Malvina Papanastasiou

HX-MS provides structural insights into the roles of mutations in structurally disordered regions of cancer-relevant proteins

Malvina Papanastasiou1, Amanda L. Waterbury2, Hui Si Kwok2, Ceejay Lee2,Allyson M. Freedy2, Cindy Su2, Will Hawkins1, Andrew Reiter1, Samuel M. Hoenig2, Michael E. Vinyard2, Brian B. Liau1,2 and Steven A. Carr1

1 Broad Institute, Cambridge, MA; 2Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA

Introduction: The histone demethylase LSD1 has emerged as a promising target for the treatment of cancers, with numerous LSD1 inhibitors currently in clinical trials, particularly for AML. Recently, the Liau group employed CRISPR-suppressor scanning to identify single-guide RNAs (sgRNAs) that target LSD1 and can promote drug resistance to LSD1 inhibitors (Vinyard, Liau, 2019). As anticipated, the most highly enriched drug-enriched sgRNAs were found to target the active site. Unexpectedly, multiple sgRNAs were discovered to target the N-terminus IDR. Recent experiments using LSD1 mutants have demonstrated that deletions in the LSD1 IDR inhibit AML differentiation and further promote in vitro condensate formation. To provide structural insights into the role of LSD1 IDR that eludes from high resolution structures, we employed HX-MS.

Methods: For HX-MS experiments, proteins were diluted in deuterium (5-fold) for various labeling periods and temperatures and were subsequently quenched with ice chilled guanidine hydrochloride. Samples were prepared either manually or using a Trajan Parallel Extended autosampler, with lipid filtration capabilities, coupled to a Thermo Q-Exactive HF Orbitrap MS. Samples were digested using a combination of proteases (Pepsin:Protease XIII, NovaBioassays). Generated peptides were desalted using a C18 trap (Acclaim PepMap, Thermo) and separated on a Hypersil Gold C18 analytical column (Thermo) employing a 18 min gradient. Deuterium content at the peptide level was measured in HDExaminer (Sierra Analytics) and bimodal distributions were analyzed using HXExpress v3.

Preliminary data: For HX-MS experiments we utilized full-length wild-type LSD1/CoREST, two mutants with short amino acid deletions in their IDR (NΔ1, NΔ2), and a mutant lacking the N-terminus IDR ΝΔ(1-170). More than 800 distinct peptides were identified for LSD1, including 35 peptides spanning the N-terminus IDR, and more than 300 peptides for CoREST, resulting in complete sequence coverage for both proteins. We performed HX-MS at two temperatures (0 oC and 30 oC) to expand the sampling window across many orders of magnitude and capture potential differences between wild-type and mutants in rapidly exchanging amide hydrogens in the N-terminus IDR and in slowly exchanging amide hydrogens in the folded SWIRM, amine-oxidase (AOD), and Tower domains. Differences in D-uptake between wild-type and mutants were identified in peptides encompassing the entire IDR sequence, the AOD, and the intrinsically disordered C-terminus, only at time scales faster than 10 sec and 0 oC. Along the entire length of the IDR, extensive bimodal distributions were observed, pointing to an ensemble of interconverting conformers that transiently populate folded states. Our findings demonstrate that N-terminus deletions enhance the dynamics and accessibility of LSD1 by shifting the IDR conformational landscape towards an ‘open state’. Our results corroborate recent findings demonstrating that these mutants promote in vitro condensate formation, paradoxically, not via IDR-IDR interactions, but likely as a result of increased interactions with newly exposed epitopes in folded regions of the LSD1/CoREST.

Novel aspect: HX-MS offers novel insights into the role of LSD1 IDR, which eludes high resolution structures, in condensate formation in vitro.

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