Ab Initio Modeling and Experimental Assessment of Janus Kinase 2 (JAK2) Kinase-Pseudokinase Complex Structure

Abstract

The Janus Kinase 2 (JAK2) plays essential roles in transmitting signals from multiple cytokine receptors, and constitutive activation of JAK2 results in hematopoietic disorders and oncogenesis. JAK2 kinase activity is negatively regulated by its pseudokinase domain (JH2), where the gain-of-function mutation V617F that causes myeloproliferative neoplasms resides. In the absence of a crystal structure of full-length JAK2, how JH2 inhibits the kinase domain (JH1), and how V617F hyperactivates JAK2 remain elusive. We modeled the JAK2 JH1–JH2 complex structure using a novel informatics-guided protein-protein docking strategy. A detailed JAK2 JH2-mediated auto-inhibition mechanism is proposed, where JH2 traps the activation loop of JH1 in an inactive conformation and blocks the movement of kinase αC helix through critical hydrophobic contacts and extensive electrostatic interactions. These stabilizing interactions are less favorable in JAK2-V617F. Notably, several predicted binding interfacial residues in JH2 were confirmed to hyperactivate JAK2 kinase activity in site-directed mutagenesis and BaF3/EpoR cell transformation studies. Although there may exist other JH2-mediated mechanisms to control JH1, our JH1–JH2 structural model represents a verifiable working hypothesis for further experimental studies to elucidate the role of JH2 in regulating JAK2 in both normal and pathological settings.

Molecular basis for pseudokinase-dependent autoinhibition of JAK2 tyrosine kinase

“…Our JAK2 JH2–JH1 model is fundamentally different from models proposed previously 23,29,30 , in which only V617F among the many MPN mutations is present in the respective JH2–JH1 interfaces (Supplementary Fig. 5b).…”

“…2a). E592R, however, was not activated, which was unexpected because mutation to alanine (E592A) was shown previously to be partially activating 23 . MD simulations of E592R could rationalize the experimental result: Arg592 can form a salt bridge with pSer523, along with Arg947 (Supplementary Fig.…”

“…Arg947 also interacted with pSer523 during the simulation. Notably, the mutation R588A was shown previously to be partially activating 23 .…”

Dynamic architecture of a protein kinase

“…For our community analysis, we computed Cartesian mutual information values instead of dihedral mutual information values, as the former better capture correlated motions involving semirigid regions, such as secondary structure elements (93), whereas the latter are more suitable for analyzing couplings between surface sites where binding and posttranslational modification often occur (30,94). We calculated the Cartesian mutual information for backbone Cα atoms, as well as for a representative side-chain atom (SI Appendix, Table S2) of each residue.…”

Activating Janus kinase pseudokinase domain mutations in myeloproliferative and other blood cancers

“…The second is an asymmetric model [41,42], whereby the N-terminal lobe of JH2 binds to the C-terminal lobe of JH1, possibly inhibiting JH1. This model is based on the mechanism of activation described for EGFR (epidermal growth factor receptor) kinase in a dimer where one kinase is the 'activator' and the other kinase binding with its N-terminal lobe being the 'receiver' [43,44].…”