Kinase Domain Insertions Define Distinct Roles of CLK Kinases in SR Protein Phosphorylation

Bullock, Alex N.; Das, Sumit Kumar; Debreczeni, J.; Rellos, P.; Fedorov, O.; Niesen, F.; Guo, K.; Papagrigoriou, E.; Amos, A.; Cho, Suhyung; Turk, Benjamin E.; Ghosh, Gourisankar; Knapp, Stefan

doi:10.1016/j.str.2008.12.023

Cited by 112 publications

(174 citation statements)

References 41 publications

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“…A number of hydrophobic anchor points present in the ␤ hairpin are conserved as well, but because most interactions are mediated by main chain contacts, there is little sequence conservation in this region. Interestingly, a ␤-hairpin insert located between strands ␤7 and ␤8 also is present in Cdc2-like kinases (CLKs) (15), but there it is oriented differently and packs against the opposite side of the lower kinase lobe (Fig. S4).…”

Section: Resultsmentioning

confidence: 99%

Structure and functional characterization of the atypical human kinase haspin

Eswaran

Patnaik

Filippakopoulos

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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The protein kinase haspin/Gsg2 plays an important role in mitosis, where it specifically phosphorylates Thr-3 in histone H3 (H3T3). Its protein sequence is only weakly homologous to other protein kinases and lacks the highly conserved motifs normally required for kinase activity. Here we report structures of human haspin in complex with ATP and the inhibitor iodotubercidin. These structures reveal a constitutively active kinase conformation, stabilized by haspin-specific inserts. Haspin also has a highly atypical activation segment well adapted for specific recognition of the basic histone tail. Despite the lack of a DFG motif, ATP binding to haspin is similar to that in classical kinases; however, the ATP ␥-phosphate forms hydrogen bonds with the conserved catalytic loop residues Asp-649 and His-651, and a His651Ala haspin mutant is inactive, suggesting a direct role for the catalytic loop in ATP recognition. Enzyme kinetic data show that haspin phosphorylates substrate peptides through a rapid equilibrium random mechanism. A detailed analysis of histone modifications in the neighborhood of H3T3 reveals that increasing methylation at Lys-4 (H3K4) strongly decreases substrate recognition, suggesting a key role of H3K4 methylation in the regulation of haspin activity.ATP binding ͉ histone modification ͉ phosphorylation mechanism ͉ germ cell-specific gene 2 (Gsg2) ͉ mitosis E ukaryotic protein kinases (ePKs) constitute a large group of enzymes that regulate a vast diversity of cellular processes. Kinase activity depends on a number of highly conserved sequence motifs required for ATP/Mg 2ϩ binding and catalysis. About 10% of human ePKs lack one or more essential catalytic motifs and have been initially classified as inactive pseudokinases (1). However, a number of such proteins are active kinases, including haspin [haploid germ cell-specific nuclear protein kinase, encoded by Germ cell-specific gene 2 (Gsg2)]. Haspin lacks both the conserved ATP/Mg 2ϩ binding motif Asp-Phe-Gly (DFG), which is replaced by Asp-Tyr-Thr (DYT), and the Ala-Pro-Glu (APE) motif usually found at the C terminus of the activation segment (2). In addition, haspin shares only weak sequence homology with ePKs and contains a highly divergent kinase domain with several unique inserts (2, 3). Haspin mRNA is abundant in testis and also present in somatic cells (4, 5), and orthologues are found throughout eukaryotic phyla (2). Ectopically expressed haspin localizes to the nucleus in interphase and to the chromosomes in mitosis (4, 6), while depletion of haspin leads to premature chromatid separation, failure of chromosome alignment, and a block in mitosis in a prometaphase-like state (6, 7).Haspin autophosphorylates in vitro (4, 6, 8) and phosphorylates its only currently known substrate, histone H3, at threonine-3 (H3T3ph) both in vitro and in cells (6). H3T3 phosphorylation occurs specifically during mitosis and is particularly prominent at inner centromere regions (6, 7, 9). Recently, H3T3ph has been suggested to relieve an inhibitory effect of ...

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Section: Resultsmentioning

confidence: 99%

Structure and functional characterization of the atypical human kinase haspin

Eswaran

Patnaik

Filippakopoulos

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

155

167

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“…The dual specificity kinases autophosphorylate on tyrosine residues, but the target substrates for both CLK1 and DYRK1A are serine or threonine residues. [31][32][33] The results, shown in Figure 8(C), show that these two kinases have a surface like the serine/ threonine kinases, with a substrate recognition site similar to that of PKA and no extended area for tyrosine at the phosphorylation site. No peptide substrate is co-crystallized, so the question remains as to how these proteins accommodate the bulky tyrosine for autophosphorylation.…”

Section: Resultsmentioning

confidence: 95%

Comparative surface geometry of the protein kinase family

et al. 2009

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Identifying conserved pockets on the surfaces of a family of proteins can provide insight into conserved geometric features and sites of protein-protein interaction. Here we describe mapping and comparison of the surfaces of aligned crystallographic structures, using the protein kinase family as a model. Pockets are rapidly computed using two computer programs, FADE and Crevasse. FADE uses gradients of atomic density to locate grooves and pockets on the molecular surface. Crevasse, a new piece of software, splits the FADE output into distinct pockets. The computation was run on 10 kinase catalytic cores aligned on the aF-helix, and the resulting pockets spatially clustered. The active site cleft appears as a large, contiguous site that can be subdivided into nucleotide and substrate docking sites. Substrate specificity determinants in the active site cleft between serine/threonine and tyrosine kinases are visible and distinct. The active site clefts cluster tightly, showing a conserved spatial relationship between the active site and aF-helix in the C-lobe. When the aC-helix is examined, there are multiple mechanisms for anchoring the helix using spatially conserved docking sites. A novel site at the top of the N-lobe is present in all the kinases, and there is a large conserved pocket over the hinge and the aC-b4 loop. Other pockets on the kinase core are strongly conserved but have not yet been mapped to a protein-protein interaction. Sites identified by this algorithm have revealed structural and spatially conserved features of the kinase family and potential conserved intermolecular and intramolecular binding sites.

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“…Haspin and CLK1, both wild-type and all mutants, have been purified as described (18,25). Briefly, the recombinant proteins were purified using Co 2+ /Ni 2+ affinity chromatography.…”

Section: Protein Purificationmentioning

confidence: 99%

Halogen-aromatic π interactions modulate inhibitor residence time

Heroven

Georgi

Ganotra

et al. 2018

Preprint

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Prolonged drug residence times may result in longer lasting drug efficacy, improved pharmacodynamic properties and "kinetic selectivity" over off-targets with fast drug dissociation rates. However, few strategies have been elaborated to rationally modulate drug residence time and thereby to integrate this key property into the drug development process. Here, we show that the interaction between a halogen moiety on an inhibitor and an aromatic residue in the target protein can significantly increase inhibitor residence time.By using the interaction of the serine/threonine kinase haspin with 5-iodotubercidin (5-iTU) derivatives as a model for an archetypal active state (type I) kinase-inhibitor binding mode, we demonstrate that inhibitor residence times markedly increase with the size and polarizability of the halogen atom. This key interaction is dependent on the interactions with an aromatic residue in the gate keeper position and we observe this interaction in other kinases with an aromatic gate keeper residue. We provide a detailed mechanistic characterization of the halogen-aromatic π interactions in the haspin-inhibitor complexes by means of kinetic, thermodynamic, and structural measurements along with binding energy calculations. Since halogens are frequently used in drugs and aromatic residues are often present in the binding sites of proteins, our results provide a compelling rationale for introducing aromatic-halogen interactions to prolong drug-target residence times.. CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was . http://dx.doi.org/10.1101/255513 doi: bioRxiv preprint first posted online Jan. 29, 2018; Heroven et al. 3 SignificanceThe time that a drug resides on its target protein has emerged as an important parameter for drug development. A longer drug-target residence time can lead to improved drug efficacy and reduced adverse side effects. However, general strategies for the rational design of drugs with long target residence times are lacking. Here, by experimental and computational characterization of a set of inhibitor-kinase complexes, we show how interactions between inhibitor halogen atoms and protein aromatic residues can increase target residence times. Our results provide a simple strategy for the rational design of inhibitors with prolonged target residence times.

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Kinase Domain Insertions Define Distinct Roles of CLK Kinases in SR Protein Phosphorylation

Cited by 112 publications

References 41 publications

Structure and functional characterization of the atypical human kinase haspin

Structure and functional characterization of the atypical human kinase haspin

Comparative surface geometry of the protein kinase family

Halogen-aromatic π interactions modulate inhibitor residence time

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