Abstract
CysE and CysK, the last two enzymes of the cysteine biosynthetic pathway, engage in a bienzyme complex, cysteine synthase, with yet incompletely characterized three-dimensional structure and regulatory function. Being absent in mammals, the two enzymes and their complex are attractive targets for antibacterial drugs. We have used hydrogen/deuterium exchange MS to unveil how complex formation affects the conformational dynamics of CysK and CysE. Our results support a model where CysE is present in solution as a dimer of trimers, and each trimer can bind one CysK homodimer. When CysK binds to one CysE monomer, intratrimer allosteric communication ensures conformational and dynamic symmetry within the trimer. Furthermore, a long-range allosteric signal propagates through CysE to induce stabilization of the interface between the two CysE trimers, preparing the second trimer for binding the second CysK with a nonrandom orientation. These results provide new molecular insights into the allosteric formation of the cysteine synthase complex and could help guide antibacterial drug design.
Highlights
CysE and CysK, the last two enzymes of the cysteine biosynthetic pathway, engage in a bienzyme complex, cysteine synthase, with yet incompletely characterized three-dimensional structure and regulatory function
In order to explore the dynamic properties of CysK and CysE upon forming the cysteine synthase (CS) complex, we performed Hydrogen/deuterium exchange MS (HDX-MS) experiments where each protein component alone and in complex was labeled across a range of well-defined time points: 0.03, 1.67, 16.67, 60, and 720 min
Examples of peptides belonging to regions of the protein that undergo significant changes in dynamics upon binding CysE are labeled in Figure 2, and the regions are mapped onto the CysK crystal structure in Figure 4, A and B (PDB codes: 1D6S and 1OAS)
Summary
In Brief We have used hydrogen/ deuterium exchange MS to unveil the allosteric changes occurring during complex formation of CysE and CysK, the last two enzymes of cysteine biosynthetic pathway in bacteria. A long-range allosteric signal propagates through CysE to induce stabilization of the interface between the two CysE trimers, preparing the second trimer for binding the second CysK with a nonrandom orientation These results provide new molecular insights into the allosteric formation of the cysteine synthase complex and could help guide antibacterial drug design. We demonstrate that complex formation induces allosteric cross talk between monomers of CysE and further causes long-range allosteric changes at the dimer-of-trimers interface that could play a regulatory role
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