Structural and functional characterization of Clp1, a eukaryotic RNA‐specific polynucleotide kinase

Polynucleotide kinases (PNKs) are crucial enzymes involved in DNA and RNA repair, RNA maturation, as well as in RNA degradation processes. These enzymes have a conserved PNK domain, showing structural homology to the classical fold of P-loop kinases. PNKs catalyse the transfer of the γ-phosphate group of ATP molecule to the 5’-hydroxyl group of polynucleotide substrates. Depending on their in vivo function, PNKs show different substrate specificity. Interestingly, eukaryotic PNKs have recently been identified that specifically phosphorylate RNA substrates. These novel RNA-specific PNKs constitute the Clp1 subfamily of PNKs named after their first identified member. Human Clp1 was shown to participate in various RNA maturation pathways: (i) cleavage and polyadenylation of RNA polymerase II pre-mRNA transcripts, (ii) tRNA-splicing, and (iii) phosphorylation of synthetic siRNAs. Despite extensive studies on Clp1, the structural elements involved in RNA-specificity and the mechanism of the phosphoryl transfer reaction have remained elusive so far. This thesis, therefore, aims for the structural and functional characterization of the RNA-specific Clp1 PNK. During this work, the three-dimensional structure of Clp1 from Caenorhabditis elegans was described for the first time at atomic resolution. Clp1 is a multi-domain protein that consists of a central PNK domain sandwiched by dditional N- and C-terminal domains. Clp1 was crystallized with various RNA substrates that differ in length and sequence. Based on these results, the structural features of Clp1’s RNA-specificity were elucidated. Clp1 uses an “RNA-sensor” that recognizes the 2’-hydroxyl group of the RNA at the ultimate position. Additionally, Clp1 was also crystallized in enzymatically relevant states such as an inhibited substrate bound state, a transition state analog and a product bound state. A general model for enzyme catalysis of PNKs was derived from these structures. In contrast to other described PNKs, Clp1’s ATP-binding site within the PNK domain is obstructed by the N-terminal domain. The crystal structures as well as activity assays with truncated variants of Clp1 showed a contribution of the N-terminal domain to ATP-binding by interactions with the nucleobase. The phosphate groups of the ATP molecule are anchored in the active site tunnel, which is formed by the common P-loop motif, a divalent metal cofactor (Mg2+), and an α-helical LID module. Structure-guided mutational analysis identified the essential role of the Walker A lysine for enzyme catalysis. Moreover, Clp1 crystal structures revealed a non-canonical Walker A lysine in an “arrested” conformation that acts as a molecular switch. Activation of the switch is only achieved in the transition state complex. In contrast to other nucleotide kinases, Clp1 seems to apply a substrate-gating mechanism that prevents futile ATP hydrolysis. In this context, the classical Walker A lysine seems to have a so far underestimated regulatory function. Such a molecular switch mechanism of the Walker A lysine is not restricted to Clp1 exclusively, since the PDB database provides a significant number of crystal structures showing a similar “arrested“ conformation of the Walker A lysine. Thus, an additional function of the Walker A lysine as a molecular switch in enzyme catalysis is suggested. In conclusion, this thesis provides the first crystal structures of Clp1, elucidating its RNA-specificity as well as the phosphoryl transfer reaction mechanism