Archna Archna, Scrima Andrea
Structural Biology of Autophagy Group, Department of Structure and Function of Proteins, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany.
Acta Crystallogr F Struct Biol Commun. 2017 Oct 1;73(Pt 10):560-567. doi: 10.1107/S2053230X17013280. Epub 2017 Sep 28.
ATG16L1 plays a major role in autophagy. It acts as a molecular scaffold which mediates protein-protein interactions essential for autophagosome formation. The ATG12ATG5-ATG16L1 complex is one of the key complexes involved in autophagosome formation. Human ATG16L1 comprises 607 amino acids with three functional domains named ATG5BD, CCD and WD40, where the C-terminal WD40 domain represents approximately 50% of the full-length protein. Previously, structures of the C-terminal WD40 domain of human ATG16L1 as well as of human ATG12ATG5 in complex with the ATG5BD of ATG16L1 have been reported. However, apart from the ATG5BD, no structural information for the N-terminal half, including the CCD, of human ATG16L1 is available. In this study, the authors aimed to structurally characterize the N-terminal half of ATG16L1. ATG16L1 in complex with ATG5 has been purified and crystallized in two crystal forms. However, both crystal structures revealed degradation of ATG16L1, resulting in crystals comprising only full-length ATG5 and the ATG5BD of ATG16L1. The structures of ATG5-ATG5BD in two novel crystal forms are presented, further supporting the previously observed dimerization of ATG5-ATG16L1. The reported degradation points towards a high instability at the linker region between the ATG5BD and the CCD in ATG16L1. Based on this observation and further biochemical analysis of ATG16L1, a stable 236-amino-acid subfragment comprising residues 72-307 of the N-terminal half of ATG16L1, covering the residual, so far structurally uncharacterized region of human ATG16L1, was identified. Here, the identification, purification, biochemical characterization and crystallization of the proteolytically stable ATG16L1 subfragment are reported.
自噬相关蛋白16样蛋白1（ATG16L1）在自噬过程中发挥着重要作用。它作为一种分子支架，介导自噬体形成所必需的蛋白质 - 蛋白质相互作用。ATG12ATG5 - ATG16L1复合物是参与自噬体形成的关键复合物之一。人类ATG16L1由607个氨基酸组成，具有三个功能结构域，分别命名为ATG5结合结构域（ATG5BD）、卷曲螺旋结构域（CCD）和WD40结构域，其中C末端的WD40结构域约占全长蛋白的50%。此前，已报道了人类ATG16L1 C末端WD40结构域以及与ATG16L1的ATG5BD结合的人类ATG12ATG5的结构。然而，除了ATG5BD外，关于人类ATG16L1 N末端一半（包括CCD）的结构信息尚无报道。在本研究中，作者旨在对ATG16L1的N末端一半进行结构表征。与ATG5结合的ATG16L1已被纯化并以两种晶体形式结晶。然而，两种晶体结构均显示ATG16L1发生了降解，导致晶体仅包含全长ATG5和ATG16L1的ATG5BD。本文展示了两种新型晶体形式下ATG5 - ATG5BD的结构，进一步支持了先前观察到的ATG5 - ATG16L1二聚化现象。报道的降解现象表明ATG16L1中ATG5BD和CCD之间的连接区域具有高度不稳定性。基于这一观察结果以及对ATG16L1的进一步生化分析，鉴定出了一个稳定的236个氨基酸的亚片段，该亚片段包含ATG16L1 N末端一半的72 - 307位残基，覆盖了人类ATG16L1目前在结构上尚未表征的剩余区域。在此，报道了该蛋白水解稳定的ATG16L1亚片段的鉴定、纯化、生化表征及结晶过程。
