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Title: Decoding the Interactions Regulating the Active State Mechanics of Eukaryotic Protein Kinases}
Article:
Abstract:
Eukaryotic protein kinases regulate most cellular functions by phosphorylating targeted protein substrates through a highly conserved catalytic core. In the active state, the catalytic core oscillates between open, intermediate, and closed conformations. Currently, the intramolecular interactions that regulate the active state mechanics are not well understood. Here, using cAMP-dependent protein kinase as a representative model coupled with biochemical, biophysical, and computational techniques, we define a set of highly conserved electrostatic and hydrophobic interactions working harmoniously to regulate these mechanics. These include the previously identified salt bridge between a lysine from the β3-strand and a glutamate from the αC-helix as well as an electrostatic interaction between the phosphorylated activation loop and αC-helix and an ensemble of hydrophobic residues of the Regulatory spine and Shell. Moreover, for over three decades it was thought that the highly conserved β3-lysine was essential for phosphoryl transfer, but our findings show that the β3-lysine is not required for phosphoryl transfer but is essential for the active state mechanics.
Introduction:
Eukaryotic protein kinases( EPKs) were first discovered in 1943, and their functional role in phosphorylation was elucidated in 1956[1–3]. In 1968, cAMP-dependent protein kinase( PKA) was the second protein kinase to be discovered, and in 1969, it was shown that phosphorylation is not tissue- or species-specific[4]. PKA has since served as the prototype for our understanding of EPK structure and function. EPKs are ubiquitously expressed in all eukaryotes, and approximately 2% of the human genome encodes for EPKs[5, 6]. EPKs are involved in most biological processes and have been associated with numerous human diseases, making EPKs key candidates for therapeutic intervention[7]. EPKs share a highly conserved catalytic core that mediates the transfer of the γ-phosphate of adenosine triphosphate( ATP) to a protein substrate[8]. Structurally, the core consists of two lobes, the N-lobe and C-lobe( Fig 1A)[9]. Within the core there are two nonlinear hydrophobic motifs known as the Catalytic( C)-spine and the Regulatory( R)-spine that span both lobes( Fig 1B)[10]. The R-spine includes two residues from the C-lobe( RS1[Y164] from the Y/Hrd motif in the catalytic loop[CL] and RS2[F185] from the dFg motif in the activation loop[AL]) and two from the N-lobe( RS3[L95] from the αC-helix and RS4[L106] from the β4-strand)( Fig 1C). The R-spine is anchored to the αF-helix through a highly conserved aspartate( RS0[D220])[11]. The R-spine is supported by an ensemble of conserved hydrophobic residues referred to as the Shell( Sh1[V104] and Sh2[gatekeeper, M120] from the αC-β4 loop and Sh3[M118] from the β5-strand)[11, 12]. EPKs are typically in equilibrium between the inactive and active states in which the R-spine is disassembled and assembled, respectively[11, 13]. After the assembly of the R-spine, the active state of an EPK toggles between open, intermediate, and closed conformations as it traverses the catalytic cycle[9, 14–16]. The transition from the open to the closed conformation is initiated by the binding of ATP, which transitions the core from the open to an intermediate conformation( Fig 1D). This transition is primarily driven by the interaction of the adenine ring of ATP with the C-spine and the hinge region, a process that fuses the N- and C-lobe portions of the C-spine[17]. The binding of a substrate allows the final transition from the intermediate to the closed conformation[18]. The C-lobe portion of the active site cleft of most EPKs has a negatively charged electrostatic surface and thus requires two magnesium ions( Mg2+) to fully neutralize the negative charge of the phosphates( Fig 1E)[15]. These Mg2+ ions bind to two conserved residues from the C-lobe—an aspartate( D184 in PKA) from the DFG motif in the AL and an asparagine( N171) from the CL—to position the γ-phosphate of ATP for phosphoryl transfer( Fig 1F)[19]. The α/β-phosphates of ATP interact with the highly conserved lysine residue in the β3-strand( β3K[K72]), which forms a salt bridge with a conserved glutamate from the αC-helix( αCE[E91])[20, 21]. It was initially assumed that β3K was required for ATP binding and the salt bridge with αCE was required for stabilizing β3K. However, subsequent biochemical data showed that mutation of these residues abolished catalytic activity but had no impact on ATP binding, and therefore the precise role of these residues in catalysis remained unclear[22, 23]. The Glycine-rich Loop( G-loop) of the N-lobe—which interacts with ATP, substrate, and the C-lobe—seals the active site chamber in the closed conformation. When the substrate( serine, threonine, or tyrosine) is positioned by the conserved aspartate from the HRD motif in the CL( D166), phosphoryl transfer is achieved[24]. Finally, the phosphorylated substrate and adenosine diphosphate( ADP) are released and the EPK transitions back to an open conformation, where it is poised to bind another ATP molecule to repeat this process[25]. Although the end points of the phosphoryl transfer process have been elucidated and each step in the catalytic cycle has been captured in a crystal lattice, the interactions that facilitate the catalytic cycle remain unclear. Using an Escherichia coli expression system, site-directed mutagenesis, western blotting, a radioactive phosphoryl transfer assay, molecular dynamics( MD) simulations, molecular modeling, and a thermal shift assay, we have deciphered the interactions required for the mechanics of the catalytic cycle as well as phosphoryl transfer. Using PKA as a model system, we show that the β3K-αCE salt bridge is required for connecting the αC-helix with the β-sheet region of the N-lobe during the catalytic cycle and that in the absence of the salt bridge, the mechanics can be restored by strengthening the hydrophobicity of the R-spine and Shell residues. The positive charge of β3K is required for neutralizing the negative charge on αCE, and in the absence of β3K, our MD simulation data indicates that the negative charge of αCE interacts with the Mg2+ ions, causing misalignment of the γ-phosphate, which inhibits phosphoryl transfer. Surprisingly, although mutating β3K on its own is inactive, removing both β3K and αCE simultaneously partially restores catalytic activity.
Discussion:
According to our spine hypothesis and reinforced by NMR studies[10, 14, 16], the binding of the adenine ring of ATP drives the transition of the EPK core from the open to the intermediate conformation by assembling the C-spine. This transition also coordinates the electrostatic interaction of β3K with αCE and the α/β-phosphates of ATP. Breaking the β3K–αCE salt bridge by mutating αCE to an alanine destabilizes the active conformation that is associated with the assembled spine, where it cannot undergo cis-autophosphorylation, trans-autophosphoryaltion, or steady-state catalysis. The assembly of the R-spine, however, can be partially restored by coexpressing αCE/A with PDK1, which introduces an interaction between the phosphorylated activation loop( pT197) and H87 from the αC-Helix. These concerted effects associated with phosphorylation of the activation loop allow the αC-helix to be positioned in an “active” state where it can now transition from the open to the intermediate and closed conformations. Our results show, however, that even though αCE/A+PDK1 regained catalytic activity, the steady-state catalysis remained low, most likely because of the inability of the EPK core to unhinge the αC-helix from the AL. To ameliorate this deficiency, we mutated H87 to an alanine so that the αC-helix was no longer electrostatically anchored to the AL. The αCE/A+H87A( PDK1) double mutant had a ~50% increase in steady-state catalysis, confirming that assembly of the R-spine by phosphorylation is due to the linking of the AL phosphate with the HRD motif and not to the interaction with H87. Following AL phosphorylation, the active conformation is not stabilized independently of H87; instead, H87 appears to negatively regulate the steady-state phosphoryl transfer by impeding opening of the catalytic cleft. These results indicate that the salt bridge is required for closing as well as opening. In the absence of the salt bridge as well as the electrostatic interaction between the activation loop and the αC-helix, the active state mechanics can be regulated through the R-spine and Shell. By enhancing the hydrophobicity of RS3 from a leucine to a phenylalanine, we were able to connect the β-sheet region and the αC-helix through the hydrophobic interaction between the Sh2 and Sh3 from the β-sheet region and RS3 from the αC-helix. The αCE/A+H87A+RS3L/F( PDK1) is ~2-fold more catalytically active than the WT-C. These results indicate that the active state mechanics of EPKs are harmoniously regulated by both electrostatic and hydrophobic interactions. Our results also show that β3K is required for sensing ATP and enabling the transition of the EPK core from the open to the intermediate conformation, and any mutation of β3K significantly reduces this conformational shift. The inability of the β3K/A mutant to transition from the open to the intermediate conformation is rescued to ~4-fold of WT-C by introducing a lysine on the G-loop, which mimics WNK-kinase. In contrast, the other β3K mutants were unable to achieve similar catalytic activities because of the clamped G-loop conformation induced by β3K/M, the inactive-like clockwise twisting of the lobes in the shorter β3K/H mutant, and the counter-clockwise twisting of the N-lobe in the bulkier β3K/R mutant, based on our MD simulation data. For more than a quarter century, it was believed that β3K was directly involved in phosphoryl transfer by positioning the γ-phosphate of ATP[13]; however, our MD simulation data indicated that in the absence of the positive charge on β3K, the free negative charge on the partnering αCE starts interacting with the Mg2+, resulting in the delocalization of the γ-phosphate. By removing both of the charges on β3K/A and αCE/A, we were able to partially rescue the catalytic activity that was lost in the single β3K/A mutant. In conjunction with previous findings, our results unambiguously indicate that β3K is not required for binding or positioning ATP and is not essential for phosphoryl transfer. To make sure that β3K is not only involved in connecting the αC-helix with the β-sheet region but also sensing ATP, we introduced the enhanced R-spine + Shell system into the double mutant( β3K/A+ αCE/A+H87/A+RS3L/F), and the catalytic activity was only rescued to ~12% of the WT-C as compared to αCE/A+H87/A+RS3L/F, which is ~2-fold more active than WT-C, indicating that β3K is required for connecting the different regions of the N-lobe in the presence ATP but not required for phosphoryl transfer. Currently, any variation of β3K is a key feature used for identifying pseudokinases[5], but our results indicate that there are other mechanisms that can compensate for these variations. We therefore believe that an in-depth analysis of all the catalytic components is required prior to definitively labeling an EPK as a pseudokinase. Furthermore, EPK mutations have been reported in numerous diseases, but one of the major challenges is differentiating between causative and noncausative mutations without extensive experimental analysis[44]. This study opens a new avenue of predicting disease-causing mutations that do not generate a constitutively active or inactive EPK but enhance the rate of catalytic activity once the EPK has been activated. It is also currently known that EPKs have noncatalytic roles in biological processes, and β3K mutants are widely utilized to decipher these noncanonical functions[45]. Our findings show that these mutations should be designed with caution, as the conformational differences between the different mutants may interfere with these noncanonical functions as well.
Eukaryotic protein kinases ( EPKs ) regulate over a third of the human proteome by transferring the γ-phosphate from adenosine triphosphate ( ATP ) to a protein substrate in a process known as protein phosphorylation . Biochemical and biophysical studies have shown that EPKs undergo multiconformational rearrangements in which the catalytic core is oscillating between open , intermediate , and closed conformations when active . Presently , the intramolecular interactions that regulate this dynamic process are not well understood . In this paper , we show how a set of conserved electrostatic and hydrophobic interactions harmoniously regulate the active state mechanics . The electrostatic interactions involve the highly conserved salt bridge between the lysine from subdomain-II and glutamate from subdomain-III as well as an interaction between the activation loop and αC-helix . The hydrophobic interactions include the nonlinear motifs known as the Regulatory spine and Shell that traverse both lobes of the catalytic core . Furthermore , our findings show that the highly conserved “catalytic lysine” is not directly required for phosphoryl transfer but rather serves as a hub that aligns and positions the dynamic core elements required for catalysis .