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

• Protein–protein interactions are crucial in biology and play roles in for example, the immune system, signaling 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 center 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.

Ämnesord

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

Nyckelord

computational protein redesign

non-hotspot-centric interactions

proteinaceous inhibitor

protein–protein complex

surface plasmon resonance

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art (ämneskategori)

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