Biology subjects

Ahn, N. G.

Publications and source records attributed to Ahn, N. G..

3 recordsLinked to original sources

bioRxiv2023

Conformation Selection by ATP-competitive Inhibitors and Allosteric Communication in ERK2

Activation of the extracellular signal regulated kinase-2 (ERK2) by phosphorylation has been shown to involve changes in protein dynamics, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and NMR relaxation dispersion measurements. These can be described by a global exchange between two conformational states of the active kinase, named "L" and "R", where R is associated with a catalytically productive ATP-binding mode. An ATP-competitive ERK1/2 inhibitor, Vertex-11e, has properties of conformation selection for the R-state, revealing movements of the activation loop that are allosterically coupled to the kinase active site. However, the features of inhibitors important for R-state selection are unknown. Here we survey a panel of ATP-competitive ERK inhibitors using HDX-MS and NMR and identify 14 new molecules with properties of R-state selection. They reveal effects propagated to distal regions in the P+1 and helix F segments surrounding the activation loop, as well as helix L16. Crystal structures of inhibitor complexes with ERK2 reveal systematic shifts in the Gly loop and helix C, mediated by a Tyr-Tyr ring stacking interaction and the conserved Lys-Glu salt bridge. The findings suggest a model for the R-state involving small movements in the N-lobe that promote compactness within the kinase active site and alter mobility surrounding the activation loop. Such properties of conformation selection might be exploited to modulate the protein docking interface used by ERK substrates and effectors.

biochemistry↗

bioRxiv2023

Activation loop plasticity and active site coupling in the MAP kinase, ERK2

Changes in the dynamics of the protein kinase, ERK2, have been shown to accompany its activation by dual phosphorylation. However, our knowledge about the conformational changes represented by these motions is incomplete. Previous NMR relaxation dispersion studies showed that active, dual-phosphorylated ERK2 undergoes global exchange between at least two energetically similar conformations. These findings, combined with measurements by hydrogen exchange mass spectrometry (HX-MS), suggested that the global conformational exchange involves motions of the activation loop (A-loop) that are coupled to regions surrounding the kinase active site. In order to better understand the contribution of dynamics to the activation of ERK2, we applied long conventional molecular dynamics (MD) simulations starting from crystal structures of active, phosphorylated (2P), and inactive, unphosphorylated (0P) ERK2. Individual trajectories were run for (5 to 25) {micro}s and totaled 727 {micro}s. The results showed that the A-loop is unexpectedly flexible in both 2P- and 0P-ERK2, and able to adopt multiple long-lived (>5 {micro}s) conformational states. Simulations starting from the X-ray structure of 2P-ERK2 (2ERK) revealed A-loop states corresponding to restrained dynamics within the N-lobe, including regions surrounding catalytic residues. One A-loop conformer forms lasting interactions with the C-terminal L16 segment and shows reduced RMSF and greater compaction in the active site. By contrast, simulations starting from the most common X-ray conformation of 0P-ERK2 (5UMO) reveal frequent excursions of A-loop residues away from a C-lobe docking site pocket and towards a new state that shows greater dynamics in the N-lobe and disorganization around the active site. Thus, the A-loop in ERK2 appears to switch between distinct conformational states that reflect allosteric coupling with the active site, likely occurring via the L16 segment. Analyses of crystal packing interactions across many structural datasets suggest that the A-loop observed in X-ray structures of ERK2 may be driven by lattice contacts and less representative of the solution structure. The novel conformational states identified by MD expand our understanding of ERK2 regulation, by linking the activated state of the kinase to reduced dynamics and greater compaction surrounding the catalytic site.

biophysics↗

bioRxiv2019

Activation loop dynamics are controlled by conformation-selective inhibitors of ERK2

Modulating the dynamics of protein kinases expands the inhibitory mechanisms for small molecules. NMR measurements of the MAP kinase, ERK2, have shown that activation by dual-phosphorylation induces global motions involving exchange between two states, \"L\" and \"R\". We show that ERK inhibitors Vertex-11e and SCH772984 exploit the small energetic difference between L and R to shift the equilibrium in opposing directions, while inhibitor GDC-0994 and ATP analogue AMP-PNP retain L{rightleftharpoons}R exchange. An X-ray structure of active 2P-ERK2 complexed with AMP-PNP reveals a shift in the Gly-rich loop along with domain closure to position the nucleotide in a more catalytically productive conformation relative to inactive 0P-ERK2:ATP. X-ray structures of 2P-ERK2 complexed with Vertex-11e or GDC-0994 recapitulate this closure, which is blocked in a complex with a SCH772984 analogue. Thus, the L[->]R shift in 2P-ERK2 is associated with movements needed to form a competent active site. Solution measurements by hydrogen-exchange mass spectrometry (HX-MS) reveal distinct binding modes for Vertex-11e, GDC-0994 and AMP-PNP to active vs inactive ERK2, where the extent of HX protection matches their degree of R-state formation. In addition, Vertex-11e and SCH772984 show opposite effects on HX near the activation loop, suggesting that L{rightleftharpoons}R exchange involves coupling between the activation loop and the active site. Consequently, these inhibitors differentially affect MAP kinase phosphatase activity towards 2P-ERK2. We conclude that global motions in ERK2 promote productive nucleotide binding, and couple with the activation loop to allow control of dephosphorylation by conformation-selective inhibitors.\n\nSIGNIFICANCE STATEMENTProtein kinases in the RAF/MKK/ERK signaling pathway are dysregulated in cancer and are important targets for inhibitor development. Catalytic activation of the MAP kinase, ERK2, induces global motions involving exchange between two conformational states. Using nuclear magnetic resonance (NMR) and hydrogen-exchange mass spectrometry, we show that inhibitors exploit these motions to trap ERK2 in distinct states. Our findings reveal motions of the activation loop coupled to the active site. Inhibitor binding can control these activation loop dynamics to alter its rate of dephosphorylation by MAP kinase phosphatase.

biochemistry↗