1. Volume 54, Issue 6, Pages 975-986 (June 2014)
RNA Specificity and Regulation of Catalysis in the Eukaryotic Polynucleotide Kinase Clp1 
Aytac Dikfidan, Bernhard Loll, Cathleen Zeymer, Iris Magler, Tim Clausen, Anton Meinhart 
Molecular Cell 
Volume 54, Issue 6, Pages (June 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 54, 975-986DOI: (10.1016/j.molcel.2014.04.005)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1 RPNK Activity and Overall Architecture of ceClp1
(A) The kinase activity of ceClp1 was characterized in a gel-based phosphorylation assay. ceClp1 was incubated with various RNA and DNA oligonucleotide substrates (Table S6). Substrate molecules are either a single-stranded oligonucleotide, a double-stranded oligonucleotide containing a blunt end, or a double-stranded oligonucleotide with a 3′ overhang. T4 PNK reaction samples are shown as positive controls (Ctrl). ceClp1 has significant kinase activity upon incubation with ssRNA, whereas ssDNA was not efficiently phosphorylated. Notably, after 1 hr incubation we observed phosphorylation of ssDNA as well (Figure S1A). ceClp1 displays enzymatic activity on double-stranded oligonucleotides with blunt ends; however, RNA is more efficiently phosphorylated. Double-stranded RNA oligonucleotides containing a 3′ overhang were poor substrates for ceClp1, and respective DNA molecules were not phosphorylated. Note that the double band of RNA results from partial rehybridization during PAGE.
(B) Architecture of ceClp1⋅GC⋅AMP-PNP⋅Mg2+. The left panel shows a ribbon representation of ceClp1 with the NtD (green), PNK (cyan), and CtD (red). Structurally important motifs for ligand binding and/or catalysis are colored: P loop (purple), clasp (orange), lid (blue), and Walker B Asp151, the catalytic base (black). The ATP and the GC dinucleotide are shown in the yellow stick representation, and the Mg2+ ion (purple) and coordinating water molecules (red) are shown as spheres. See also Figure S2.
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4. Figure 2 RNA-Binding and Minimal Substrate Requirements of ceClp1
(A) Steady-state enzyme kinetics performed at constant ATP and protein concentrations. The RNA dinucleotide (GC) (KM = 109 ± 11 μM and kcat = 2.9 ± 0.1 s−1) is phosphorylated as efficiently as the longer GAAAA ribonucleotide (KM = 99 ± 25 μM and kcat = 2.6 ± 0.3 s−1). Black lines illustrate the fits to the data.
(B) The active site of ceClp1⋅GC⋅AMP-PNP⋅Mg2+ showing residues in stick representation important for substrate binding and enzyme catalysis. Motifs for ligand binding and/or catalysis are color coded as in Figure 1. The 5′ base of the RNA (yellow) is bound by the clasp residue Trp233. Moreover, the bridging phosphate group is recognized by Gln154, Arg293, and Arg297. Opposite of the RNA binding site, the adenosine moiety of AMP-PNP interacts mainly with residues Glu16, Phe39, and Arg56 of the NtD (light gray), whereas the triphosphate group is bound in a narrow pocket bordered by P loop, the clasp, and the lid motif. Hydrogen bonds and salt bridges discussed in the manuscript are shown as black dashed lines. The coordination of the magnesium ion is highlighted by dotted lines.
(C) RNA-binding site in the structure of ceClp1⋅GAAA⋅ADP⋅Mg2+. The RNA tetranucleotide GAAA is shown in the yellow stick representation. The base of guanine is sandwiched between the adjacent adenine and Trp233 (orange), which is part of the clasp. Thr191 acts as an RNA discriminator, sensing the 2′-hydroxyl group of the terminal ribose. To illustrate the clasp-like RNA binding, W233 and the first two bases of GAAA are additionally shown as surface representation. See also Figures S3 and S4A.
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5. Figure 3
Structure of ceClp1 Bound to the Ribonucleotide GC and AMP-PNP-Mg2+
A cross-section of ceClp1 showing the conserved nucleotide and RNA-binding site. The amino acid sequence conservation of Clp1 from higher eukaryotes (Figure S2) is mapped onto the molecular surface of ceClp1. The bound RNA and AMP-PNP are shown in the yellow stick representation.
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6. Figure 4
Crystal Structures Capturing Different RNA- and Nucleotide-Bound States along the Reaction Pathway of ceClp1
(A–E) Close-up views of the active site of ceClp1. Putative energy diagram of the reaction pathway catalyzed by ceClp1 (A). Schematic illustration of the active site in complex with GC⋅ATP⋅Mg2+ (B). ceClp1 in the inhibited, substrate-bound state: ceClp1⋅GC⋅AMP-PNP⋅Mg2+ (C). ceClp1 bound to a transition-state analog: ceClp1⋅GC⋅ADP-AlF4-⋅Mg2+ (D). ceClp1 in the RNA-released state: ceClp1⋅ADP⋅Mg2+ (E). Prior to and after the transition-state complex, the Walker A lysine (Lys127) is arrested outside the active site. Only in the transition state, Lys127 adopts an active conformation. The color code is the same as in Figure 1. Hydrogen bonds and salt bridges are shown as black dashed lines. The coordination of the magnesium ion is highlighted by dotted lines. See Figure S5.
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7. Figure 5 Model of the Phosphoryl Transfer Reaction Catalyzed by Clp1
The 5′-hydroxyl group of the RNA is deprotonated by the general base, the Walker B Asp151 (2.6 Å). The color code is the same as in Figure 1. Prior to and after the transition-state complex, Lys127 does not interact with AMP-PNP. It is fully stretched and arrested by hydrogen bonds formed to P loop (Gly121; 3.0 Å) and the clasp (Thr230; 3.0 Å, 2.9 Å). Characteristic for the transition-state analog is an apically oriented AlF4−, consistent with an in-line transfer mechanism. Lys127 has moved into the active site in the transition-state complex and interacts with ADP (2.7 Å) and the AlF4− (2.6 Å). Interactions of Arg288 and Arg293 are involved both in the binding of the RNA and in compensating developing negative charges during the transfer reaction. In the RNA-released state, Arg293 interacts with Asp124 (2.7 and 2.9 Å), and therefore it is no longer available in the active site.
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8. Figure 6 Model for Binding of Double-Stranded RNA Substrates to ceClp1
An ideal dsA-RNA oligomer is superimposed with the sugar-phosphate backbone of the ssRNA of the ceClp1⋅GAAA⋅ADP⋅Mg2+ structure. The ribbon representation is shown beneath a transparent molecular surface. The dsRNA is shown as a ribbon model, with the phosphate backbone in orange and the nucleotide bases in blue.
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9. Figure 7 Structural Comparison of P Loop Conformations
(A and B) A selection of structures with conventional (A) and nonconventional (B) conformations of the Walker A lysine is depicted. PDB entries 1–10 (A) and 61–72 (B), listed in Table S3, are shown. Superposition of the P loop region (GXXXXGK[S/T]) of respective structures and ceClp1 was performed as described in the Supplemental Experimental Procedures. The Walker A lysine and the nucleotide are shown in the yellow model, whereas the P loop motif is shown as a ribbon diagram.
Molecular Cell  , DOI: ( /j.molcel )
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