Mechanistic Insights of IscU Conformation Regulation for Fe–S Cluster Biogenesis Revealed by Variable-Temperature Electrospray Ionization Native Ion Mobility Mass Spectrometry

: Iron-sulfur (Fe-S) cluster cofactors are required for the function of many critical cellular processes. These cofactors are assembled and inserted into apo target proteins by conserved Fe-S cluster biosynthetic pathways. In the ISC system of E. coli, the scaffold protein IscU assembles Fe-S cluster intermediates from iron, electrons, and inorganic sulfur, which is provided by the cysteine desulfurase enzyme IscS. IscU also binds to Zn, which mimics and competes for binding with the Fe-S cluster. Crystallographic and nuclear magnetic resonance (NMR) spectroscopic studies reveal that IscU is a metamorphic protein that exists in multiple conformational states, which include at least a structured form and a disordered form. The structured form of IscU is favored by metal binding and is stable in a narrow temperature range, undergoing both cold and hot denaturation. However, there is controversy over whether the structured or disordered form of IscU binds to IscS and functions in Fe-S cluster assembly. The recent development of variable-temperature electrospray ionization (vT-ESI) native ion mobility mass spectrometry (nIM-MS) enables probing the temperature dependence of macromolecular conformation and binding events in a single experiment. Here, vT-ESI nIM-MS results established that IscU exists in structured, intermediate, and disordered conformations. IscU samples shift towards high-charge states that have more extended conformations under extreme temperatures, consistent with cold/heat denaturation. A comparison of Zn-IscU and apo-IscU reveals that Zn(II) binding (i) attenuates the cold/heat-denaturation of IscU and promotes the refolding of IscU; (ii) favors the structured aand intermediate conformations and inhibits the disordered high charge states; and (iii) attenuates collisional induced unfolding (CIU) of intermediate conformations. Moreover, IscS was shown to alter the local conformation around the IscU active site, resulting in weakened Zn(II) affinity. Overall, these findings provide structural rationalization of the role of Zn(II) on stabilizing IscU conformation and the role of IscS on altering the IscU active site to prepare for Zn(II) release and cluster synthesis. This work highlights how vT-ESI-nMS can be applied as a powerful tool in mechanistic enzymology by providing details of relationships among temperature, protein conformations, and ligand/protein binding.