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Conformational heterogeneity of the BTK PHTH domain drives multiple regulatory states

ABSTRACT: Full-length BTK has been refractory to structural analysis. The nearest full-length structure of BTK to date consists of the autoinhibited SH3-SH2-kinase core. Precisely how the BTK N-terminal domains (the Pleckstrin homology/Tec homology (PHTH) domain and the long linker that includes proline-rich regions (PRR)) contribute to BTK regulation remains unclear. Here we produce crystals of full-length BTK for the first time. Despite efforts to stabilize the autoinhibited state, the diffraction data still reveals only the SH3-SH2-kinase core with no electron density visible for the PHTH-PRR segment. CryoEM data, on the other hand, provides a glimpse of the PHTH domain. CryoEM reconstructions support conformational heterogeneity in the PHTH-PRR region; the globular PHTH domain adopts a range of states arrayed around the autoinhibited SH3-SH2-kinase core. Upon disassembly of the SH3-SH2-kinase core, an autoinhibitory site on the kinase domain becomes available for PHTH domain binding. This PHTH/kinase autoinhibitory contact is then lost upon interaction of PHTH with PIP3. Membrane-induced dimerization activates BTK and here we solve a structure of an activation loop swapped BTK kinase domain dimer that likely represents the conformational state leading to trans-autophosphorylation. Together, these data provide the first structural insight into full-length BTK and allow a deeper understanding of allosteric control over the BTK kinase domain during distinct stages of activation.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Thomas Wales

LAB HEAD: Thomas E. Wales

PROVIDER: PXD041657 | Pride | 2024-05-24

REPOSITORIES: Pride

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Conformational heterogeneity of the BTK PHTH domain drives multiple regulatory states.

Lin David Yin-Wei DY Kueffer Lauren E LE Juneja Puneet P Wales Thomas E TE Engen John R JR Andreotti Amy H AH

eLife 20240108

Full-length Bruton's tyrosine kinase (BTK) has been refractory to structural analysis. The nearest full-length structure of BTK to date consists of the autoinhibited SH3-SH2-kinase core. Precisely how the BTK N-terminal domains (the Pleckstrin homology/Tec homology [PHTH] domain and proline-rich regions [PRR] contain linker) contribute to BTK regulation remains unclear. We have produced crystals of full-length BTK for the first time but despite efforts to stabilize the autoinhibited state, the d ...[more]

PMID: 38189455

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Project description:Src family kinases are often overexpressed and constitutively active in cancer cells, where they represent promising targets for therapeutic intervention. In myeloid hematopoietic cells, Hck, Lyn, and Fgr are associated with both chronic and acute forms of myeloid leukemia. All three of these kinases are expressed in acute myeloid leukemia (AML), where high-level expression correlates with poor survival. The pyrrolopyrimidine Src-family kinase inhibitor A-419259 (also known as RK-20449) has been shown to significantly inhibit patient-derived AML bone marrow cell growth in immunocompromised mice. Recent work has identified an N-phenylbenzamide kinase inhibitor, known as TL02-59, which potently inhibited Fgr kinase activity in vitro and reversed orthotopic engraftment of AML cells in the bone marrow and spleen of immunocompromised mice following oral administration. The inhibitory mechanism of this compound with Fgr or other Src-family members has not been reported. Both wild-type Fgr and a tail mutant (i.e., replacement of Tyr527 with Phe) were shown to produce equal numbers of transformed colonies when expressed in rodent fibroblasts, whereas wild-type Hck transforming activity was completely repressed. In both Hck and Fgr, however, the tail tyrosine residue was shown to be phosphorylated, and hydrogen deuterium exchange mass spectrometry (HDX MS) of near-full-length Fgr was consistent with the closed conformation in solution, with the SH3 domain bound to the SH2-kinase linker and the tail bound to SH2. Here we present the first X-ray crystal structures of near-full-length Fgr bound to the ATP site inhibitors A-419259 and TL02-59. Structural changes induced by the inhibitors in the crystalline state were validated in solution using HDX MS, with A-419259 stabilizing the closed conformation and TL02-59 enhancing exposure of the SH3 domain to solvent. These new structures help to explain the constitutive activity of wild-type Fgr in cells as well as its unique sensitivity to TL02-59, which is currently in development for AML and other maladies.

			Action	DRS
	01_FLBTK_10m.raw.zip	Raw
	01_FLBTK_10s.raw.zip	Raw
	01_FLBTK_1h.raw.zip	Raw
	01_FLBTK_4h.raw.zip	Raw
	01_FLBTK_UND1.raw.zip	Raw