Identification of a hidden strain switch provides clues to an ancient structural mechanism in protein kinases

Abstract

The protein kinase catalytic domain contains several conserved residues of unknown functions. Here, using a combination of computational and experimental approaches, we show that the function of some of these residues is to maintain the backbone geometry of the active site in a strained conformation. Specifically, we find that the backbone geometry of the catalytically important HRD motif deviates from ideality in high-resolution structures and the strained geometry results in favorable hydrogen bonds with conserved noncatalytic residues in the active site. In particular, a conserved aspartate in the F-helix hydrogen bonds to the strained HRD backbone in diverse eukaryotic and eukaryotic-like protein kinase crystal structures. Mutations that alter this hydrogen-bonding interaction impair catalytic activity in Aurora kinase. Although the backbone strain is present in most active conformations, several inactive conformations lack the strain because of a peptide flip in the HRD backbone. The peptide flip is correlated with loss of hydrogen bonds with the F-helix aspartate as well as with other interactions associated with kinase regulation. Within protein kinases that are regulated by activation loop phosphorylation, the strained residue is an arginine, which coordinates with the activation loop phosphate. Based on analysis of strain across the protein kinase superfamily, we propose a model in which backbone strain co-evolved with conserved residues for allosteric control of catalytic activity. Our studies provide new clues for the design of allosteric protein kinase inhibitors.allostery | conformational strain | DFG flip | hydrophobic spine | Ramachandran plot

Deciphering the Structural Basis of Eukaryotic Protein Kinase Regulation

“…To examine if this polar interaction is required to maintain the proper assembly of the R-spine, we mutated RS0 to alanine (RS0A), and Western blot results show that catalytic activity was abolished (Figure 2B,G). This is consistent with recent studies on Aurora kinase, where mutation of RS0 to an alanine (RS0A) abolished Aurora kinase activity [18]. To test if loss of this polar interaction could be rescued through a hydrophobic interaction, we replaced RS0 with a leucine (RS0L); results show that some catalytic activity was rescued (Figure 2B,G).…”

KinView: a visual comparative sequence analysis tool for integrated kinome research

“…Although members of the PKC family of kinases have recently been shown to function as tumor suppressors through characterization of cancer variants 87, 88 , the functional impact of D523N PKCβ (D220N PKA ) has not been established. Since D220 PKA anchors the regulatory spine to the αF-helix and plays a critical role in kinase functions 89, 90 , we predicted that the D523N PKCβ would decrease PKC activity. To test this hypothesis, we first transfected mCherry tagged wild-type or D523N PKCβ PKCβII in COS7 cells and examined its phosphorylation status, a marker of correct processing and an event that requires the catalytic function of the enzyme 91 .…”

Computational and Experimental Characterization of Patient Derived Mutations Reveal an Unusual Mode of Regulatory Spine Assembly and Drug Sensitivity in EGFR Kinase

“…30–32 The RS is formed by a contiguous network of hydrophobic interactions (RS1, RS2, RS3, and RS4) that are structurally assembled in the active state, but disassembled in the inactive state (Figure 1), 30,32,33 though some exceptions to this rule have been noted. 34,35 Large-scale statistical comparisons of protein kinase sequences and crystal structures have also identified additional structural and conformational features associated with RS assembly and kinase activity. For example, a conformational “strain” switch in the catalytic site was shown to be correlated with RS assembly and kinase activity.…”

“…For example, a conformational “strain” switch in the catalytic site was shown to be correlated with RS assembly and kinase activity. 34 Likewise, extension of the RS through family and group- specific residues have been suggested to contribute to unique modes of allosteric regulation in some kinases. 36,37 A molecular dynamics (MD) based structural network modeling approach identified the RS as a central hub for structural stability and allosteric communication.…”