. Here, we found that RTP801 interacts with HSPC117, DDX1 and CGI-99, three members of the tRNA ligase complex (tRNA-LC), which ligates the excised exons of intron-containing tRNAs and the mRNA exons of the transcription factor XBP1 during the unfolded protein response (UPR). We also found that RTP801 modulates the mRNA ligase activity of the complex in vitro since RTP801 knockdown promoted XBP1 splicing and the expression of its transcriptional target, SEC24D. Conversely, RTP801 overexpression inhibited the splicing of XBP1. Similarly, in human AD postmortem hippocampal samples, where RTP801 is upregulated, we found that XBP1 splicing was dramatically decreased. In the 5xFAD mouse model of AD, silencing RTP801 expression in hippocampal neurons promoted Xbp1 splicing and prevented the accumulation of intron-containing pre-tRNAs. Finally, the tRNA-enriched fraction obtained from 5xFAD mice promoted abnormal dendritic arborization in cultured hippocampal neurons, and RTP801 silencing in the source neurons prevented this phenotype. Altogether, these results show that elevated RTP801 impairs RNA processing in vitro and in vivo in the context of AD and suggest that RTP801 inhibition could be a promising therapeutic approach.

Regulation of Archease by the mTOR-vATPase axis.

Larval terminal cells of the Drosophila tracheal system generate extensive branched tubes, requiring a huge increase in apical membrane. We discovered that terminal cells compromised for apical membrane expansion - mTOR-vATPase axis and apical polarity mutants - were invaded by the neighboring stalk cell. The invading cell grows and branches, replacing the original single intercellular junction between stalk and terminal cell with multiple intercellular junctions. Here, we characterize disjointed, a mutation in the same phenotypic class. We find that disjointed encodes Drosophila Archease, which is required for the RNA ligase (RtcB) function that is essential for tRNA maturation and for endoplasmic reticulum stress-regulated nonconventional splicing of Xbp1 mRNA. We show that the steady-state subcellular localization of Archease is principally nuclear and dependent upon TOR-vATPase activity. In tracheal cells mutant for Rheb or vATPase loci, Archease localization shifted dramatically from nucleus to cytoplasm. Further, we found that blocking tRNA maturation by knockdown of tRNAseZ also induced compensatory branching. Taken together, these data suggest that the TOR-vATPase axis promotes apical membrane growth in part through nuclear localization of Archease, where Archease is required for tRNA maturation.

Thioredoxin regulates the redox state and the activity of the human tRNA ligase complex.

The mammalian tRNA ligase complex (tRNA-LC) catalyzes the splicing of intron-containing pre-tRNAs in the nucleus and the splicing of XBP1 mRNA during the unfolded protein response (UPR) in the cytoplasm. We recently reported that the tRNA-LC coevolved with PYROXD1, an essential oxidoreductase that protects the catalytic cysteine of RTCB, the catalytic subunit of the tRNA-LC, against aerobic oxidation. In this study, we show that the oxidoreductase Thioredoxin (TRX) preserves the enzymatic activity of RTCB under otherwise inhibiting concentrations of oxidants. TRX physically interacts with oxidized RTCB, and reduces and reactivates RTCB through the action of its redox-active cysteine pair. We further show that TRX interacts with RTCB at late stages of UPR. Since the interaction requires oxidative conditions, our findings suggest that prolonged UPR generates reactive oxygen species. Thus, our results support a functional role for TRX in securing and repairing the active site of the tRNA-LC, thereby allowing pre-tRNA splicing and UPR to occur when cells encounter mild, but still inhibitory levels of reactive oxygen species.

Structures of RNA ligase RtcB in complexes with divalent cations and GTP.

Pyrococcus horikoshii (Pho) RtcB exemplifies a family of binuclear transition metal- and GTP-dependent RNA ligases that join 3'-phosphate and 5'-OH ends via RtcB-(histidinyl-N)-GMP and RNA3'pp5'G intermediates. We find that guanylylation of PhoRtcB is optimal with manganese and less effective with cobalt and nickel. Zinc and copper are inactive and potently inhibit manganese-dependent guanylylation. We report crystal structures of PhoRtcB in complexes with GTP and permissive (Mn, Co, Ni) or inhibitory (Zn, Cu) metals. Zinc and copper occupy the M1 and M2 sites adjacent to the GTP phosphates, as do manganese, cobalt, and nickel. The identity/positions of enzymic ligands for M1 (His234, His329, Cys98) and M2 (Cys98, Asp95, His203) are the same for permissive and inhibitory metals. The differences pertain to: (i) the coordination geometries and phosphate contacts of the metals; and (ii) the orientation of the His404 nucleophile with respect to the GTP α-phosphate and pyrophosphate leaving group. M2 metal coordination geometry correlates with metal cofactor activity, whereby inhibitory Zn2 and Cu2 assume a tetrahedral configuration and contact only the GTP γ-phosphate, whereas Mn2, Co2, and Ni2 coordination complexes are pentahedral and contact the ß- and γ-phosphates. The His404-Nε-Pα-O(α-ß) angle is closer to apical in Mn (179°), Co (171°), and Ni (169°) structures than in Zn (160°) and Cu (155°) structures. The octahedral Mn1 geometry in our RtcB•GTP•Mn2+ structure, in which Mn1 contacts α-, ß-, and γ-phosphates, transitions to a tetrahedral configuration after formation of RtcB•(His404)-GMP•Mn2+ and departure of pyrophosphate.

Insights into the structure and function of the RNA ligase RtcB.

To be functional, some RNAs require a processing step involving splicing events. Each splicing event necessitates an RNA ligation step. RNA ligation is a process that can be achieved with various intermediaries such as self-catalysing RNAs, 5'-3' and 3'-5' RNA ligases. While several types of RNA ligation mechanisms occur in human, RtcB is the only 3'-5' RNA ligase identified in human cells to date. RtcB RNA ligation activity is well known to be essential for the splicing of XBP1, an essential transcription factor of the unfolded protein response; as well as for the maturation of specific intron-containing tRNAs. As such, RtcB is a core factor in protein synthesis and homeostasis. Taking advantage of the high homology between RtcB orthologues in archaea, bacteria and eukaryotes, this review will provide an introduction to the structure of RtcB and the mechanism of 3'-5' RNA ligation. This analysis is followed by a description of the mechanisms regulating RtcB activity and localisation, its known partners and its various functions from bacteria to human with a specific focus on human cancer.

RNA specificity and regulation of catalysis in the eukaryotic polynucleotide kinase Clp1.

RNA-specific polynucleotide kinases of the Clp1 subfamily are key components of various RNA maturation pathways. However, the structural basis explaining their substrate specificity and the enzymatic mechanism is elusive. Here, we report crystal structures of Clp1 from Caenorhabditis elegans (ceClp1) in a number of nucleotide- and RNA-bound states along the reaction pathway. The combined structural and biochemical analysis of ceClp1 elucidates the RNA specificity and lets us derive a general model for enzyme catalysis of RNA-specific polynucleotide kinases. We identified an RNA binding motif referred to as "clasp" as well as a conformational switch that involves the essential Walker A lysine (Lys127) and regulates the enzymatic activity of ceClp1. Structural comparison with other P loop proteins, such as kinases, adenosine triphosphatases (ATPases), and guanosine triphosphatases (GTPases), suggests that the observed conformational switch of the Walker A lysine is a broadly relevant mechanistic feature.

Caveat mutator: alanine substitutions for conserved amino acids in RNA ligase elicit unexpected rearrangements of the active site for lysine adenylylation.

Naegleria gruberi RNA ligase (NgrRnl) exemplifies the Rnl5 family of adenosine triphosphate (ATP)-dependent polynucleotide ligases that seal 3'-OH RNA strands in the context of 3'-OH/5'-PO4 nicked duplexes. Like all classic ligases, NgrRnl forms a covalent lysyl-AMP intermediate. A two-metal mechanism of lysine adenylylation was established via a crystal structure of the NgrRnl•ATP•(Mn2+)2 Michaelis complex. Here we conducted an alanine scan of active site constituents that engage the ATP phosphates and the metal cofactors. We then determined crystal structures of ligase-defective NgrRnl-Ala mutants in complexes with ATP/Mn2+. The unexpected findings were that mutations K170A, E227A, K326A and R149A (none of which impacted overall enzyme structure) triggered adverse secondary changes in the active site entailing dislocations of the ATP phosphates, altered contacts to ATP, and variations in the numbers and positions of the metal ions that perverted the active sites into off-pathway states incompatible with lysine adenylylation. Each alanine mutation elicited a distinctive off-pathway distortion of the ligase active site. Our results illuminate a surprising plasticity of the ligase active site in its interactions with ATP and metals. More broadly, they underscore a valuable caveat when interpreting mutational data in the course of enzyme structure-function studies.

Healing for destruction: tRNA intron degradation in yeast is a two-step cytoplasmic process catalyzed by tRNA ligase Rlg1 and 5'-to-3' exonuclease Xrn1.

In eukaryotes and archaea, tRNA splicing generates free intron molecules. Although ∼ 600,000 introns are produced per generation in yeast, they are barely detectable in cells, indicating efficient turnover of introns. Through a genome-wide search for genes involved in tRNA biology in yeast, we uncovered the mechanism for intron turnover. This process requires healing of the 5' termini of linear introns by the tRNA ligase Rlg1 and destruction by the cytoplasmic tRNA quality control 5'-to-3' exonuclease Xrn1, which has specificity for RNAs with 5' monophosphate.

Systematic minimization of RNA ligase ribozyme through large-scale design-synthesis-sequence cycles.

Template-directed RNA ligation catalyzed by an RNA enzyme (ribozyme) is a plausible and important reaction that could have been involved in transferring genetic information during prebiotic evolution. Laboratory evolution experiments have yielded several classes of ligase ribozymes, but their minimal sequence requirements remain largely unexplored. Because selection experiments strongly favor highly active sequences, less active but smaller catalytic motifs may have been overlooked in these experiments. We used large-scale DNA synthesis and high-throughput ribozyme assay enabled by deep sequencing to systematically minimize a previously laboratory-evolved ligase ribozyme. After designing and evaluating >10 000 sequences, we identified catalytic cores as small as 18 contiguous bases that catalyze template-directed regiospecific RNA ligation. The fact that such a short sequence can catalyze this critical reaction suggests that similarly simple or even simpler motifs may populate the RNA sequence space which could have been accessible to the prebiotic ribozymes.

Atomic structures of the RNA end-healing 5'-OH kinase and 2',3'-cyclic phosphodiesterase domains of fungal tRNA ligase: conformational switches in the kinase upon binding of the GTP phosphate donor.

Fungal tRNA ligase (Trl1) rectifies RNA breaks with 2',3'-cyclic-PO4 and 5'-OH termini. Trl1 consists of three catalytic modules: an N-terminal ligase (LIG) domain; a central polynucleotide kinase (KIN) domain; and a C-terminal cyclic phosphodiesterase (CPD) domain. Trl1 enzymes found in all human fungal pathogens are untapped targets for antifungal drug discovery. Here we report a 1.9 Å crystal structure of Trl1 KIN-CPD from the pathogenic fungus Candida albicans, which adopts an extended conformation in which separate KIN and CPD domains are connected by an unstructured linker. CPD belongs to the 2H phosphotransferase superfamily by dint of its conserved central concave ß sheet and interactions of its dual HxT motif histidines and threonines with phosphate in the active site. Additional active site motifs conserved among the fungal CPD clade of 2H enzymes are identified. We present structures of the Candida Trl1 KIN domain at 1.5 to 2.0 Å resolution-as apoenzyme and in complexes with GTP•Mg2+, IDP•PO4, and dGDP•PO4-that highlight conformational switches in the G-loop (which recognizes the guanine base) and lid-loop (poised over the nucleotide phosphates) that accompany nucleotide binding.