1. Volume 11, Issue 11, Pages 1329-1337 (November 2003)
Structures of P. falciparum PfPK5 Test the CDK Regulation Paradigm and Suggest Mechanisms of Small Molecule Inhibition 
Simon Holton, Anais Merckx, Darren Burgess, Christian Doerig, Martin Noble, Jane Endicott 
Structure 
Volume 11, Issue 11, Pages (November 2003)
DOI: /j.str

2. Figure 1 PfPK5 Sequence and Fold
(A) Alignment of PfPK5 with selected CDKs. PfPK5 (Swissprot accession number Q07785) was aligned with selected CDK sequences (human CDK2 [P24941], S. pombe Cdc2 [P04551], S. cerevisiae CDC28 [P00546], H. sapien CDK6 [Q00534], H. sapien CDK5 [Q00535], and T. brucei tbcrk1 [s05853]), using the program CLUSTAL-W (Thompson et al., 1994), and rendered with the program Alscript (Barton, 1993) (numbered as for PfPK5). Key sequence motifs are highlighted: glycines of the kinase GXGXXG motif, magenta; residues subject to regulatory phosphorylation in CDK1, red; CDK6 residues that contact p16INK4A, light blue (Russo et al., 1998); residues delineating the activation loop, cyan; residues equivalent to CDK5 153, a key CDK2/CDK5 sequence difference, salmon (Tarricone et al., 2001); GDSEID motif, involved in both CKS1 (Bourne et al., 1996) and KAP binding (Song et al., 2001), dark blue; other residues that contact KAP or CKS proteins, turquoise; the kinase insert region, and CDK insert region, areas of hypervariability between kinases are boxed (Hanks and Hunter, 1995). Residues shaded in yellow are highly conserved between all CDKs.
(B) The monomeric PfPK5 fold. The N-terminal domain (residues 1–82) is colored white and the C-terminal domain (residues 83–288) gold. The glycine-rich loop (residues 10–19), the C helix (residues 39–56), and the activation loop (residues 143–170 from the conserved DFG to APE motifs) are colored magenta, red, and cyan respectively. PfPK5 residues Asp125, Asn130, and Asp143 are drawn in ball-and-stick mode and are discussed in the main text.
(C) Overlay of the structures of monomeric PfPK5Thr198Ala and CDK2 in the vicinity of the activation loop. CDK2 has been superimposed, colored green (De Bondt et al., 1993). The CDK2 activation loop forms a β hairpin that turns across the end of the glycine loop, while the activation loop of PfPK5Thr198Ala adopts an extended structure as it stretches away from αL12 into a short α helix of 2.5 turns.
Structure  , DOI: ( /j.str )

3. Figure 2 Phosphorylation and Kinase Activity of PfPK5
(A) PfPK5 histone H1 kinase activity. 15 pmol of PfPK5WT or PfPK5T158A protein were preincubated at 30°C for 30 min either alone (lanes 1 and 3) or with 15 pmol of bovine cyclinA3 (lanes 2 and 4). After the addition of 0.5 μg histone H1, 0.1 mM ATP, 1 μCi [32P]ATP, 15 mM MgCl2, 2 mM MnCl2, and 25 mM Tris-HCl, pH 7.4, samples were incubated for a further 30 min at 30°C. The final reaction volume was 10 μl. Reactions were stopped by the addition of loading dye, and analyzed on a 12% Tris-glycine SDS-PAGE and then by autoradiography. Lanes 5 and 6 are negative controls. Results are discussed in the main text.
(B) Phosphorylation of the PfPK5 activation segment. 15 pmol of substrate (either CDK2 or PfPK5) was incubated with 2 pmol of enzyme (human CDK7/CyclinH, X. laevis CDK7/CyclinH, or S. cerevisiae GST-CIV1, all kind gifts of Dr. N. Brown) for 30 min at 30°C. Reaction buffer was 0.1 mM ATP, 1 μCi [32P] ATP, 15 mM MgCl2, 2 mM MnCl2, and 25 mM Tris-HCl, pH 7.5. Reactions were stopped by addition of loading dye and then analyzed by SDS-PAGE and autoradiography. All CAKs phosphorylate CDK2 with differing efficacy (lanes 2–4), but do not phosphorylate PfPK5 (lanes 6–8). CDK2 and PfPK5 do not autophosphorylate under these conditions (lanes 1 and 5).
Structure  , DOI: ( /j.str )

4. Figure 3
Comparative Analysis of Sequence Conservation between PfPK5 and CDK2 and Known Sites of CDK2-Protein Interaction
The structures of complexes of T160-phosphorylated CDK2/cyclin A/AMP-PNP/substrate peptide (Brown et al., 1999a) and CDK2/CKS1 (Bourne et al., 1996) were superimposed within the program O (Jones et al., 1991). AMP-PNP and the substrate peptide (sequence HHASPRK) are drawn in ball-and-stick mode with carbon atoms colored green. CKS1 and cyclin A are rendered in ribbon representation and colored dark purple and green respectively. The CDK2 molecular surface is colored cyan for those residues where the CDK2 and PfPK5 sequences are similar in character as defined by a score greater than 0 in the Blosum65 matrix (Henikoff and Henikoff, 1992) or red where the pair score is less than or equal to 0. The view in (A) is similar to that in Figures 1B and (B) is rotated around the y axis.
Structure  , DOI: ( /j.str )

5. Figure 4 Inhibitor Binding to PfPK5
(A) Stereo pair of NU6102 and purvalanol B bound within the PfPK5 active site. All residues in the PfPK5 structure bound to NU6102 within a 7 Å sphere of the inhibitor are drawn. The inhibitor and PfPK5 structures are drawn in ball-and-stick mode with carbon atoms of PfPK5, NU6102, and Purvalanol B colored white, yellow, and cyan respectively. To illustrate the minor differences between the PfPK5 structures in the glycine loop and at residue Lys88, residues Ile10 and Lys88 from the PfPK5/Purvalanol B structure have also been drawn with carbon atoms colored white. Hydrogen bonds between the NU6102 sulfamoyl substituent and PfPK5 are shown as dotted lines.
(B) Sequence differences between CDK1, CDK2, CDK4/6, and PfPK5 close to the ATP nucleotide binding site.
Structure  , DOI: ( /j.str )

6. Figure 5 Indirubin-5-Sulphonate Binding to PfPK5
Stereo pair. The structures of monomeric PfPK5 (magenta), T160-phosphorylated CDK2/cyclin A (gold/green), and PfPK5/indirubin-5-sulphonate (cyan) were superimposed as described and rendered in ribbon representation. The side chains of PfPK5 residues Lys32, Glu50, and Asp143 and indirubin5-sulphonate are drawn in ball-and-stick mode with carbon atoms colored cyan and green respectively. Model phased 2Fobs-Fcalc·αcalc electron density for the inhibitor is contoured at 0.2 e−Å−3. The view highlights the different orientations of the C helix relative to the rest of the PfPK5 fold in the PfPK5 and PfPK5/indirubin-5-sulphonate structures.
Structure  , DOI: ( /j.str )