Zinc binding regulates amyloid-like aggregation of GAPR-1

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, we demonstrate that GAPR-1 binds zinc ions. Zn 2+ binding causes a slight but significant conformational change as revealed by circular dichroism, tryptophan fluorescence and trypsin digestion. The Zn 2+ -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 transmission electron microscopy. Molecular dynamic simulations show binding of Zn 2+ to His54 and His103. Mutation of these two highly conserved residues resulted in strongly diminished amyloid-like aggregation. Finally, we show that proteins from the CRISP subfamily are also able to form ThT-positive structures in vitro in a heparin- and Zn 2+ -dependent manner, suggesting that oligomerization regulated by metal ions could be a common structural property of the CAP domain.