Spontaneous Self-Assembly of Engineered Armadillo Repeat Protein Fragments into a Folded Structure

Repeat proteins are built of modules, each of which constitutes a structural motif. We have investigated whether fragments of a designed consensus armadillo repeat protein (ArmRP) recognize each other. We examined a split ArmRP consisting of an N-capping repeat (denoted Y), three internal repeats (M), and a C-capping repeat (A). We demonstrate that the C-terminal MA fragment adopts a fold similar to the corresponding part of the entire protein. In contrast, the N-terminal YM2 fragment constitutes a molten globule. The two fragments form a 1:1 YM2:MA complex with a nanomolar dissociation constant essentially identical to the crystal structure of the continuous YM3A protein. Molecular dynamics simulations show that the complex is structurally stable over a 1 μs timescale and reveal the importance of hydrophobic contacts across the interface. We propose that the existence of a stable complex recapitulates possible intermediates in the early evolution of these repeat proteins. [Display omitted] •Armadillo repeat proteins can be expressed as two soluble complementary fragments•When mixed the fragments recognize each other and form a complex with high affinity•The structure of the complex resembles the entire full-length protein•One fragment forms a template onto which the other fragment folds and binds Watson et al. use solution NMR and other biophysical methods to investigate the noncovalent interactions between two fragments of an Armadillo repeat protein (ArmRP). They show that two fragments recognize each other and form a complex that structurally resembles the single-chain protein.