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Loss of ATP-binding: The case of VRK3

Tags VRK3 inactive active kinases mutations ATP binding
By: matthieu chartier  
October 17 2012

Around 50 human protein kinase domains are predicted to be enzymatically inactive (pseudokinases). VRK3 is one of them.

The VRK family is part of the CK1 kinase group a small group of kinases that are very similar to each other in sequence, but very distinct from other kinase groups.

VRK1 VRK2 and VRK3 are part of the VRK family. Of the three of them, only VRK3 is known to be inactive.

  • VRK1 is an active nuclear kinase whose substrates include p53, ATF, Jun, BAF, and histone H3, and is involved in cell cycle, chromatin condensation, and transcriptional regulation.

  • VRK2 has two splice forms that localize either to the nucleus and cytoplasm or to the ER and mitochondria.

  • VRK3 is the only VRK to lack enzymatic activity.

VRK1, VRK2 and VRK3 were monitered during thermal denaturation with and without ATP. VRK1 and VRK2 showed a shift in melting temperature unlike VRK3. This suggests that VRK3 has no binding affinity with ATP.

Interestingly, VRK3 had the highest native Tm of the three proteins. This supports the notion that VRK3 is stable and rigid even in the absence of ATP.

The G-loop

The G-loop, usually glycine-rich, provides conformational flexibility enabling hydrogen bond formation between the backbone of the loop and the y-phosphate of ATP. The lack of ATP binding in VRK3 is likely to be caused in part by degradation of the G-loop motif.

In the G-loop of VRK3, Q177 would form steric clashes with ATP. Also, D175 occupies the ATP binding site (near the region where the phosphate would be), mimicking an ATP phosphate. These and other changes produce a highly acidic ATP binding pocket that is likely to repel rather than accept the negatively charged phosphates of ATP.

The residues present in the G-loop of kinases can be good indicators of protein inactivity/activity.

The adenine ring binding region

In the adenine ring binding region of CK1 kinases, residue D86 usually accepts a hydrogen bond from ATP via its backbone carbonyl. In VRK3 the equivalent proline (P260) has an altered backbone conformation and can no longer bind ATP.

L88 in CK1 donates a hydrogen bond to ATP via its backbone amine. In VRK3, L262 has shifted conformation such that the side chain would sterically clash with the adenine ring.

Furthermore, VRK3 has a conserved substitution to a large hydrophobic residue (F313) in the C-terminal lobe, a position that is conserved as a smaller hydrophobic residue in active VRKs and CK1s. The combined affect of these changes is to fill in much of the region where the adenine ring would normally bind.

1. Scheeff, E. D., Eswaran, J., Bunkoczi, G., Knapp, S., & Manning, G. (2009). Structure of the Pseudokinase VRK3 Reveals a Degraded Catalytic Site, a Highly Conserved Kinase Fold, and a Putative Regulatory Binding Site. Structure (London, England : 1993), 17(1), 128–138. doi:10.1016/j.str.2008.10.018

Comments (1)
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Rafael Najmanovich - October 18 2012, 14:50:39
Were these thermal denaturation studies performed on the entire proteins or isolated kinase domains?