An ultra-high affinity protein-protein interface displaying sequence-robustness

Marie Sofie Møller*, Sita Vaag Olesen, Ingemar André

*Corresponding author for this work

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Protein–protein interactions are crucial in biology and play roles in for example, the immune system, signalling pathways, and enzyme regulation. Ultra‐high affinity interactions (Kd, <0.1 nM) occur in these systems, however, structures and energetics behind stability of ultra‐high affinity protein–protein complexes are not well understood. Regulation of the starch debranching barley limit dextrinase (LD) and its endogenous cereal type inhibitor (LDI) exemplifies an ultra‐high affinity complex (Kd of 42 pM). In this study the LD–LDI complex is investigated to unveil how robust the ultra‐high affinity is to LDI sequence variation at the protein–protein interface and whether alternative sequences can retain the ultra‐high binding affinity. The interface of LD–LDI was engineered using computational protein redesign aiming at identifying LDI variants predicted to retain ultra‐high binding affinity. These variants present a very diverse set of mutations going beyond conservative and alanine substitutions typically used to probe interfaces. Surface plasmon resonance analysis of the LDI variants revealed that high affinity of LD–LDI requires interactions of several residues at the rim of the protein interface, unlike the classical hotspot arrangement where key residues are found at the centre of the interface. Notably, substitution of interface residues in LDI, including amino acids with functional groups different from the wild‐type, could occur without loss of affinity. This demonstrates that ultra‐high binding affinity can be conferred without hotspot residues, thus making complexes more robust to mutational drift in evolution. The present mutational analysis also demonstrates how energetic coupling can emerge between residues at large distances at the interface.
Original languageEnglish
JournalProtein Science
Issue number6
Pages (from-to)1144-1156
Number of pages13
Publication statusPublished - 2021


  • Protein-protein interaction
  • Protein complex
  • Surface plasmon resonance (SPR)
  • Enzyme inhibitor
  • Protein evolution
  • Protein design
  • Non-hotspot-centric interaction


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