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Structural, Thermodynamic, and Kinetic Effects of a Phosphomimetic Mutation in Dynein Light Chain LC8

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Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331
*Corresponding authors. Phone: (541) 737-4143. Fax: (541) 737-0481. E-mail: [email protected]
Cite this: Biochemistry 2009, 48, 48, 11381–11389
Publication Date (Web):October 28, 2009
https://doi.org/10.1021/bi901589w
Copyright © 2009 American Chemical Society

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    Abstract

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    Dynein light chain LC8 is a small, dimeric, very highly conserved globular protein first identified as an integral part of the dynein and myosin molecular motors but now recognized as a dimerization hub with wider significance. Phosphorylation at Ser88 is thought to be involved in regulating LC8 in the apoptotic pathway. The phosphomimetic Ser88Glu mutation weakens dimerization of LC8 and thus its overall ligand-binding affinity, because only the dimer binds ligands. The 1.9 Å resolution crystal structure of dimeric LC8S88E bound to a fragment of the ligand Swallow (Swa) presented here shows that the tertiary structure is identical to that of wild-type LC8/Swa, with Glu88 well accommodated sterically at the dimer interface. NMR longitudinal magnetization exchange spectroscopy reveals remarkably slow association kinetics (kon ∼ 1 s−1 mM−1) in the monomer−dimer equilibrium of both wild-type LC8 and LC8S88E, possibly due to the strand-swapped architecture of the dimer. The Ser88Glu mutation raises the dimer dissociation constant (KD) through a combination of a higher koff and lower kon. Using a minimal model of titration linked to dimerization, we dissect the thermodynamics of dimerization of wild-type LC8 and LC8S88E in their various protonation states. When both Glu88 residues are protonated, the LC8S88E dimer is nearly as stable as the wild-type dimer, but deprotonation of one Glu88 residue raises KD by a factor of 400. We infer that phosphorylation of one subunit of wild-type LC8 raises KD by at least as much to prevent dimerization of LC8 at physiological concentrations. Some LC8 binding partners may bind tightly enough to promote dimerization even when one subunit is phosphorylated; thus linkage between phosphorylation and dimerization provides a mechanism for differential regulation of binding of LC8 to its diverse partners.

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    KD, kon, and koff measurements for wild-type LC8 and LC8S88E (shown in Figure 4). This material is available free of charge via the Internet at http://pubs.acs.org.

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    Cited By

    This article is cited by 11 publications.

    1. Fei Xiao, Jingwei Weng, Kangnian Fan, and Wenning Wang . Mechanism of Ser88 Phosphorylation-Induced Dimer Dissociation in Dynein Light Chain LC8. The Journal of Physical Chemistry B 2010, 114 (47) , 15663-15672. https://doi.org/10.1021/jp1048869
    2. Aidan B. Estelle, August George, Elisar J. Barbar, Daniel M. Zuckerman, . Quantifying cooperative multisite binding in the hub protein LC8 through Bayesian inference. PLOS Computational Biology 2023, 19 (4) , e1011059. https://doi.org/10.1371/journal.pcbi.1011059
    3. Zentaro Kiuchi, Yukino Nishibori, Satoru Kutsuna, Masashi Kotani, Ichiro Hada, Toru Kimura, Toshiyuki Fukutomi, Daisuke Fukuhara, Noriko Ito-Nitta, Akihiko Kudo, Takanobu Takata, Yasuhito Ishigaki, Naohisa Tomosugi, Hirotoshi Tanaka, Satsuki Matsushima, Shinya Ogasawara, Yoshiaki Hirayama, Hiromu Takematsu, Kunimasa Yan. GLCCI1 is a novel protector against glucocorticoid‐induced apoptosis in T cells. The FASEB Journal 2019, 33 (6) , 7387-7402. https://doi.org/10.1096/fj.201800344RR
    4. John C. Williams, Amanda E. Siglin, Christine M. Lightcap, Amrita Dawn. Structural analysis of dynein intermediate and light chains. 2018, 52-87. https://doi.org/10.1016/B978-0-12-809470-9.00003-5
    5. Enora Moutin, Vincent Compan, Fabrice Raynaud, Caroline Clerté, Nathalie Bouquier, Gilles Labesse, Matthew L. Ferguson, Laurent Fagni, Catherine A. Royer, Julie Perroy. The stoichiometry of scaffold complexes in living neurons – DLC2 functions as a dimerization engine for GKAP. Journal of Cell Science 2014, 127 (16) , 3451-3462. https://doi.org/10.1242/jcs.145748
    6. Thanh Trung Thach, Jun-Goo Jee, Sang-Ho Lee. Effects of a Phosphomimetic Mutant of RAP80 on Linear Polyubiquitin Binding Probed by Calorimetric Analysis. Bulletin of the Korean Chemical Society 2012, 33 (4) , 1285-1289. https://doi.org/10.5012/bkcs.2012.33.4.1285
    7. Enora Moutin, Fabrice Raynaud, Laurent Fagni, Julie Perroy. GKAP–DLC2 interaction organizes the postsynaptic scaffold complex to enhance synaptic NMDA receptor activity. Journal of Cell Science 2012, 125 (8) , 2030-2040. https://doi.org/10.1242/jcs.098160
    8. Virgil Muresan, Zoia Muresan. Unconventional functions of microtubule motors. Archives of Biochemistry and Biophysics 2012, 520 (1) , 17-29. https://doi.org/10.1016/j.abb.2011.12.029
    9. John C. Williams, Amanda E. Siglin, Christine M. Lightcap, Amrita Dawn. Structural Analysis of Dynein Intermediate and Light Chains. 2012, 156-189. https://doi.org/10.1016/B978-0-12-382004-4.10005-6
    10. Péter Rapali, Áron Szenes, László Radnai, Anita Bakos, Gábor Pál, László Nyitray. DYNLL/LC8: a light chain subunit of the dynein motor complex and beyond. The FEBS Journal 2011, 278 (17) , 2980-2996. https://doi.org/10.1111/j.1742-4658.2011.08254.x
    11. László Radnai, Péter Rapali, Zsuzsa Hódi, Dániel Süveges, Tamás Molnár, Bence Kiss, Bálint Bécsi, Ferenc Erdödi, László Buday, József Kardos, Mihály Kovács, László Nyitray. Affinity, Avidity, and Kinetics of Target Sequence Binding to LC8 Dynein Light Chain Isoforms. Journal of Biological Chemistry 2010, 285 (49) , 38649-38657. https://doi.org/10.1074/jbc.M110.165894

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