The Imidazole Glycerol Phosphate Synthase complex for the thermophilic bacteria Thermotoga maritima - kinetics of the dimerization and thermodynamics of the thermal stability

• Grant number: 25/11314-3
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: August 01, 2025
• End date: July 31, 2030
• Field of knowledge: Biological Sciences - Biochemistry - Enzymology
• Principal Investigator: Sandro Roberto Marana
• Grantee: Livia Ranieri Esteves dos Santos
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 21/10577-0 - Biology of Bacteria and Bacteriophages Research Center, AP.CEPID

Abstract

The Imidazole Glycerol Phosphate Synthase heterodimer (IGPS), formed by the enzymes HisF and HisH, is part of the pathway that produces histidine and purines in bacteria, but it is absent in mammals. In the thermophile bacteria Thermotoga maritima, the HisF-HisH complex (PDB 1GPW; UniProt entries Q9X0C8 and Q9X0C6, respectively) shows a dimerization interface of about 1110 Å2, comprising 37 residues from HisF and 27 from HisH. Interestingly, conformational changes are essential for the allosteric contact between HisF and HisH active sites. Another notably aspect of the IGPS complex is the thermal stability. For instance, the isolate HisF, which folds as a (b/a)8 barrel, has a melting temperature (Tm) above 95°C.Hence, the IGPS heterodimer is a good model to study fundamental protein properties as the thermal stability and protein-protein interactions.Approach 1The HisF and HisH binding is usually studied from the perspective of a simple binding equilibrium involving rigid bodies. However, conformational dynamics is an essential characteristic of the allosteric contact between their active sites. This clear contrast suggests indeed that the simple binding equilibrium may be an oversimplification. Hence this first approach aims at to analyze the HisF and HisH binding from the perspective of a two steps process combining binding and structural changes in any order.Approach 2As already mentioned, HisF has a Tm higher than 95°C. On the other hand, a mutant HisF containing the replacement Y182A (PDB 6VDG) is clearly less thermostable than the wild-type protein, showing a Tm of 84°C. Studies based on the molecular dynamics simulation and HSQC indicated that at high temperature (70°C) the Y182A mutant is folded, but it is enriched in unconcerted motion, whereas the wild-type HisF exhibits a higher proportion of correlated motions between residues. Hence the substitution Y182A altered the distribution of thermal energy through the HisF structure, leaving the mutant HisF in the brink of denaturation at temperatures in which the wild-type protein is still stable. Based on that observation, we proposed that in a same temperature in which both proteins are folded, the structure of the mutant HisF visits more frequently "open" conformations, in which its core residues are more exposed to the solvent.Hence the goal of the approach 2 is to evaluate the degree of exposition to the solvent of the wild-type HisF and mutant HisF Y182A residues by determining their ratio of H/D exchange in 15N - 1H TROSY-2D NMR experiments. (AU)