Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands.
Biomolecular Imaging, Bijvoet Center, Utrecht University, Utrecht, the Netherlands.
Biosci Rep. 2019 Feb 12;39(2). doi: 10.1042/BSR20182345. Print 2019 Feb 28.
Members of the CAP superfamily (Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins) are characterized by the presence of a CAP domain that is defined by four sequence motifs and a highly conserved tertiary structure. A common structure-function relationship for this domain is hitherto unknown. A characteristic of several CAP proteins is their formation of amyloid-like structures in the presence of lipids. Here we investigate the structural modulation of Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1) by known interactors of the CAP domain, preceding amyloid-like aggregation. Using isothermal titration calorimetry (ITC), we demonstrate that GAPR-1 binds zinc ions. Zn binding causes a slight but significant conformational change as revealed by CD, tryptophan fluorescence, and trypsin digestion. The Zn-induced conformational change was required for the formation of GAPR-1 oligomers and amyloid-like assemblies in the presence of heparin, as shown by ThT fluorescence and TEM. Molecular dynamics simulations show binding of Zn to His and His Mutation of these two highly conserved residues resulted in strongly diminished amyloid-like aggregation. Finally, we show that proteins from the cysteine-rich secretory protein (CRISP) subfamily are also able to form ThT-positive structures in a heparin- and Zn-dependent manner, suggesting that oligomerization regulated by metal ions could be a common structural property of the CAP domain.
CAP 超家族（富含半胱氨酸的分泌蛋白、抗原 5 和与发病机制相关的 1 蛋白）的成员的特征是存在 CAP 结构域，该结构域由四个序列基序和高度保守的三级结构定义。该结构域的常见结构-功能关系尚不清楚。几个 CAP 蛋白的特征之一是在存在脂质的情况下形成类似淀粉样的结构。在这里，我们研究了 CAP 结构域的已知相互作用物对植物高尔基体相关发病机制相关蛋白 1（GAPR-1）的结构调制，在形成类似淀粉样的聚集之前。使用等温滴定量热法（ITC），我们证明 GAPR-1 结合锌离子。CD、色氨酸荧光和胰蛋白酶消化表明，Zn 结合导致轻微但显著的构象变化。在肝素存在下，Zn 诱导的构象变化是形成 GAPR-1 寡聚物和类似淀粉样组装所必需的，如 ThT 荧光和 TEM 所示。分子动力学模拟表明 Zn 与 His 和 His 结合这两个高度保守的残基的突变导致类似淀粉样聚集的强烈减少。最后，我们表明富含半胱氨酸的分泌蛋白 (CRISP) 亚家族的蛋白质也能够以肝素和 Zn 依赖性的方式形成 ThT 阳性结构，这表明由金属离子调节的寡聚化可能是 CAP 结构域的共同结构特性。
