A litmus test for classifying recognition mechanisms of transiently binding proteins

Partner recognition in protein binding is critical for all biological functions, and yet, delineating its mechanism is challenging, especially when recognition happens within microseconds. We present a theoretical and experimental framework based on straight-forward nuclear magnetic resonance relaxation dispersion measurements to investigate protein binding mechanisms on sub-millisecond timescales, which are beyond the reach of standard rapid-mixing experiments. This framework predicts that conformational selection prevails on ubiquitin's paradigmatic interaction with an SH3 (Src-homology 3) domain. By contrast, the SH3 domain recognizes ubiquitin in a two-state binding process. Subsequent molecular dynamics simulations and Markov state modeling reveal that the ubiquitin conformation selected for binding exhibits a characteristically extended C-terminus. Our framework is robust and expandable for implementation in other binding scenarios with the potential to show that conformational selection might be the design principle of the hubs in protein interaction networks. The authors provide a litmus test for the recognition mechanism of transiently binding proteins based on nuclear magnetic resonance and find a conformational selection binding mechanism through concentration-dependent kinetics of ubiquitin and SH3.

Automated summary

The study presents a theoretical and experimental framework to investigate protein binding mechanisms on sub-millisecond timescales. Using nuclear magnetic resonance and molecular dynamics simulations, the authors find that the binding mechanism between ubiquitin and the SH3 domain is based on conformational selection.