SKEMPI 2.0: An updated benchmark of changes in protein-protein binding energy, kinetics and thermodynamics upon mutation

Justina Jankauskaite; Brian Jiménez-García; Justas Dapkunas; Juan Fernández-Recio; Iain H. Moal

Range: The changes in binding free energy upon mutation range from -12.4 to 12.4 kcal.mol −1 , as in SKEMPI 1.1, with ∆ log 10 k on ranging from -3.6 to 2.4, ∆ log 10 k off ranging from -6.0 to 6.8, ∆∆H ranging from -18.3 to 26.5 kcal.mol −1 , and ∆∆S ranging from -61 to 80 cal.mol −1 .K −1 . Around 60 mutant are very destabilising, reducing binding energy by 8 kcal.mol −1 or more. These are all in enzyme/inhibitor complexes such as the inhibition of acetylcholinesterase by the snake venom fasciculin, or the inhibition of enzymes which would be detrimental should they unbind and become active in the wrong location, such as nucleases (barnase / barstar, colicin E9 DNase / Im9, RNase A / angiogenin) and proteases (such as trypsin / BPTI). These interactions tend to be around picomolar affinity and are at the upper limit of what can be detected, due to the time The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/341735 doi: bioRxiv preprint required to reach equilibrium and the low concentrations required by the mass action law to probe informative regions of the binding curve. These very destabilising mutations reduce affinity into the micromolar range, near the lower limit of what can be quantified using standard methods. As a consequence of both mutant and wild-type affinities being near detection thresholds, errors in these entries are typically large. Further, while some mutations may cause changes in affinity larger than seven orders of magnitude, the absence of affinities for such mutations in the benchmark can be explained by the fact that such mutations would involve affinities beyond the upper or lower limit. Indeed, there are new entries in which single or double substitutions reduce binding from tens of picomolar to having no detectable binding. For many of the highly destabilising mutations a crystal structure for the mutant has also been solved, and the 30 most stabilising mutations in the database (∆∆G ¡ -5 kcal.mol −1 ) consist of the reverse mutation applied to these structures. These are mostly single or double mutants, but include mutations to up to 27 residues of the non-cognate Colicin E2/Im9 complex, which move it toward the cognate E9/Im9 in sequence space [40] .