Periodic Reporting for period 1 - DEAllAct (Dissecting and Engineering Allosteric Activation in a Biosynthetic Enzyme)
Période du rapport: 2020-11-16 au 2022-11-15
Enzymes are biological catalysts that speed up the rate of a specific chemical reaction in the cell, performing important functions and supporting life. Enzymes can be allosterically modulated, i.e. altering their reaction rate and/or substrate affinity upon binding to or mutation of a site remote from the active site of the enzyme. These processes are fundamental regulatory mechanisms of biochemical reactions that often involve a complex network of interactions. The enzyme ATP phosphoribosyltransferase (ATPPRT) from the cold-adapted bacterium Psychrobacter arcticus, is responsible for the first and flux-controlling step in histidine biosynthesis and is subject to complex allosteric control. ATPPRT is constituted of two parts, the protein possessing the catalytic domains (HisGs) and a catalytically inactive regulatory (HisZ). HisZ has a dual function: it allosterically enhances catalysis by HisGs, and it binds histidine and mediates allosteric inhibition, making it a model system for understanding the allosteric regulation of catalysis.
Overcoming histidine inhibition in ATPPRT is key in synthetic biology efforts toward histidine production in bacteria. The DEAllAct project aimed to understand the molecular details of the catalytic and allosteric mechanism of ATPPRT enzyme to predict and test mutations at the protein-protein interface that enhance the catalytic activity of the protein containing the catalytic domains (HisGs) without the regulatory domain (HisZ) by mimicking its allosteric activation. The project also aimed to explore the design of a computational tool to dissect allostery by combining computational simulations and biophysical experimental studies that can be used to predict specific mutations at the protein-protein interface of allosterically regulated complexes.
Conclusions of the action:
1. Successful identification of interaction networks that lead to allosteric activation of HisGs upon binding to the regulatory protein.
2. Demonstrated the involvement of two key residues at the binding domain that can rescue each other for the catalytic activation of the enzyme upon binding of the regulatory protein.
3. Developed a tool capable of identifying residues of key importance for binding interactions between the enzyme and its regulatory protein.
4. Identified and computationally validated 5 hotspots at the binding interface of HisGs that can potentially enhance its stand-alone activity.
Overcoming histidine inhibition in ATPPRT is key in synthetic biology efforts toward histidine production in bacteria. The DEAllAct project aimed to understand the molecular details of the catalytic and allosteric mechanism of ATPPRT enzyme to predict and test mutations at the protein-protein interface that enhance the catalytic activity of the protein containing the catalytic domains (HisGs) without the regulatory domain (HisZ) by mimicking its allosteric activation. The project also aimed to explore the design of a computational tool to dissect allostery by combining computational simulations and biophysical experimental studies that can be used to predict specific mutations at the protein-protein interface of allosterically regulated complexes.
Conclusions of the action:
1. Successful identification of interaction networks that lead to allosteric activation of HisGs upon binding to the regulatory protein.
2. Demonstrated the involvement of two key residues at the binding domain that can rescue each other for the catalytic activation of the enzyme upon binding of the regulatory protein.
3. Developed a tool capable of identifying residues of key importance for binding interactions between the enzyme and its regulatory protein.
4. Identified and computationally validated 5 hotspots at the binding interface of HisGs that can potentially enhance its stand-alone activity.
By performing molecular dynamics (MD) simulations of the wild-type enzyme and Arg56Ala mutant and analysing the interactions and the residues with the highest contributions to the communication pathways, it was demonstrated that HisZ binding increases intermonomer communication across the two subunits of HisGs. The project aimed to get atomic details into the allosteric rescue of the Arg56Ala variant upon binding the regulatory protein, while the corresponding variant in the nonactivated system was completely supressing its catalytic activity. A shift in the distance between Arg56 and the substrate PRPP was shown, enabling a stabilizing interaction in the activated system, and displaying a bimodal distribution of distances in the nonactivated. A second binding domain residue (Arg32) was shown to display similar conformational changes, being able to rescue its catalytic activity in the absence of Arg56 when the regulatory protein was present. The hypothesis that Arg32 and Arg56 can compensate for the absence of the other in the presence of HisZ was further validated through experimental testing. These results have been accepted for publication (“Allosteric rescue of catalytically impaired ATP phosphoribosyltransferase variants links protein dynamics to active-site electrostatic preorganisation”).
A novel approach was used to screen at the protein interface, succeeding in the identification of residues of key importance for binding interactions between the enzyme and its regulatory protein, and started the creation of the toolkit dCPL. 6 hotspots at the binding interface of HisGs were identified, 5 of which have been computationally validated by MD simulations, showing the aforementioned shift in the distances of Arg56 and Arg32. Experimental validation of the two best candidates is currently in process. These results, as well as the toolkit, will be published and released, as soon as the data is completed.
Simultaneous work was done on understanding catalysis into another enzyme from the same histidine biosynthetic pathway, namely HisA. Such enzyme has three decorating loops playing an important role in regulating their specificity and evolvability. Empirical valence bond (EVB) was applied to model the corresponding catalytic reaction in both open and closed states, highlighting its impact in regulating the catalytic reaction. The results of this work deviation were published in the journal JACS Au (“Complex Loop Dynamics Underpin Activity, Specificity, and Evolvability in the (βα)8 Barrel Enzymes of Histidine and Tryptophan Biosynthesis”).
During the DEAllAct project these conferences have been crucial to disseminate the results to the wide scientific community:
• Advances in Protein Folding, Evolution and Design 2022 (APFED22). Bayreuth (Germany), April 2022.
• Speaker at the internal Biochemistry retreat at BMC, Uppsala University. Uppsala (Sweden), May 2022.
• Speaker at the Girona Seminar. Girona (Spain), June 2022.
• Gordon Research Conference (GRC) in Computational Chemistry. Castelldefels (Spain), July 2022.
• Protein Society 36th Annual Symposium. San Francisco (USA), July 2022.
• Speaker at an internal seminar at the School of Biology, University of St. Andrews. St. Andrews (UK), November 2022.
During the lifetime of the DEAllAct project, we published a review related to computational enzyme design by means of enzyme evolution, where we review the latest successful computational tools and approaches used for the prediction and design of improved enzymes learning from natural evolution. The review was published in Trends in Biochemical Sciences (“Exploiting enzyme evolution for computational protein design”).
A novel approach was used to screen at the protein interface, succeeding in the identification of residues of key importance for binding interactions between the enzyme and its regulatory protein, and started the creation of the toolkit dCPL. 6 hotspots at the binding interface of HisGs were identified, 5 of which have been computationally validated by MD simulations, showing the aforementioned shift in the distances of Arg56 and Arg32. Experimental validation of the two best candidates is currently in process. These results, as well as the toolkit, will be published and released, as soon as the data is completed.
Simultaneous work was done on understanding catalysis into another enzyme from the same histidine biosynthetic pathway, namely HisA. Such enzyme has three decorating loops playing an important role in regulating their specificity and evolvability. Empirical valence bond (EVB) was applied to model the corresponding catalytic reaction in both open and closed states, highlighting its impact in regulating the catalytic reaction. The results of this work deviation were published in the journal JACS Au (“Complex Loop Dynamics Underpin Activity, Specificity, and Evolvability in the (βα)8 Barrel Enzymes of Histidine and Tryptophan Biosynthesis”).
During the DEAllAct project these conferences have been crucial to disseminate the results to the wide scientific community:
• Advances in Protein Folding, Evolution and Design 2022 (APFED22). Bayreuth (Germany), April 2022.
• Speaker at the internal Biochemistry retreat at BMC, Uppsala University. Uppsala (Sweden), May 2022.
• Speaker at the Girona Seminar. Girona (Spain), June 2022.
• Gordon Research Conference (GRC) in Computational Chemistry. Castelldefels (Spain), July 2022.
• Protein Society 36th Annual Symposium. San Francisco (USA), July 2022.
• Speaker at an internal seminar at the School of Biology, University of St. Andrews. St. Andrews (UK), November 2022.
During the lifetime of the DEAllAct project, we published a review related to computational enzyme design by means of enzyme evolution, where we review the latest successful computational tools and approaches used for the prediction and design of improved enzymes learning from natural evolution. The review was published in Trends in Biochemical Sciences (“Exploiting enzyme evolution for computational protein design”).
ATPPRT enzyme is responsible for the first flux-controlling step in histidine biosynthesis. The engineering of HisGs enzyme with stand-alone activity is particularly important to furnish a small, activated, and histidine-insensitive enzyme for histidine production in bacteria. Furthermore, the final outcome of the project, dCPL, after final refinement and release, has the potential to be used for any scientific group or company interested in understanding allosteric interactions and the identification of potential hotspots to target allosteric activation or inactivation. We will publicly release our code and protocols under liberal licenses, in line with Open Science best practices, and for maximum impact of our approach. The study and understanding of allostery is an increasingly important field, especially in drug discovery, since it can facilitate drug-target selectivity as allosteric sites tend to be less conserved than active sites across homologous proteins. It is also of increasing interest in enzyme engineering and synthetic biology, where allosteric control may need to be introduced or eliminated.