Sequence Determinants of Allosteric Back-to-front Control of the Arf Nucleotide Switch

Unlike AI-based protein structure prediction, the sequence determinants of protein dynamics, and thus function, remain elusive. The nucleotide switch in Arf GTPases involves a massive structural change, which we showed recently in Arf1 is facilitated by a dynamic molten globule ensemble. Here we investigate the unresolved sequence-dynamics paradigm by comparing Arf1 and Arf6 using a combination of high-pressure NMR and other biophysical methods. We show that, as for Arf1, the Arf6 nucleotide switch implicates a functional molten globule ensemble, suggesting that this mechanism is a general feature of Arf and Arf-like GTPases. Comparison of the local stability distributions identifies key sequence determinants for the differences in switching between Arf1 and Arf6. Remarkably, these determinants are distant from the nucleotide-binding site, revealing back-to-front allosteric coupling controlling the switch. Evolutionary covariance analysis suggests that this back-to-front allosteric coupling is a fundamental characteristic of the Arf and Arf-like family, and may extend to the entire small GTPase kingdom. Our study thus establishes notable sequence-dynamics relationships with implications for signaling and diseases.