Allosteric communication and subunit interaction specificity in glutamine amidotransferases
Structural Biology
Final Report Abstract
We have studied functional interactions between the synthase and the glutaminase subunits of glutamine amidotransferases (GATases) and the structural determinants of these allosteric interactions. In the first work package, steady-state enzyme kinetics, multi-dimensional NMR spectroscopy and crystal structure analysis were used to characterize the allosteric communication pathway between the synthase (cyclase) subunit HisF and the glutaminase subunit HisH of imidazole glycerol phosphate synthase (ImGP-S). The results showed that the simultaneous binding of the HisF substrate N’-[(5’-phosphoribulosyl)-formimino]-5-aminoimidazole-4-carboxamidribonucleotide (PRFAR) and the HisH substrate glutamine (Gln) are required to transform the inactive into the active form of ImGP-S. This transition from the inactive to the active conformation involves a conformational readjustment of loop 1 in HisF, rearrangements in the HisF core, the closing of the cyclase:glutaminase interface, and the formation of an oxyanion hole at the active site of HisH. Moreover, we have performed crystal structure analysis and used site-directed mutagenesis to unravel how the glutaminase PabA is stimulated by the synthase PabB within 4-amino-4-deoxychorismate synthase (ADC-S). Specifically, we found that incubation with glutamine leads to a synthase-glutaminase subunit interface expansion by one third of its original area. These changes lead to an occlusion of the glutaminase active site for sequestered catalysis and enable subsequent transport of volatile ammonia to the synthase active site. In the second work package, we have used protein engineering to retrace the evolution of the orthogonal TrpEx:TrpG (anthranilate synthase, AS) / PabB:PabA (ADC-S) systems found in “modern” γ-proteobacteria from the TrpE:PabA (AS) / PabB:PabA (ADC-S) systems found in more “ancient” bacteria. For this purpose, TrpG-specific residues were grafted into PabA by two different approaches, namely interface design based on the Rosetta software and, alternatively, a data-driven approach that exploits the differential conservation of interface residues in PabA and TrpG. Gel filtration chromatography and activity titrations showed that both approaches resulted in PabA variants that bound to TrpEx with higher affinity than to the native interaction partner PabB. Hence, we have accomplished a reprogramming of proteinprotein interaction specificity that provides novel insights into the evolutionary adaptation of protein interfaces.
Publications
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Significance of the Protein Interface Configuration for Allostery in Imidazole Glycerol Phosphate Synthase. Biochemistry, 59(29), 2729-2742.
Kneuttinger, Andrea C.; Rajendran, Chitra; Simeth, Nadja A.; Bruckmann, Astrid; König, Burkhard & Sterner, Reinhard
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Molecular basis for the allosteric activation mechanism of the heterodimeric imidazole glycerol phosphate synthase complex. Nature Communications, 12(1).
Wurm, Jan Philip; Sung, Sihyun; Kneuttinger, Andrea Christa; Hupfeld, Enrico; Sterner, Reinhard; Wilmanns, Matthias & Sprangers, Remco
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Reprogramming the Specificity of a Protein Interface by Computational and Data-Driven Design. Structure, 29(3), 292-304.e3.
Hertle, Regina; Nazet, Julian; Semmelmann, Florian; Schlee, Sandra; Funke, Franziska; Merkl, Rainer & Sterner, Reinhard
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Mechanistic analysis and antibiotic target validation of aminodeoxychorismate synthase and anthranilate synthase. Doctoral thesis, University of Regensburg.
Funke, Franziska
