Structural studies of intrinsically disordered MLL-fusion protein AF9 in complex with peptidomimetic inhibitors

Yang, Yuting; Ahmad, Ejaz; Premkumar, Vidhya; Liu, Alicen; Ashikur Rahman, S. M.; Nikolovska‐Coleska, Zaneta

doi:10.1002/pro.5019

Title: Structural studies of intrinsically disordered MLL-fusion protein AF9 in complex with peptidomimetic inhibitors

Journal Article · 15 May 2024 · Protein Science

DOI: https://doi.org/10.1002/pro.5019 · OSTI ID:2470167

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^[1]; Premkumar, Vidhya ^[1]; Liu, Alicen ^[1];

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Univ. of Michigan, Ann Arbor, MI (United States). Medical School

AF9 (MLLT3) and its paralog ENL(MLLT1) are members of the YEATS family of proteins with important role in transcriptional and epigenetic regulatory complexes. These proteins are two common MLL fusion partners in MLL-rearranged leukemias. The oncofusion proteins MLL-AF9/ENL recruit multiple binding partners, including the histone methyltransferase DOT1L, leading to aberrant transcriptional activation and enhancing the expression of a characteristic set of genes that drive leukemogenesis. The interaction between AF9 and DOT1L is mediated by an intrinsically disordered C-terminal ANC1 homology domain (AHD) in AF9, which undergoes folding upon binding of DOT1L and other partner proteins. We have recently reported peptidomimetics that disrupt the recruitment of DOT1L by AF9 and ENL, providing a proof-of-concept for targeting AHD and assessing its druggability. Intrinsically disordered proteins, such as AF9 AHD, are difficult to study and characterize experimentally on a structural level. In this study, we present a successful protein engineering strategy to facilitate structural investigation of the intrinsically disordered AF9 AHD domain in complex with peptidomimetic inhibitors by using maltose binding protein (MBP) as a crystallization chaperone connected with linkers of varying flexibility and length. The strategic incorporation of disulfide bonds provided diffraction-quality crystals of the two disulfide-bridged MBP–AF9 AHD fusion proteins in complex with the peptidomimetics. These successfully determined first series of 2.1–2.6 Å crystal complex structures provide high-resolution insights into the interactions between AHD and its inhibitors, shedding light on the role of AHD in recruiting various binding partner proteins. We show that the overall complex structures closely resemble the reported NMR structure of AF9 AHD/DOT1L with notable difference in the conformation of the β-hairpin region, stabilized through conserved hydrogen bonds network. These first series of AF9 AHD/peptidomimetics complex structures are providing insights of the protein–inhibitor interactions and will facilitate further development of novel inhibitors targeting the AF9/ENL AHD domain.

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Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF)

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 2470167

Journal Information:: Protein Science, Journal Name: Protein Science Journal Issue: 6 Vol. 33; ISSN 0961-8368

Publisher:: Wiley -- The Protein SocietyCopyright Statement

Country of Publication:: United States

Language:: English

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37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
AF9
DOT1L
MBP fusion
disulfide bridge
intrinsically disordered proteins
peptidomimetrics
x-ray crystallography

Title: Structural studies of intrinsically disordered MLL-fusion protein AF9 in complex with peptidomimetic inhibitors

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