Dissecting peripheral protein-membrane interfaces

Peripheral membrane proteins (PMPs) include a wide variety of proteins that have in common to bind transiently to the chemically complex interfacial region of membranes through their interfacial binding site (IBS). In contrast to protein-protein or protein-DNA/RNA interfaces, peripheral protein-membrane interfaces are poorly characterized. We collected a dataset of PMP domains representative of the variety of PMP functions: membrane-targeting domains (Annexin, C1, C2, discoidin C2, PH, PX), enzymes (PLA, PLC/D) and lipid-transfer proteins (START). The dataset contains 1328 experimental structures and 1194 AphaFold models. We mapped the amino acid composition and structural patterns of the IBS of each protein in this dataset, and evaluated which were more likely to be found at the IBS compared to the rest of the domains' accessible surface. In agreement with earlier work we find that about two thirds of the PMPs in the dataset have protruding hydrophobes (Leu, Ile, Phe, Tyr, Trp and Met) at their IBS. The three aromatic amino acids Trp, Tyr and Phe are a hallmark of PMPs IBS regardless of whether they protrude on loops or not. This is also the case for lysines but not arginines suggesting that, unlike for Arg-rich membrane-active peptides, the less membrane-disruptive lysine is preferred in PMPs. Another striking observation was the over-representation of glycines at the IBS of PMPs compared to the rest of their surface, possibly procuring IBS loops a much-needed flexibility to insert in-between membrane lipids. The analysis of the 9 superfamilies revealed amino acid distribution patterns in agreement with their known functions and membrane-binding mechanisms. Besides revealing novel amino acids patterns at protein-membrane interfaces, our work contributes a new PMP dataset and an analysis pipeline that can be further built upon for future studies of PMPs properties, or for developing PMPs prediction tools using for example, machine learning approaches. Author summary Peripheral membrane proteins (PMPs) are soluble proteins that bind transiently to the surface of cell membranes. Having the ability to exist in both a soluble and a membrane-bound form their membrane-binding region is constrained to retain a fine balance of polar and hydrophobic character, which makes it difficult to distinguish it from the rest of their surface. As a result peripheral membrane-binding sites are notoriously difficult to predict. We collected and curated a dataset containing 2500 structures of PMPs and compared their membrane-binding sites to the rest of their solvent-accessible surfaces, in order to reveal features of PMPs'membrane-binding sites. We find that, among positively charged amino acids, lysines are significantly more present than arginines. Protruding hydrophobes are a landmark of the interfacial binding sites of ca. 2/3 of peripheral membrane binding proteins, indicating that a majority of PMPs takes advantage of the hydrophobic effect while a non-negligeable minority (1/3) most likely relies on electrostatics interactions or other mechanisms. The IBS of peripheral membrane proteins contain significantly more glycines than the rest of their surface. These findings and the collected dataset will be useful for the development of prediction models for membrane-binding sites of PMPs.