Sequence Determinants of a Specific Inactive Protein Kinase Conformation
How to predict if a protein kinase can bind ligands type II inhibitors in the DFG-out conformation
This is a précis of the Chemistry and Biology paper by Hari, Merritt and Maly. The abstract is as follows:
“Only a small percentage of protein kinases have been shown to adopt a distinct inactive ATP-binding site conformation, called the Asp-Phe-Gly-out (DFG-out) conformation. Given the high degree of homology within this enzyme family, we sought to understand the basis of this disparity on a sequence level. We identified two residue positions that sensitize mitogen-activated protein kinases (MAPKs) to inhibitors that stabilize the DFG-out inactive conformation. After characterizing the structure and dynamics of an inhibitor-sensitive MAPK mutant, we demonstrated the generality of this strategy by sensitizing a kinase (apoptosis signal-regulating kinase 1) not in the MAPK family to several DFG-out stabilizing ligands, using the same residue positions. The use of specific inactive conformations may aid the study of noncatalytic roles of protein kinases, such as binding partner interactions and scaffolding effects.”
The authors introduce their research by repeating a fundamental truism in the study of kinase mechanics: Active protein kinases are quite similar in conformation but any PK can adopt an infinite number of inactive conformations (an observation also mentioned in a Mol Cell paper by Jura et al). The latter is true even if only a few repeatable inactive conformations have been crystallized. This introduction quickly zooms onto one specific inactive conformation — the DFG-out conformation that allows binding of type II inhibitors such as imatinib. Most PKs that bind such inhibitors are tyrosine kinases. Amongst S/T kinases, the MAPK p38α can also be inhibited by type II inhibitors but the same is not true for p38δ. Interestingly, the authors omit mentioning (or even looking into?) p38β and p38γ. The former is known to bind type 2 inhibitors and not the latter (AFAIK). (Cite PDB).
The essence of the paper is about identifying two residues key to binding type 2 inhibitors. The first position is no surprise — the gatekeeper. It is generally true that this has to be threonine or smaller in order to create a hydrophobic pocket large enough to gain access to the larger back pocket. There are exceptions (cite them later). The second residue is positioned directly before the DFG triad. That this is critical to DFG adopting the out conformation is no surprise, given its proximity!
To investigate the importance of these two residues, Hari et al studied 4 MAPKs: p38α, p38δ, Jnk3 and Erk2. Amongst them, only p38α is known to bind type II inhibitors. It is also the only one with a threonine gatekeeper (position highlighted in blue above). Both 38δ and Jnk3 became capable of strongly binding type II inhibitors when their gatekeepers were mutated (to Thr in 38δ and Ile in Jnk3). But this was not true of Erk2, which only became sensitive when a second mutation was carried out in the position before the DFG motif to convert the cysteine (see red above) into leucine.
Hari et al also point out that the N-terminally flanking position of DFG has been previously investigated for its role in allowing the DFG motif to adopt the out conformation. In that study, however, Martin et al were investigating inhibitors that only occupy the ATP pocket.
