UC San Diego

Since the crystal structure of PKA was solved over thirty years ago, the overall kinase core has been well characterized; various structures have elucidated the overall catalytic cycle and internal interactions required for an active kinase. However, the function and regulation of full-length (FL) kinases and their complexes that include conserved, critically important intrinsically disordered regions (IDRs), are relatively poorly understood. Here we analyze p90 ribosomal S6 kinase (RSK) which is a downstream effector kinase of the Ras/MAPK signaling pathway. RSK contains two distinct, functional kinase domains, an N-terminal kinase (NTK) and a C-terminal kinase (CTK), and flexible IDRs with conserved motifs that mediate interactions necessary for complex assembly with its activating kinase ERK1/2 and to propagate the multistep RSK activation pathway. A flexible Linker connects the NTK and CTK, and a C-terminal tail contains motif that mediate high affinity ERK2 binding. RSK has an important role in cardiac function and is mis-regulated in neurodegenerative disorders and various forms of cancer. We utilize a combination of computational, biophysical, and structural techniques to study FL RSK and the RSK-ERK complex. Molecular dynamics simulations and network analysis are utilized to better understand the interfaces that comprise the CTK-ERK2 heterodimeric complex, and to identify residues that are critical for the stability of the complex but would be difficult to appreciate using other classical techniques. We utilized hydrogen deuterium exchange mass spectrometry (HDXMS) to measure solvent accessibility of the FL RSK2-ERK2 complex to determine flexible and unstructured regions in the complex. And we explored how the flexible IDRs of RSK mediate additional steps in RSK activation by peptide arrays. A non-catalytic viral protein ORF45, was found to stabilize the FL RSK2-ERK2 complex. With HDXMS and peptide arrays, we identified that ORF45 docks onto the NTK, and indirectly, enhances the RSK2-ERK2 interface. Finally, we used cryoEM on the RSK2-ERK2-ORF45 complex and solved low resolution reconstructions of free ERK2, RSK2, and the RSK2-ERK2-ORF45 complex to evaluate their overall size and shape. A major continuing goal is to improve the resolution of our reconstructions to determine the orientation of all three kinase domains relative to one another, and identify how the IDRs of RSK are positioned to mediate additional steps in RSK activation.