RNA Specificity and Regulation of Catalysis in the Eukaryotic Polynucleotide Kinase Clp1

• ceClp1 structures along the reaction pathway provide a molecular movie of catalysis
• Structural basis for substrate specificity of RNA-specific polynucleotide kinases
• Tracking catalytically important residues in ceClp1 that are missing in scClp1
• ceClp1 regulates enzyme catalysis by a conformational switch of the Walker A lysine

SummaryRNA-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.

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