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

Abstract

Protein-protein interactions are crucial in biology and play roles in for example, the immune system, signaling pathways, and enzyme regulation. Ultrahigh affinity interactions (K d <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 (K d 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.

Structure, Function and Protein Engineering of Cereal-Type Inhibitors Acting on Amylolytic Enzymes

“…Because a large number of different CM-proteins and posttranslationally modified forms thereof are present in seeds, it is difficult to purify any of the proteins to a highly homogenous state from natural sources for characterization of structure and function. Therefore, selected CM-proteins have been produced recombinantly in microbial hosts, Escherichia coli (CM2, CM3, CM16, 0.28, corn Hageman factor inhibitor, bifunctional αamylase/trypsin inhibitor, and rye BIII) and Pichia pastoris Stahl et al, 2007;Jensen et al, 2011;Møller et al, 2015Møller et al, , 2021 Emmer (Triticum dicoccon) TMA α-amylase from red flour beetle Hageman factor (Factor XIIa) (K i = 1.0 nM), Factor XIa (K i = 5.4 µM), bovine pancreatic trypsin (K i = 2.1 nM), mammalian trypsins, trypsin from yellow mealworm α-amylases from rice weevil 1BEA (Mahoney et al, 1984;Chong and Reeck, 1987;Chen et al, 1992;Behnke et al, 1998;Korneeva et al, 2014) Ragi/Indian finger millet (Eleusine coracana)…”

“…This is in most cases because the proteins were purified from their original source; hence, either the purity or the yield or both have been low. Recombinant protein production has enabled mutational analysis of structure/function relationships of a few CM-protein inhibitors (García-Maroto et al, 1991;Alam et al, 2001;Møller et al, 2015Møller et al, , 2021) and three-dimensional structures have been determined for four members including some in complex with target enzymes, namely wheat 0.19 dimeric inhibitor (Oda et al, 1997), ragi bifunctional a-amylase/trypsin inhibitor (RBI) (Strobl et al, 1995(Strobl et al, , 1998Gourinath et al, 2000), corn Hageman factor/αamylase inhibitor (CHFI) (Behnke et al, 1998), and barley limit dextrinase inhibitor (LDI) (Møller et al, 2015) (Table 1). Most characterized CTIs that inhibit hydrolases, except LDI, target α-1,4-glucan endo-acting a-amylases from GH13.…”

“…Moreover, site-directed mutagenesis established that a hydrophobic cluster situated on the second LDI α-helix flanked by ionic interactions at the proteinprotein interface was important for the picomolar affinity of the enzyme complex (Møller et al, 2015). Furthermore, computerguided thorough mutational analysis of the complex revealed that LDI-limit dextrinase intermolecular contacts as well as intramolecular interactions in LDI play a role for the ultra-high affinity (Møller et al, 2021). Remarkably, the inhibitor-enzyme complexation does not rely on an interface-centered hotspot constituted by a few residues, as in the case of the other CTI αamylase inhibitors.…”

“…Remarkably, the inhibitor-enzyme complexation does not rely on an interface-centered hotspot constituted by a few residues, as in the case of the other CTI αamylase inhibitors. Rather LDI residues across the protein interface contributed importantly to binding, hence making the complex more robust to mutational drift in evolution (Møller et al, 2021).…”

“…Among the CTIs targeting hydrolases, only LDI and RBI have been subjected to protein engineering attempts. The complex structure between LDI and limit dextrinase provided as mentioned above a starting point for rational and computerguided engineering of LDI showing the potential of LDI as a backbone for engineering (Møller et al, 2015(Møller et al, , 2021. Limit dextrinase plays a key role in malting and mashing together with other endogenous amylolytic enzymes in germinated barley seeds.…”