Crystal structure of EML1 reveals the basis for Hsp90 dependence of oncogenic EML4-ALK by disruption of an atypical β-propeller domain

Mark W. Richards; Edward W. P. Law; La’Verne P. Rennalls; Sara Busacca; Laura O’Regan; Andrew M. Fry; Dean A. Fennell; Richard Bayliss

doi:10.1073/pnas.1322892111

Edited by Charles David Stout, The Scripps Research Institute, La Jolla, CA, and accepted by the Editorial Board February 24, 2014 (received for review December 9, 2013)

Significance

Echinoderm microtubule-associated protein (EMAP)-like (EML) proteins normally function in the cytoskeleton. In some lung cancers, genetic abnormalities generate the oncogenic fusion protein EML4-anaplastic lymphoma kinase (ALK) on which the cancer cells depend for survival. We have determined the molecular structure of a conserved, tubulin-binding region of EML1 that reveals an unexpected protein fold. This region is disrupted in ∼70% of EML4-ALK fusions found in patients, causing them to be sensitive to drugs that target Hsp90, a cellular factor that stabilizes misfolded protein. Our findings will potentially enable more effective, stratified therapy of EML4-ALK nonsmall cell lung cancer and suggest that the truncation of a globular domain at the translocation breakpoint may prove generally predictive of Hsp90 inhibitor sensitivity in cancers driven by fusion oncogenes.

Abstract

Proteins of the echinoderm microtubule-associated protein (EMAP)-like (EML) family contribute to formation of the mitotic spindle and interphase microtubule network. They contain a unique hydrophobic EML protein (HELP) motif and a variable number of WD40 repeats. Recurrent gene rearrangements in nonsmall cell lung cancer fuse EML4 to anaplastic lymphoma kinase (ALK), causing expression of several fusion oncoprotein variants. We have determined a 2.6-Å crystal structure of the representative ∼70-kDa core of EML1, revealing an intimately associated pair of β-propellers, which we term a TAPE (tandem atypical propeller in EMLs) domain. One propeller is highly atypical, having a discontinuous subdomain unrelated to a WD40 motif in place of one of its blades. This unexpected feature shows how a propeller structure can be assembled from subdomains with distinct folds. The HELP motif is not an independent domain but forms part of the hydrophobic core that joins the two β-propellers. The TAPE domain binds α/β-tubulin via its conserved, concave surface, including part of the atypical blade. Mapping the characteristic breakpoints of each EML4-ALK variant onto our structure indicates that the EML4 TAPE domain is truncated in many variants in a manner likely to make the fusion protein structurally unstable. We found that the heat shock protein 90 (Hsp90) inhibitor ganetespib induced degradation of these variants whereas others lacking a partial TAPE domain were resistant in both overexpression models and patient-derived cell lines. The Hsp90-sensitive EML4-ALK variants are exceptions to the rule that oncogenic fusion proteins involve breakpoints in disordered regions of both partners.

Footnotes

↵¹To whom correspondence should be addressed. E-mail: richard.bayliss{at}le.ac.uk.

Author contributions: M.W.R., E.W.P.L., L.O., A.M.F., D.A.F., and R.B. designed research; M.W.R., E.W.P.L., L.P.R., and S.B. performed research; M.W.R., E.W.P.L., S.B., D.A.F., and R.B. analyzed data; and M.W.R., A.M.F., D.A.F., and R.B. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. C.D.S. is a guest editor invited by the Editorial Board.
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 4ci8).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1322892111/-/DCSupplemental.

View Full Text