Activation Loop Dynamics Are Coupled to Core Motions in Extracellular Signal-Regulated Kinase-2.

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Protein kinases regulate cellular processes via phosphorylation.
• The activation loop of kinases is crucial for enzyme activity and regulation.
• Extracellular signal-regulated kinase 2 (ERK2) activation involves dual phosphorylation of its activation loop.

Purpose of the Study:

• To investigate the conformational dynamics of the dual-phosphorylated activation loop in ERK2.
• To determine if activation loop dynamics are coupled to the kinase core and catalytic site.
• To understand the role of activation loop dynamics in ERK2 function and allosteric regulation.

Main Methods:

• X-ray crystallography to determine static structures.
• Nuclear Magnetic Resonance (NMR) Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments to probe dynamics.
• Site-directed mutagenesis to assess the impact of mutations on dynamics and function.

Main Results:

• Despite stabilizing interactions, the phosphorylated ERK2 activation loop exhibits microsecond to millisecond timescale mobility.
• Activation loop dynamics are coupled to global motions in the kinase core and catalytic site.
• Mutations in either the activation loop or the core region affect the dynamics of the other, demonstrating allosteric coupling.

Conclusions:

• The active conformation of dual-phosphorylated ERK2 is not static but involves dynamic exchange between conformers.
• The activation loop acts as part of a dynamic allosteric network, influencing nucleotide binding and catalytic function.
• Understanding these coupled dynamics is key to comprehending kinase regulation and function.