Noncovalent binding of a cyclic peptide inhibitor to the peptidyl-prolyl isomerase Pin1, explored by hydrogen exchange mass spectrometry

Pin1 is a peptidyl-prolyl isomerase (PPIase) that plays a central role in eukaryotic cell cycle regulation, making this protein an interesting target for cancer therapy. Pin1 exhibits high specificity for substrates where proline is preceded by phosphoserine or phosphothreonine. The protein comprises an N-terminal WW (tryptophan–tryptophan) domain and a C-terminal PPIase domain. The cyclic peptide [CRYPEVEIC] (square brackets are used to denote the cyclic structure) represents a lead compound for a new class of nonphosphorylated Pin1 inhibitors. Unfortunately, it has not been possible thus far to characterize the Pin1–[CRYPEVEIC] complex by X-ray crystallography. Thus, the exact binding mode remains unknown. The current work employs hydrogen/deuterium exchange mass spectrometry for gaining insights into the Pin1–[CRYPEVEIC] interactions. The WW domain shows extensive conformational dynamics, both in the presence and in the absence of ligand. In contrast, profound changes in deuteration kinetics are observed in the PPIase domain after the addition of [CRYPEVEIC]. The secondary structure elements β2, α3, and α4 exhibit markedly reduced deuteration, consistent with their postulated involvement in ligand binding. Unexpectedly, [CRYPEVEIC] destabilizes the range of residues 61–86, a segment that comprises basic side chains that normally interact with the substrate phosphate. This destabilization is likely caused by steric clashes with Y3 or E5 of the inhibitor. Ligand-induced destabilization has previously been reported for a few other proteins, but effects of this type are not very common. Our findings suggest that future crystallization trials on Pin1 variants deleted for residues in the 61–86 range might provide a path towards high-resolution X-ray structures of Pin1 bound to cyclic peptide inhibitors.

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

10.1101/639567 ◽

2019 ◽

Author(s):

Laurel M. Pegram ◽

Jennifer C. Liddle ◽

Yao Xiao ◽

Maria Hoh ◽

Johannes Rudolph ◽

...

Keyword(s):

Mass Spectrometry ◽

Map Kinase ◽

Active Site ◽

Hydrogen Exchange ◽

Activation Loop ◽

X Ray ◽

Exchange Mass Spectrometry ◽

Map Kinase Phosphatase ◽

Loop Dynamics ◽

Hydrogen Exchange Mass Spectrometry

ABSTRACTModulating 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⇌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⇌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.SIGNIFICANCE 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.

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