Conformational transitions in BTG1 antiproliferative protein and their modulation by disease mutants

Published:April 21, 2022DOI:


      B cell translocation gene 1 (BTG1) protein belongs to the BTG/transducer of ERBB2 (TOB) family of antiproliferative proteins whose members regulate various key cellular processes such as cell cycle progression, apoptosis, and differentiation. Somatic missense mutations in BTG1 are found in ∼70% of a particularly malignant and disseminated subtype of diffuse large B cell lymphoma (DLBCL). Antiproliferative activity of BTG1 has been linked to its ability to associate with transcriptional cofactors and various enzymes. However, molecular mechanisms underlying these functional interactions and how the disease-linked mutations in BTG1 affect these mechanisms are currently unknown. To start filling these knowledge gaps, here, using atomistic molecular dynamics (MD) simulations, we explored structural, dynamic, and kinetic characteristics of BTG1 protein, and studied how various DLBCL mutations affect these characteristics. We focused on the protein region formed by α2 and α4 helices, as this interface has been reported not only to serve as a binding hotspot for several cellular partners but also to harbor sites for the majority of known DLBCL mutations. Markov state modeling analysis of extensive MD simulations revealed that the α2-α4 interface in the wild-type (WT) BTG1 undergoes conformational transitions between closed and open metastable states. Importantly, we show that some of the mutations in this region that are observed in DLBCL, such as Q36H, F40C, Q45P, E50K (in α2), and A83T and A84E (in α4), either overstabilize one of these two metastable states or give rise to new conformations in which these helices are distorted (i.e., kinked or unfolded). Based on these results, we conclude that the rapid interconversion between the closed and open conformations of the α2-α4 interface is an essential component of the BTG1 functional dynamics that can prime the protein for functional associations with its binding partners. Disruption of the native dynamic equilibrium by DLBCL mutants leads to the ensemble of conformations in BTG1 that are unlikely structurally and/or kinetically to enable productive functional interactions with the binding proteins.
      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Biophysical Journal
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Rouault J.P.
        • Rimokh R.
        • Magaud J.
        • et al.
        Btg1, a member of a new family of antiproliferative genes.
        Embo J. 1992; 11: 1663-1670
        • Winkler G.S.
        The mammalian anti-proliferative BTG/Tob protein family.
        J. Cell Physiol. 2010; 222: 66-72
        • Yuniati L.
        • Scheijen B.
        • van Leeuwen F.N.
        • et al.
        Tumor suppressors BTG1 and BTG2: beyond growth control.
        J. Cell Physiol. 2019; 234: 5379-5389
        • Kanda M.
        • Oya H.
        • Kodera Y.
        • et al.
        Diversity of clinical implication of B-cell translocation gene 1 expression by histopathologic and anatomic subtypes of gastric cancer.
        Dig. Dis Sci. 2015; 60: 1256-1264
        • Kanda M.
        • Sugimoto H.
        • Kodera Y.
        • et al.
        B-cell translocation gene 1 serves as a novel prognostic indicator of hepatocellular carcinoma.
        Int. J. Oncol. 2015; 46: 641-648
        • Kawakubo H.
        • Brachtel E.
        • Maheswaran S.
        • et al.
        Loss of B-cell translocation gene-2 in estrogen receptor-positive breast carcinoma is associated with tumor grade and overexpression of cyclin D1 protein.
        Cancer Res. 2006; 66: 7075-7082
        • Struckmann K.
        • Schraml P.
        • Moch H.
        • et al.
        Impaired expression of the cell cycle RegulatorBTG2Is common in clear cell renal cell carcinoma.
        Cancer Res. 2004; 64: 1632-1638
        • Ficazzola M.A.
        • Fraiman M.
        • Walden P.D.
        • et al.
        Antiproliferative B cell translocation gene 2 protein is downregulated post-transcriptionally as an early event in prostate carcinogenesis.
        Carcinogenesis. 2001; 22: 1271-1279
        • Almasmoum H.A.
        • Airhihen B.
        • Winkler G.S.
        • et al.
        Frequent loss of BTG1 activity and impaired interactions with the Caf1 subunit of the Ccr4-Not deadenylase in non-Hodgkin lymphoma.
        Leuk. Lymphoma. 2021; 62: 281-290
        • Mlynarczyk C.
        • Teater M.
        • Melnick A.
        • et al.
        BTG1 mutation promotes aggressive lymphoma development by lowering the threshold to MYC activation and generating "Super-Competitor" B cells.
        Blood. 2021; 138: 359
        • Busson M.
        • Carazo A.
        • Cabello G.
        • et al.
        Coactivation of nuclear receptors and myogenic factors induces the major BTG1 influence on muscle differentiation.
        Oncogene. 2005; 24: 1698-1710
        • Horiuchi M.
        • Takeuchi K.
        • Inagaki F.
        • et al.
        Structural basis for the antiproliferative activity of the tob-hCaf1 complex.
        J. Biol. Chem. 2009; 284: 13244-13255
        • Amine H.
        • Ripin N.
        • Mauxion F.
        • et al.
        A conserved motif in human BTG1 and BTG2 proteins mediates interaction with the poly(A) binding protein PABPC1 to stimulate mRNA deadenylation.
        Rna Biol. 2021; 18: 2450-2465
        • Lin W.J.
        • Gary J.D.
        • Herschman H.R.
        • et al.
        The mammalian immediate-early TIS21 protein and the leukemia-associated BTG1 protein interact with a protein-arginine -methyltransferase.
        J. Biol. Chem. 1996; 271: 15034-15044
        • Rouault J.P.
        • Prevot D.
        • Corbo L.
        • et al.
        Interaction of BTG1 and p53-regulated BTG2 gene products with mCaf1, the murine homolog of a component of the yeast CCR4 transcriptional regulatory complex.
        J. Biol. Chem. 1998; 273: 22563-22569
        • Prevot D.
        • Voeltzel T.
        • Corbo L.
        • et al.
        The leukemia-associated protein Btg1 and the p53-regulated protein Btg2 interact with the homeoprotein Hoxb9 and enhance its transcriptional activation.
        J. Biol. Chem. 2000; 275: 147-153
        • Hwang S.S.
        • Lim J.
        • Flavell R.A.
        • et al.
        mRNA destabilization by BTG1 and BTG2 maintains T cell quiescence.
        Science. 2020; 367: 1255-1260
        • Luo E.C.
        • Nathanson J.L.
        • Yeo G.W.
        • et al.
        Large-scale tethered function assays identify factors that regulate mRNA stability and translation.
        Nat. Struct. Mol. Biol. 2020; 27: 989-1000
        • Hata K.
        • Nishijima K.
        • Mizuguchi J.
        Role for Btg1 and Btg2 in growth arrest of WEHI-231 cells through arginine methylation following membrane immunoglobulin engagement.
        Exp. Cell Res. 2007; 313: 2356-2366
        • Doidge R.
        • Mittal S.
        • Winkler G.S.
        • et al.
        The anti-proliferative activity of BTG/TOB proteins is mediated via the Caf1a (CNOT7) and Caf1b (CNOT8) deadenylase subunits of the Ccr4-not complex.
        PLoS One. 2012; 7: e51331
        • Ezzeddine N.
        • Chen C.Y.A.
        • Shyu A.B.
        Evidence providing new insights into TOB-promoted deadenylation and supporting a link between TOB's deadenylation-enhancing and antiproliferative activities.
        Mol. Cell Biol. 2012; 32: 1089-1098
        • Pasternak M.
        • Pfender S.
        • Schuh M.
        • et al.
        The BTG4 and CAF1 complex prevents the spontaneous activation of eggs by deadenylating maternal mRNAs.
        Open Biol. 2016; 6: 160184
        • Yu C.
        • Ji S.Y.
        • Fan H.Y.
        • et al.
        BTG4 is a meiotic cell cycle-coupled maternal-zygotic transition licensing factor in oocytes.
        Nat. Struct. Mol. Biol. 2016; 23: 387-394
        • Ikematsu N.
        • Yoshida Y.
        • Yamamoto T.
        • et al.
        Tob2, a novel anti-proliferative Tob/BTG1 family member, associates with a component of the CCR4 transcriptional regulatory complex capable of binding cyclin-dependent kinases.
        Oncogene. 1999; 18: 7432-7441
        • Prevot D.
        • Morel A.P.
        • Corbo L.
        • et al.
        Relationships of the antiproliferative proteins BTG1 and BTG2 with CAF1, the human homolog of a component of the yeast CCR4 transcriptional complex.
        J. Biol. Chem. 2001; 276: 9640-9648
        • Yoshida Y.
        • Hosoda E.
        • Yamamoto T.
        • et al.
        Association of ANA, a member of the antiproliferative tob family proteins, with a cafl component of the CCR4 transcriptional regulatory complex.
        Jpn. J. Cancer Res. 2001; 92: 592-596
        • Yang X.
        • Morita M.
        • Rao Z.
        • et al.
        Crystal structures of human BTG2 and mouse TIS21 involved in suppression of CAF1 deadenylase activity.
        Nucleic Acids Res. 2008; 36: 6872-6881
        • Rodier A.
        • Rochard P.
        • Cabello G.
        • et al.
        Identification of functional domains involved in BTG1 cell localization.
        Oncogene. 2001; 20: 2691-2703
        • Suarez E.
        • Wiewiora R.P.
        • Zuckerman D.M.
        • et al.
        What markov state models can and cannot do: correlation versus path-based observables in protein-folding models.
        J. Chem. Theor. Comput. 2021; 17: 3119-3133
        • Pande V.S.
        • Beauchamp K.
        • Bowman G.R.
        Everything you wanted to know about Markov State Models but were afraid to ask.
        Methods. 2010; 52: 99-105
        • Noé F.
        • Clementi C.
        Kinetic distance and kinetic maps from molecular dynamics simulation.
        J. Chem. Theor. Comput. 2015; 11: 5002-5011
        • Molgedey L.
        • Schuster H.G.
        Separation of a mixture of independent signals using time delayed correlations.
        Phys. Rev. Lett. 1994; 72: 3634-3637
        • Noe F.
        • Schutte C.
        • Weikl T.R.
        • et al.
        Constructing the equilibrium ensemble of folding pathways from short off-equilibrium simulations.
        Proc. Natl. Acad. Sci. U S A. 2009; 106: 19011-19016
        • E W.
        • Vanden-Eijnden E.
        Towards a theory of transition paths.
        J. Stat. Phys. 2006; 123: 503-523
        • Phillips J.C.
        • Braun R.
        • Schulten K.
        • et al.
        Scalable molecular dynamics with NAMD.
        J. Comput. Chem. 2005; 26: 1781-1802
        • Essmann U.
        • Perera L.
        • Pedersen L.G.
        • et al.
        A smooth particle mesh ewald method.
        J. Chem. Phys. 1995; 103: 8577-8593
        • Evans D.J.
        • Holian B.L.
        The nose-hoover Thermostat.
        J. Chem. Phys. 1985; 83: 4069-4074
        • Harvey M.J.
        • Giupponi G.
        • De Fabritiis G.
        ACEMD: accelerating biomolecular dynamics in the microsecond time scale.
        J. Chem. Theor. Comput. 2009; 5: 1632-1639
        • Khelashvili G.
        • Stanley N.
        • Weinstein H.
        • et al.
        Spontaneous inward opening of the dopamine transporter is triggered by PIP2-regulated dynamics of the N-terminus.
        Acs Chem. Neurosci. 2015; 6: 1825-1837
        • Eastman P.
        • Swails J.
        • Pande V.S.
        • et al.
        OpenMM 7: rapid development of high performance algorithms for molecular dynamics.
        Plos Comput. Biol. 2017; 13: e1005659
        • Lee J.
        • Cheng X.
        • Im W.
        • et al.
        CHARMM-GUI input generator for NAMD, gromacs, amber, openmm, and CHARMM/OpenMM simulations using the CHARMM36 additive force field.
        Biophys. J. 2016; 110: 641a
        • Scherer M.K.
        • Trendelkamp-Schroer B.
        • Noe F.
        • et al.
        PyEMMA 2: a software package for estimation, validation, and analysis of markov models.
        J. Chem. Theor. Comput. 2015; 11: 5525-5542
        • Röblitz S.
        • Weber M.
        Fuzzy spectral clustering by PCCA+: application to Markov state models and data classification.
        Adv. Data Anal. Classification. 2013; 7: 147-179
        • Kabsch W.
        • Sander C.
        Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features.
        Biopolymers. 1983; 22: 2577-2637
        • McGibbon Robert T.
        • Beauchamp K.
        • Pande V.
        • et al.
        MDTraj: a modern open library for the analysis of molecular dynamics trajectories.
        Biophys. J. 2015; 109: 1528-1532
        • Visiers I.
        • Braunheim B.B.
        • Weinstein H.
        Prokink: a protocol for numerical evaluation of helix distortions by proline.
        Protein Eng. 2000; 13: 603-606