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dc.contributor.advisorBettati, Stefano-
dc.contributor.authorRosa, Brenda-
dc.description.abstractCysteine is intimately related to bacterial functions concerning their fitness and pathogenicity, therefore enzymes involved in its biosynthetic pathway have been proposed as targets for the development of new antibiotics or antibiotic enhancers. Among these, serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase A (CysK), are the two enzymes concluding the reductive sulfate assimilation pathway (RSAP) that leads to the formation of L-cysteine. CysK and CysE associate to form the so-called Cysteine Synthase (CS) complex, that was the focus of this PhD work. Many aspects of CS assembly have to be clarified yet, including its regulation, the exact biological role played in cysteine biosynthesis and the intriguing possibility to exploit it as a pharmaceutical target. Moreover, the three dimensional structure of the complex is still unavailable. This work was focused on characterization of CysK interaction with CysE, within CS assembly. Particularly, the aims of the project included: exploring protein-protein interaction (PPI) hotspots of CS complex, characterize its quaternary geometry in solution together with the binding mode of the constituent enzymes and uncover changes in dynamics upon CysK interaction with CysE on both the partners of the complex. To investigate all these aspects, a combination of different approaches was applied. The “protein painting” technique was exploited to reveal PPI hotspots of CS. First, a validation of the assay was performed on a complex of CysK with another interacting partner, a bacterial toxin (CdiA-CT). The three dimensional structure of this complex is known, and CdiA-CT shares a similar mechanism of complex formation and the same structural motif adopted by CysE to assemble with CysK. Afterwards, the experimental workflow was applied to CS complex, whose crystal structure hasn’t been solved yet. As a result, a common residue in both interprotein complexes was found to be part of the interaction interface, supporting the similarity in the mechanism by which the two partner proteins bind to CysK. Moreover, the results from protein painting experiments also supported previous findings indicating that the interaction with CysE stabilizes a “closed” conformation of the enzyme. Additionally, other two sites, located near the interdimeric interface of CysK, became buried only upon complex formation. Hence, the existence of an allosteric communication between the two monomers of the enzyme was suggested and it could be responsible for the inhibition of CysK, that however retains about 10% of its activity within the complex. Concerning the quaternary geometry of CS assembly, two different models have been hypothesized, according to the determined stoichiometry. In these alternative scenarios, one or two subunits of CysE interact with only one or both CysK active sites, respectively. To create a model of CS association in solution, Small Angle X-ray Scattering (SAXS) measurements were performed by our collaborators at Elettra Synchrotron in Trieste. Ab initio modelling generated an envelope of the complex, that was further refined using the experimental contact points, obtained from the protein painting assay, as constraints for the fitting. The resulting S-shape of the complex suggested that only one subunit of CysK dimer is bound at each side of both CysE trimers. The occupation of one CysK active site is then allosterically communicated to the second monomer, leading to inhibition of enzyme activity, in agreement with previous functional data and findings from the protein painting assay. Furthermore, binding of one CysK dimer to a trimer of CysE seemed to induce an allosteric cross-talk between the two trimers of the hexamer, dictating the orientation of the interaction of the second CysK dimer on the other side of CysE. An overview of local changes in CysK and CysE dynamics in solution upon their interaction within the CS complex was provided by Hydrogen/deuterium Exchange Mass Spectrometry (HDX-MS) experiments. These results were obtained at Copenhagen University, during a six-months secondment. A continuous labelling experiment was performed on CysK, CysE and CS complex. Different segments of both proteins underwent significant changes in HDX (i.e. dynamics), following CS assembly. In particular, several regions of CysK showed structural stabilization, after its interaction with CysE. Residues located near the active site were confirmed to bind CysE C-terminal tail. Moreover, overlapping peptides belonging to a moveable region of the N-terminal domain, that undergoes an extensive conformational change toward a close conformation upon substrate binding, showed reduced HDX as a consequence of CysK interaction with CysE. This finding further corroborated the hypothesis that CysE stabilizes a closed state of the enzyme. A structural stabilization in CS complex with respect to CysK alone was also observed for residues that have been demonstrated to be crucial for the formation of CS assembly and are located on a conserved loop. Concerning CysE partner of CS complex, it interacts with CysK inserting its C-terminal tail into the active site of the other protein, as it has been previously demonstrated, and further confirmed by observation of a decrease of HDX in this region, upon CS assembly. This binding induced a structural stabilization of residues located at the N-terminal, at the interface between the two trimers of the hexamer, but also a structural destabilization across different segments of the protein. All together these data suggested that CysK binding at CysE C-terminal developed in a pathway of communication, reaching the N-terminus, where an allosteric effect of stabilization at the interface between the two trimers occurred, as a consequence of CS complex formation. The disclosure of the three dimensional organization of bacterial CS assembly, PPI hotspots and investigation on modulation of the dynamic properties of the constituent enzymes upon binding could elucidate crucial topics concerning CS complex and help the development of new antimicrobial
dc.publisherUniversità degli studi di Parma. Dipartimento di Scienze degli alimenti e del farmacoit
dc.relation.ispartofseriesDottorato di ricerca in Scienze del farmaco, delle biomolecole e dei prodotti per la saluteit
dc.rights© Brenda Rosa, 2020it
dc.subjectcysteine biosynthesisit
dc.subjectcysteine synthase complexit
dc.subjectprotein paintingit
dc.subjectprotein dynamicsit
dc.subjectserine acetyltransferaseit
dc.subjectO-acetylserine sulfhydrylaseit
dc.titleDisclosing dynamics and structural features of cysteine synthase complexit
dc.title.alternativeCaratterizzazione delle proprietà dinamiche e strutturali del complesso cisteina sintasiit
dc.typeDoctoral thesisit
Appears in Collections:Scienze del farmaco, delle biolomolecole e dei prodotti per la salute, tesi di dottorato

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