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

• Published in: Proteins, structure, function, and bioinformatics structure, function, and bioinformatics, 2017-02, Vol.85 (2), p.312-321
• Main Authors: Holinski, Alexandra, Heyn, Kristina, Merkl, Rainer, Sterner, Reinhard
• Format: Article
• Language: English
• Subjects: Affinity; Amino acid sequence; Amino acids; Aminohydrolases - chemistry; Aminohydrolases - genetics; Aminohydrolases - metabolism; Binding; Binding Sites; Biological Evolution; Biosynthesis; Cloning, Molecular; Computer applications; Enzymes; Escherichia coli - genetics; Escherichia coli - metabolism; Fluorescence; Gene Expression; Glutaminase; Glycerol; HisF; HisH; Histidine; Homology; Imidazole; imidazole glycerol phosphate synthase; in silico mutagenesis; Inspection; Interfaces; Macromolecules; Metabolism; Mutagenesis; Mutation; Phosphate; Phosphates; Phylogeny; Protein Binding; Protein Engineering; Protein Folding; Protein interaction; Protein Interaction Domains and Motifs; Protein Structure, Secondary; Protein Subunits - chemistry; Protein Subunits - genetics; Protein Subunits - metabolism; Proteins; protein–protein interaction; Pyrobaculum - classification; Pyrobaculum - enzymology; Pyrobaculum - genetics; Pyrobaculum arsenaticum; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Reconstruction; Residues; Site-directed mutagenesis; Thermodynamics; Thermotoga maritima - classification; Thermotoga maritima - enzymology; Thermotoga maritima - genetics; Titration; Zymomonas; Zymomonas - classification; Zymomonas - enzymology; Zymomonas - genetics; Zymomonas mobilis
• ISSN: 0887-3585; 1097-0134
• DOI: 10.1002/prot.25225

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.