Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND 58108, USA.
Center for Autophagy Research, Department of Internal Medicine and Microbiology, UT Southwestern Medical Center and Howard Hughes Medical Institute, Dallas, TX 75390, USA.
Acta Crystallogr D Struct Biol. 2017 Sep 1;73(Pt 9):775-792. doi: 10.1107/S2059798317011822. Epub 2017 Aug 29.
Mammalian Golgi-associated plant pathogenesis-related protein 1 (GAPR-1) is a negative autophagy regulator that binds Beclin 1, a key component of the autophagosome nucleation complex. Beclin 1 residues 267-284 are required for binding GAPR-1. Here, sequence analyses, structural modeling, mutagenesis combined with pull-down assays, X-ray crystal structure determination and small-angle X-ray scattering were used to investigate the Beclin 1-GAPR-1 interaction. Five conserved residues line an equatorial GAPR-1 surface groove that is large enough to bind a peptide. A model of a peptide comprising Beclin 1 residues 267-284 docked onto GAPR-1, built using the CABS-dock server, indicates that this peptide binds to this GAPR-1 groove. Mutation of the five conserved residues lining this groove, H54A/E86A/G102K/H103A/N138G, abrogates Beclin 1 binding. The 1.27 Å resolution X-ray crystal structure of this pentad mutant GAPR-1 was determined. Comparison with the wild-type (WT) GAPR-1 structure shows that the equatorial groove of the pentad mutant is shallower and more positively charged, and therefore may not efficiently bind Beclin 1 residues 267-284, which include many hydrophobic residues. Both WT and pentad mutant GAPR-1 crystallize as dimers, and in each case the equatorial groove of one subunit is partially occluded by the other subunit, indicating that dimeric GAPR-1 is unlikely to bind Beclin 1. SAXS analysis of WT and pentad mutant GAPR-1 indicates that in solution the WT forms monomers, while the pentad mutant is primarily dimeric. Thus, changes in the structure of the equatorial groove combined with the improved dimerization of pentad mutant GAPR-1 are likely to abrogate binding to Beclin 1.
哺乳动物高尔基相关植物致病蛋白 1（GAPR-1）是一种负自噬调节剂，可与自噬体核形成复合物的关键组成部分 Beclin 1 结合。Beclin 1 的 267-284 残基是与 GAPR-1 结合所必需的。在这里，使用序列分析、结构建模、结合下拉测定、X 射线晶体结构测定和小角度 X 射线散射研究了 Beclin 1-GAPR-1 相互作用。五个保守残基排列在赤道 GAPR-1 表面凹槽中，该凹槽足够大可以结合肽。使用 CABS-dock 服务器构建的包含 Beclin 1 残基 267-284 的肽模型表明该肽结合到该 GAPR-1 凹槽中。突变五个排列在这个凹槽中的保守残基 H54A/E86A/G102K/H103A/N138G 会破坏 Beclin 1 的结合。该五聚体突变 GAPR-1 的 1.27 Å分辨率 X 射线晶体结构已确定。与野生型 (WT) GAPR-1 结构的比较表明，五聚体突变体的赤道凹槽较浅且带正电荷，因此可能无法有效结合包含许多疏水性残基的 Beclin 1 残基 267-284。WT 和五聚体突变 GAPR-1 均以二聚体形式结晶，在每种情况下，一个亚基的赤道凹槽都被另一个亚基部分遮挡，表明二聚体 GAPR-1 不太可能与 Beclin 1 结合。WT 和五聚体突变 GAPR-1 的 SAXS 分析表明，在溶液中，WT 形成单体，而五聚体突变体主要是二聚体。因此，赤道凹槽结构的变化以及五聚体突变体 GAPR-1 二聚化的改善可能会破坏与 Beclin 1 的结合。
