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Targeting the potent Beclin 1-UVRAG coiled-coil interaction with designed peptides enhances autophagy and endolysosomal trafficking.

ABSTRACT: The Beclin 1-Vps34 complex, known as "mammalian class III PI3K," plays essential roles in membrane-mediated transport processes including autophagy and endosomal trafficking. Beclin 1 acts as a scaffolding molecule for the complex and readily transits from its metastable homodimeric state to interact with key modulators such as Atg14L or UVRAG and form functionally distinct Atg14L/UVRAG-containing Beclin 1-Vps34 subcomplexes. The Beclin 1-Atg14L/UVRAG interaction relies critically on their coiled-coil domains, but the molecular mechanism remains poorly understood. We determined the crystal structure of Beclin 1-UVRAG coiled-coil complex and identified a strengthened interface with both hydrophobic pairings and electrostatically complementary interactions. This structure explains why the Beclin 1-UVRAG interaction is more potent than the metastable Beclin 1 homodimer. Potent Beclin 1-UVRAG interaction is functionally significant because it renders UVRAG more competitive than Atg14L in Beclin 1 binding and is critical for promoting endolysosomal trafficking. UVRAG coiled-coil mutants with weakened Beclin 1 binding do not outcompete Atg14L and fail to promote endolysosomal degradation of the EGF receptor (EGFR). We designed all-hydrocarbon stapled peptides that specifically targeted the C-terminal part of the Beclin 1 coiled-coil domain to interfere with its homodimerization. One such peptide reduced Beclin 1 self-association, promoted Beclin 1-Atg14L/UVRAG interaction, increased autophagic flux, and enhanced EGFR degradation. Our results demonstrate that the targeting Beclin 1 coiled-coil domain with designed peptides to induce the redistribution of Beclin 1 among its self-associated form or Atg14L/UVRAG-containing complexes enhances both autophagy and endolysosomal trafficking.

SUBMITTER: Wu S

PROVIDER: S-EPMC6016778 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Targeting the potent Beclin 1-UVRAG coiled-coil interaction with designed peptides enhances autophagy and endolysosomal trafficking.

Wu Shuai S He Yunjiao Y Qiu Xianxiu X Yang Wenchao W Liu Wenchao W Li Xiaohua X Li Yan Y Shen Han-Ming HM Wang Renxiao R Yue Zhenyu Z Zhao Yanxiang Y

Proceedings of the National Academy of Sciences of the United States of America 20180604 25

The Beclin 1-Vps34 complex, known as "mammalian class III PI3K," plays essential roles in membrane-mediated transport processes including autophagy and endosomal trafficking. Beclin 1 acts as a scaffolding molecule for the complex and readily transits from its metastable homodimeric state to interact with key modulators such as Atg14L or UVRAG and form functionally distinct Atg14L/UVRAG-containing Beclin 1-Vps34 subcomplexes. The Beclin 1-Atg14L/UVRAG interaction relies critically on their coile ...[more]

PMID: 29866835

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Project description:An important factor that defines the toxicity of elements such as cadmium(II), mercury(II), and lead(II) with biological macromolecules is metal ion exchange dynamics. Intriguingly, little is known about the fundamental rates and mechanisms of metal ion exchange into proteins, especially helical bundles. Herein, we investigate the exchange kinetics of Cd(II) using de novo designed three-stranded coiled-coil peptides that contain metal complexing cysteine thiolates as a model for the incorporation of this ion into trimeric, parallel coiled coils. Peptides were designed containing both a single Cd(II) binding site, GrandL12AL16C [Grand = AcG-(LKALEEK)(5)-GNH(2)], GrandL26AL30C, and GrandL26AE28QL30C, as well as GrandL12AL16CL26AL30C with two Cd(II) binding sites. The binding of Cd(II) to any of these sites is of high affinity (K(A) > 3 × 10(7) M(-1)). Using (113)Cd NMR spectroscopy, Cd(II) binding to these designed peptides was monitored. While the Cd(II) binding is in extreme slow exchange regime without showing any chemical shift changes, incremental line broadening for the bound (113)Cd(II) signal is observed when excess (113)Cd(II) is titrated into the peptides. Most dramatically, for one site, L26AL30C, all (113)Cd(II) NMR signals disappear once a 1.7:1 ratio of Cd(II)/(peptide)(3) is reached. The observed processes are not compatible with a simple "free-bound" two-site exchange kinetics at any time regime. The experimental results can, however, be simulated in detail with a multisite binding model, which features additional Cd(II) binding site(s) which, once occupied, perturb the primary binding site. This model is expanded into differential equations for five-site NMR chemical exchange. The numerical integration of these equations exhibits progressive loss of the primary site NMR signal without a chemical shift change and with limited line broadening, in good agreement with the observed experimental data. The mathematical model is interpreted in molecular terms as representing binding of excess Cd(II) to surface Glu residues located at the helical interfaces. In the absence of Cd(II), the Glu residues stabilize the three-helical structure though salt bridge interactions with surface Lys residues. We hypothesize that Cd(II) interferes with these surface ion pairs, destabilizing the helical structure, and perturbing the primary Cd(II) binding site. This hypothesis is supported by the observation that the Cd(II)-excess line broadening is attenuated in GrandL26AE28QL30C, where a surface Glu(28), close to the metal binding site, was changed to Gln. The external binding site may function as an entry pathway for Cd(II) to find its internal binding site following a molecular rearrangement which may serve as a basis for our understanding of metal complexation, transport, and exchange in complex native systems containing ?-helical bundles.

Dataset Information

Targeting the potent Beclin 1-UVRAG coiled-coil interaction with designed peptides enhances autophagy and endolysosomal trafficking.

Publications

Targeting the potent Beclin 1-UVRAG coiled-coil interaction with designed peptides enhances autophagy and endolysosomal trafficking.

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