Ginsentide-like coffeetides isolated from coffee waste are cell-penetrating and metal-binding microproteins

• Main Authors: Tam, James P., Huang, Jiayi, Loo, Shining, Li, Yimeng, Kam, Antony
• Other Authors: School of Biological Sciences
• Format: Article
• Language: English
• Published: 2023
• Subjects: Science::Biological sciences; Cysteine-Rich Peptides; Cell Penetration; Microproteins; Metal-Binding Peptides; Coffee Waste Product; Chemical Synthesis; Hevein-Like Peptide; Non-Chitin Binding Hevein; Ginsentide
• Online Access: https://hdl.handle.net/10356/170613
• Institution: Nanyang Technological University

Coffee processing generates a huge amount of waste that contains many natural products. Here, we report the discovery of a panel of novel cell-penetrating and metal ion-binding microproteins designated coffeetide cC1a–c and cL1–6 from the husk of two popular coffee plants, Coffea canephora and Coffea liberica, respectively. Combining sequence determination and a database search, we show that the prototypic coffeetide cC1a is a 37-residue, eight-cysteine microprotein with a hevein-like cysteine motif, but without a chitin-binding domain. NMR determination of cC1a reveals a compact structure that confers its resistance to heat and proteolytic degradation. Disulfide mapping together with chemical synthesis reveals that cC1a has a ginsentide-like, and not a hevein-like, disulfide connectivity. In addition, transcriptomic analysis showed that the 98-residue micrcoproten-like coffeetide precursor contains a three-domain arrangement, like ginsentide precursors. Molecular modeling, together with experimental validation, revealed a Mg2+ and Fe3+ binding pocket at the N-terminus formed by three glutamic acids. Importantly, cC1a is amphipathic with a continuous stretch of 19 apolar amino acids, which enables its cell penetration to target intracellular proteins, despite being highly negatively charged. Our findings suggest that coffee by-products could provide a source of ginsentide-like bioactive peptides that have the potential to target intracellular proteins.