Chiral inversion mutagenesis identifies geometrically constrained residues within self-associating low-complexity domains

Many protein low-complexity domains (LCDs) reversibly self-associate to enable cellular function, yet fundamental questions remain regarding how polypeptide chemical and structural features beyond side chain identity contribute to LCD-LCD interactions. For instance, the folds adopted by globular proteins emerge from constraints enforced by homo-chirality of genetically-encoded polypeptides. However, it remains unclear to what extent similar geometric constraints apply to LCD self-association. Herein, we use protein total- and semi-synthesis to probe the contribution of C stereochemistry to LCD self-association with synthetic Chiral Inversion Mutagenesis (ChIM). By introducing targeted L-to-D amino acid inversions, ChIM identifies C stereocenters under geometric constraint without modification of side-chain functionalities. We apply ChIM to the LCDs of inner nuclear lamina protein Emerin and neurofilament light chain and find that chiral inversion produces strongly position-dependent effects upon LCD self-association. Our study describes essential structural features that enable LCD self-association and chemical strategies to interrogate LCD biochemistry. Significance StatementThis study identifies a critical role for amino acid side chain chirality in low-complexity domain (LCD) self-association. Using synthetic protein chemistry, we introduced targeted L-to-D amino acid inversions in LCDs without altering side chain functionalities. Chiral inversion mutagenesis identified geometrically constrained residues in the LCDs of Emerin and neurofilament light chain that localize to self-association hotspots within these sequences. Our work demonstrates that, like globular proteins, LCDs exploit polypeptide homochirality for biochemical function. The position-dependent effects of chiral inversion indicate the formation of labile structural elements that mediate oligomerization. By employing synthetic protein chemistry to probe LCD biochemistry with high positional resolution, this work provides a powerful approach to dissect chemical principles and polypeptide structural features that govern LCD-LCD interactions.