Combining ancestral sequence reconstruction with protein design to identify an interface hotspot in a key metabolic enzyme complex

Abstract

It is important to identify hotspot residues that determine protein-protein interactions in interfaces of macromolecular complexes. We have applied a combination of ancestral sequence reconstruction and protein design to identify hotspots within imidazole glycerol phosphate synthase (ImGPS). ImGPS is a key metabolic enzyme complex, which links histidine and de novo purine biosynthesis and consists of the cyclase subunit HisF and the glutaminase subunit HisH. Initial fluorescence titration experiments showed that HisH from Zymomonas mobilis (zmHisH) binds with high affinity to the reconstructed HisF from the last universal common ancestor (LUCA-HisF) but not to HisF from Pyrobaculum arsenaticum (paHisF), which differ by 103 residues. Subsequent titration experiments with a reconstructed evolutionary intermediate linking LUCA-HisF and paHisF and inspection of the subunit interface of a contemporary ImGPS allowed us to narrow down the differences crucial for zmHisH binding to nine amino acids of HisF. Homology modeling and in silico mutagenesis studies suggested that at most two of these nine HisF residues are crucial for zmHisH binding. These computational results were verified by experimental site-directed mutagenesis, which finally enabled us to pinpoint a single amino acid residue in HisF that is decisive for high-affinity binding of zmHisH. Our work shows that the identification of protein interface hotspots can be very efficient when reconstructed proteins with different binding properties are included in the analysis. Proteins 2017; 85:312-321. © 2016 Wiley Periodicals, Inc.

Ancestral sequence reconstruction produces thermally stable enzymes with mesophilic enzyme-like catalytic properties

“…In combination with an empirical analysis of the reconstructed proteins, this method has been previously used to characterize the physical properties of ancestral proteins, including thermal stability and substrate specificity 29 , 30 , 52 – 58 . The sequence reconstruction approach has also been used to investigate the evolution of protein folds 59 and to identify key amino acid residues in a metabolic enzyme complex 60 . It also allows us to deduce information on the history of life on Earth and to estimate long-term changes in the environment of the biosphere 30 , 58 , 61 .…”

Ancestral reconstruction of duplicated signaling proteins reveals the evolution of signaling specificity

“…This approach has been powerfully applied to examine the evolution of protein-ligand interactions ( Voordeckers et al, 2012 ), such as steroid hormones and their receptors ( Bridgham et al, 2009 ; Bridgham et al, 2006 ), transcription factor-DNA interactions ( Baker et al, 2013 ; McKeown et al, 2014 ; Starr et al, 2017 ), and protein-drug interactions ( Wilson et al, 2015 ). There have been fewer studies applying ancestral protein reconstruction to protein-protein interactions ( Holinski et al, 2017 ; Laursen et al, 2021 ; Wheeler et al, 2018 ; Wheeler and Harms, 2021 ), with most focusing on resurrecting the mutations that impact protein oligomerization ( Hochberg et al, 2020 ; Pillai et al, 2020 ).…”

Analysis of allosteric communication in a multienzyme complex by ancestral sequence reconstruction

“…These sequences are determined with the help of ASR (12,13), followed by the expression of the corresponding synthetic genes and the characterization of the recombinant proteins. Since the sequences of adjacent nodes in a phylogenetic tree are generally more similar to each other than the sequences of extant proteins, the identification of residues crucial for functional differences is more straightforward (25,26). In our case of TS, the vertical approach proved to be of great value for the identification of residues that are essential for the allosteric regulation of TrpB by TrpA.…”