Final Report Summary - SULTENG (Protein engineering for the study of detoxification enzymes and hub proteins)
DNA replication in all eukaryotes is mediated by the proliferating cell nuclear antigen (PCNA), which orchestrates the replication process by binding and releasing DNA modifying enzymes. Using protein engineering, biochemical and genetic approaches, we generated and characterized PCNA mutants with increased affinities for several key proteins that are parts of the replication machinery. We found that increases in PCNA-protein interaction affinities led to severe phenotypic defects in vivo. Our results demonstrate that these defects are much more severe than those induced by complete abolishment of the respective interactions, thus highlighting the importance of obtaining a fine balance between the different PCNA-partner interaction affinities for the progression of DNA replication and repair. Thus, we found that the cost of misregulating biological processes through disruptions of protein-protein interactions can be much higher than the cost of deleting parts of the network altogether, demonstrating both the fragility and robustness of biological processes.
In addition, we have established an experimental system for monitoring the co-evolution of PCNA-partner interactions in fungi species spanning 300 million years of evolution. Using a combined bioinformatics and experimental approach, we discovered that PCNA-partner interactions tightly co-evolved in fungi species, thus leading to specific modes of recognition. We found that fungal PCNA-partner interaction networks diverged into two distinct groups due to such co-evolution and that hybrids of these groups are functionally non-compatible in the yeast Saccharomyces cerevisiae. Our results indicate that the co-evolution of protein-protein interaction networks can form functional barriers for gene transfer between species, and as such, it can promote and fix speciation. The development of an experimental approach to examine the co-evolution of protein networks can pave the way for the investigation of the co-evolution in many other biological networks.
In parallel, we explored the molecular basis for the broad specificity of cytosolic sulfotransferases (SULTs) that can detoxify a wide variety of chemicals via the transfer of a sulfate group from a universal sulfate donor. Despite extensive research into SULTs, it is still not clear how a single SULT isoform can catalyse sulfate transfer to a broad range of acceptors. In this project, we used structural, protein engineering, and kinetic approaches to deepen our understanding of the molecular basis for the broad specificity and substrate inhibition of the SULT isoforms SULT1A1 and SULT1E1. We have determined several new SULT1A1 structures in complex with different acceptors and we generated SULT1A1 and SULT1E1 mutants with increased specificities. We identified several residues as those that control the broad specificities of SULT1A1 and SULT1E1, and we showed that subtle structural changes in these enzymes can lead to dramatic changes in specificity.
In addition, we have established an experimental system for monitoring the co-evolution of PCNA-partner interactions in fungi species spanning 300 million years of evolution. Using a combined bioinformatics and experimental approach, we discovered that PCNA-partner interactions tightly co-evolved in fungi species, thus leading to specific modes of recognition. We found that fungal PCNA-partner interaction networks diverged into two distinct groups due to such co-evolution and that hybrids of these groups are functionally non-compatible in the yeast Saccharomyces cerevisiae. Our results indicate that the co-evolution of protein-protein interaction networks can form functional barriers for gene transfer between species, and as such, it can promote and fix speciation. The development of an experimental approach to examine the co-evolution of protein networks can pave the way for the investigation of the co-evolution in many other biological networks.
In parallel, we explored the molecular basis for the broad specificity of cytosolic sulfotransferases (SULTs) that can detoxify a wide variety of chemicals via the transfer of a sulfate group from a universal sulfate donor. Despite extensive research into SULTs, it is still not clear how a single SULT isoform can catalyse sulfate transfer to a broad range of acceptors. In this project, we used structural, protein engineering, and kinetic approaches to deepen our understanding of the molecular basis for the broad specificity and substrate inhibition of the SULT isoforms SULT1A1 and SULT1E1. We have determined several new SULT1A1 structures in complex with different acceptors and we generated SULT1A1 and SULT1E1 mutants with increased specificities. We identified several residues as those that control the broad specificities of SULT1A1 and SULT1E1, and we showed that subtle structural changes in these enzymes can lead to dramatic changes in specificity.