doi: 10.1016/j.str.2008.10.018.
Structure of the pseudokinase VRK3 reveals a degraded catalytic site, a highly conserved kinase fold, and a putative regulatory binding site
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- PMID:19141289
- PMCID: PMC2639636
- DOI: 10.1016/j.str.2008.10.018
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Structure of the pseudokinase VRK3 reveals a degraded catalytic site, a highly conserved kinase fold, and a putative regulatory binding site
Eric D Scheeff et al. Structure..
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Abstract
About 10% of all protein kinases are predicted to be enzymatically inactive pseudokinases, but the structural details of kinase inactivation have remained unclear. We present the first structure of a pseudokinase, VRK3, and that of its closest active relative, VRK2. Profound changes to the active site region underlie the loss of catalytic activity, and VRK3 cannot bind ATP because of residue substitutions in the binding pocket. However, VRK3 still shares striking structural similarity with VRK2, and appears to be locked in a pseudoactive conformation. VRK3 also conserves residue interactions that are surprising in the absence of enzymatic function; these appear to play important architectural roles required for the residual functions of VRK3. Remarkably, VRK3 has an "inverted" pattern of sequence conservation: although the active site is poorly conserved, portions of the molecular surface show very high conservation, suggesting that they form key interactions that explain the evolutionary retention of VRK3.
Figures
Superimposition of the Kinase Domain Structures of VRK3 (blue) and VRK2 (red) (Inset) Magnified view of highly conserved residues linking αC4 to αE.
The Altered Active Site Region of VRK3 (A) Sequence conservation logos for key motifs in VRK3s, active VRKs, and the CK1 family, based on appropriate subsets of a curated sequence/structure alignment of diverse homologs (see methods). Standard motifs seen across protein kinases are shown below the logo representation (uppercase residues are conserved nearly to identity across all protein kinases, whereas lowercase residues are partially conserved; x indicates any residue; o, hydrophobic). Numbered VRK3 residues are shown in panels B and C. Yellow diamonds highlight positions in which the VRK3s violate evolutionary constraints seen in active VRKs and CK1s. (B) Comparison of the ATP binding pockets of CK1 and VRK3. ATP from the CK1 structure is modeled into VRK3 based on structural superimposition. Yellow marks VRK3 residue positions that violate evolutionary constraints (as per panel A), whereas white marks other residues with relevant contacts. Homologous CK1 residues are colored identically to VRK3 residues. Hydrogen bonds are shown as green dotted lines. Portions of the structures are not shown to improve clarity. (C) The active site region of VRK3 compared with that of VRK2, labeled as in panel B.
The Nonfunctional ATP Binding Pocket of VRK3 (A) Electrostatic surface view of VRK2. The ATP molecule (ball-and-sticks view) is modeled from CK1. (B) Equivalent view of VRK3 shows highly acidic binding pocket and partial occlusion of modeled ATP. (C) Secondary structure representation of VRK2 showing the orientation of electrostatic representations. (D) ATP binding capability of the VRKs, from a thermal denaturation assay. ΔTm is the shift in melting temperature when purified protein is placed in solution with ligand, and is the average of three experimental measurements (see Experimental Procedures).
Changes in the VRK3 Activation Segment (A) Sequence conservation logos for the full activation segments of VRK3s, active VRKs, and the CK1 family, based on appropriate subsets of a curated sequence/structure alignment of diverse homologs (see Experimental Procedures), labeled with same conventions as Figure 2A. Subsections of the activation segment are shown below the motifs. (B) The activation segment of VRK2, shown in cyan, with 3–10 helices in violet and structure outside of the activation segment in gray. Residues are shown with same conventions as Figures 2B and 2C. Portions of the structure are not shown to improve clarity. (C) VRK3 activation segment, labeled as in panel B.
Surface Conservation Patterns for VRK3s Only, All VRKs, and CK1s (A) View of the face of the molecules opposing the active site. The top three of nine conservation bins from ConSurf (see Experimental Procedures) are shown in corresponding shades of blue (darkest blue indicates the most conserved). Results for all VRKs are mapped onto the VRK3 structure, but are valid for all family members. Results for CK1s are mapped onto the CK1 structure. Conservation scoring is scaled to each sequence grouping, and is not directly comparable between groups. The unique conserved patch present in VRKs, but not CK1s, is circled. (B) Molecules from panel A are rotated ∼120° such that the active site and substrate binding region is facing forward.
Putative Binding Site on the Back of VRK3 (A) Key residues of the putative binding patch, mapped to a surface view of VRK3. Red residues are conserved in all VRKs, and blue are conserved but with distinctive patterns in VRK3 versus VRK1/2. (B) Motif logos of site in different VRKs and the CK1 family. Sequence is a catenation of residues distributed within the sequence alignment: residues in the VRK3 structure are numbered above the alignment, and black bars indicate break points where intervening sequence was removed. The two subsets of conserved residues are indicated above the motifs.
Comment in
- Pseudokinases: functional insights gleaned from structure.Kornev AP, Taylor SS.Kornev AP, et al.Structure. 2009 Jan 14;17(1):5-7. doi: 10.1016/j.str.2008.12.005.Structure. 2009.PMID:19141276Free PMC article.
References
- Blanco S., Klimcakova L., Vega F.M., Lazo P.A. The subcellular localization of vaccinia-related kinase-2 (VRK2) isoforms determines their different effect on p53 stability in tumour cell lines. FEBS J. 2006;273:2487–2504. - PubMed
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