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Conformational Dynamics of Protein Kinase A Reveal an Allosteric Mechanism Controlling Nucleotide and Substrate Binding Cooperativity


Core Concepts
The αC-β4 loop of protein kinase A controls the allosteric cooperativity between nucleotide and substrate binding by modulating the structural coupling between the enzyme's two lobes.
Abstract
The content describes an integrated computational and experimental study on the conformational dynamics and allosteric regulation of the catalytic subunit of protein kinase A (PKA-C). Key highlights: Replica-averaged metadynamics (RAM) simulations and Markov State Models (MSMs) reveal that apo PKA-C populates a ground state (GS) and two conformationally excited states (ES1 and ES2). The GS represents the active conformation, while ES1 corresponds to a canonical inactive state with the αC helix swung out. The ES2 state features a disrupted hydrophobic packing around the αC-β4 loop, which was not observed in crystal structures. The αC-β4 loop is identified as a key regulatory element that controls the allosteric coupling between the N- and C-lobes of PKA-C. Mutations in the αC-β4 loop, such as F100A, disrupt the structural communication between the two lobes and abolish the binding cooperativity between ATP and substrate. NMR experiments on the F100A mutant validate the computational predictions, showing attenuated chemical shift perturbations and loss of correlated structural changes upon nucleotide and substrate binding. The F100A mutation interrupts the allosteric network within PKA-C, leading to a dynamically uncommitted state that is unable to efficiently couple nucleotide and substrate binding. The study highlights the pivotal role of the αC-β4 loop in regulating PKA-C function and provides insights into how mutations or insertions in this motif can affect kinase activity and drug sensitivity in other homologous kinases.
Stats
The free energy (ΔG) and relative population of the ground state and the first 6 excited states of PKA-C in different forms (apo, ATP-bound, ATP/PKI-bound) obtained from the RAM simulations are provided in Supplementary Table 1. The kinetic parameters (KM, Vmax, kcat/KM) for Kemptide phosphorylation by PKA-CWT and PKA-CF100A obtained from coupled enzyme assays are shown in Supplementary Table 2. The thermodynamic parameters (ΔG, ΔH, -TΔS, Kd) for nucleotide (ATPγN) and pseudosubstrate (PKI5-24) binding to PKA-CWT and PKA-CF100A determined by isothermal titration calorimetry are presented in Supplementary Tables 3 and 4.
Quotes
"The highly conserved αC-β4 loop emerges as a pivotal element able to control the synergistic binding between nucleotide and substrate." "The data presented here show that it is possible to abolish the binding cooperativity of a kinase by turning the dial in the opposite direction, i.e., increasing the flexibility of the αC-β4 loop and disconnecting the allosteric network between the N- and C-lobes."

Deeper Inquiries

How might the insights from this study on PKA-C be leveraged to develop allosteric modulators that selectively target the αC-β4 loop in other kinases for therapeutic applications

The insights gained from this study on PKA-C regarding the critical role of the αC-β4 loop in regulating kinase function can be instrumental in the development of allosteric modulators for therapeutic applications in other kinases. By targeting the αC-β4 loop, researchers can potentially design small molecules or peptides that modulate the conformational dynamics and interactions within this region. These allosteric modulators could selectively influence the binding cooperativity between nucleotide and substrate, thereby fine-tuning the catalytic activity of the kinase. Through structure-based drug design approaches, such as virtual screening, molecular docking, and molecular dynamics simulations, novel compounds can be designed to interact with specific residues or motifs within the αC-β4 loop. By disrupting or stabilizing the interactions within this critical regulatory element, it may be possible to develop allosteric modulators that can selectively modulate the activity of various kinases for therapeutic purposes.

What other structural elements or dynamic motifs within the kinase core could serve as potential allosteric hotspots to fine-tune substrate recognition and catalytic activity

In addition to the αC-β4 loop, several other structural elements and dynamic motifs within the kinase core could serve as potential allosteric hotspots to fine-tune substrate recognition and catalytic activity. One such element is the hydrophobic core of the kinase, which includes the catalytic spine (C-spine) and regulatory spine (R-spine). Disruption or stabilization of the hydrophobic interactions within these spines can significantly impact the overall conformational dynamics and activity of the kinase. The activation loop, which plays a crucial role in substrate binding and phosphorylation, could also be targeted to modulate the catalytic activity of the kinase. Additionally, motifs involved in ATP binding, such as the Gly-rich loop and the DFG motif, could be potential allosteric hotspots for designing modulators that influence nucleotide binding and kinase activity. By strategically targeting these structural elements and dynamic motifs, researchers can develop novel allosteric modulators that provide precise control over kinase function.

Given the importance of the αC-β4 loop in regulating kinase function, how might evolution have shaped the sequence and structural features of this motif across the kinome to enable diverse signaling functions

The sequence and structural features of the αC-β4 loop across the kinome have likely been shaped by evolution to enable diverse signaling functions and regulatory mechanisms in different kinases. Evolutionary pressures have led to the conservation of key residues within the αC-β4 loop that are essential for maintaining the structural integrity and allosteric communication of the kinase. Variations in the sequence and length of the αC-β4 loop among different kinases may confer specificity in substrate recognition, catalytic activity, and binding cooperativity. The structural plasticity of the αC-β4 loop allows kinases to adapt to different cellular signaling pathways and environmental cues, providing a mechanism for fine-tuning kinase function in response to various stimuli. By understanding the evolutionary constraints and functional significance of the αC-β4 loop, researchers can gain insights into the diverse roles of this motif across the kinome and its implications for kinase regulation and signaling pathways.
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