Computational Analysis of Threonine Ladders on Distinct Beta-Solenoid Scaffolds, with Implications for the Design of Novel Antifreeze Proteins

Cold-adapted organisms frequently express antifreeze proteins (AFPs) that facilitate their survival at low temperatures, with some especially potent insect AFPs exhibiting beta-solenoid structures with ice-binding threonine ladders. Beta-solenoids exist in nature in numerous forms and emerging protein design technologies may afford opportunities to diversify them further, suggesting the possibility of creating a variety of new AFPs by installing a threonine ladder on non-AFP natural or designed beta-solenoids. However, early attempts at such engineering, combined with differences observed between AFPs and structurally similar ice-nucleating proteins, have raised a critical question: Does the specific solenoid scaffold significantly affect a threonine ladder's structural characteristics (and thus its suitability for ice binding)? We set out to address this question by creating distinct variants of a model beta-solenoid for in silico analysis via structure prediction and molecular dynamics simulations. Our findings indicate that the concavity of the beta-solenoid face on which a threonine ladder is arrayed can substantially influence the local geometry of the threonines, with less concave examples resembling the AFP TmAFP and a more concave example losing the ordered channel waters present in well-studied natural ice-binding surfaces. Disparities in concavity and threonine hydroxyl spacings appeared initially in our AlphaFold results and were supported by the simulations, illustrating AlphaFold's possible utility for high-throughput preliminary screening of designed beta-solenoid AFP sequence candidates. We demonstrate this utility by using AlphaFold to evaluate sequences generated by the state-of-the-art inverse folding tool ProteinMPNN that can potentially be transformed into novel hyperactive AFPs to support applications ranging from biomedical cryopreservation to food science.