Olivieri Paolo, Crack Jason C, Lehmann Angelika, Le Brun Nick E, Leimkühler Silke
Institute of Biochemistry and Biology, Department of Molecular Enzymology, University of Potsdam D-14476 Potsdam Germany
Centre for Molecular and Structural Biochemistry, School of Chemistry, Pharmacy and Pharmacology, University of East Anglia Norwich Research Park Norwich NR4 7TJ UK
RSC Chem Biol. 2024 Sep 27;5(11):1165-76. doi: 10.1039/d4cb00225c.
CyaY, the frataxin homolog of , plays an important role in ISC iron-sulfur cluster assembly through interactions with the cysteine desulfurase IscS, which regulate the supply of sulfur. IscS is not exclusive for ISC Fe-S cluster assembly, as it functions as a hub for the supply of sulfur to a number of other sulfur-requiring pathways, such as for the biosynthesis of Moco and thiolated tRNAs. How the balance of sulfur supply to the various competing pathways is achieved is not fully understood, but a network of protein-protein interactions plays a key role. For example, IscU and TusA compete for binding to IscS and thus for sulfur supply to ISC and Moco/tRNA biosynthesis. Here, we show that TusA can displace CyaY from IscS and can form hetero-complexes involving IscS, CyaY and TusA. Displacement of CyaY from IscS raised the question of whether it can interact with the SUF pathway. The SUF cysteine desulfurase SufS functions as a complex with SufE. Native mass spectrometry studies showed that the SufS dimer can bind up to four SufE molecules, two at high affinity, and two at low affinity, sites. Titration of SufSE (or SufS alone) with CyaY demonstrated binding, probably at the lower affinity site in competition with SufE. Binding of CyaY dramatically reduced the activity of SufSE , and over-expression of CyaY also significantly affected total cellular desulfurase activity and Fe-S cluster assembly, with the greatest effect observed in mutant strains in which SufS was the principal desulfurase. These data point to a physiological role for CyaY in regulating the desulfurase activity of IscS and SufS and, hence, both the iron-sulfur assembly systems. They also demonstrate that TusA can displace the regulatory CyaY protein from IscS-CyaY complexes, facilitating sulfur delivery from IscS to other essential cellular processes, and increasing the likelihood of SufSE-CyaY interactions.
CyaY是[某种生物]的frataxin同源物，通过与半胱氨酸脱硫酶IscS相互作用，在ISC铁硫簇组装中发挥重要作用，IscS调节硫的供应。IscS并非ISC铁硫簇组装所特有，因为它作为一个枢纽，为许多其他需要硫的途径供应硫，比如钼辅因子（Moco）和硫醇化tRNA的生物合成。目前尚不完全清楚如何实现向各种竞争途径的硫供应平衡，但蛋白质-蛋白质相互作用网络起着关键作用。例如，IscU和TusA竞争与IscS结合，从而竞争向ISC和Moco/tRNA生物合成供应硫。在此，我们表明TusA可以将CyaY从IscS上置换下来，并能形成涉及IscS、CyaY和TusA的异源复合物。CyaY从IscS上被置换引发了一个问题，即它是否能与SUF途径相互作用。SUF半胱氨酸脱硫酶SufS与SufE形成复合物发挥作用。天然质谱研究表明，SufS二聚体最多可结合四个SufE分子，两个以高亲和力结合，两个以低亲和力结合。用CyaY滴定SufSE（或单独的SufS）表明存在结合，可能是在与SufE竞争的较低亲和力位点。CyaY的结合显著降低了SufSE的活性，CyaY的过表达也显著影响总细胞脱硫酶活性和铁硫簇组装，在SufS是主要脱硫酶的突变菌株中观察到的影响最大。这些数据表明CyaY在调节IscS和SufS的脱硫酶活性方面具有生理作用，因此，对两个铁硫组装系统都有作用。它们还表明TusA可以从IscS-CyaY复合物中置换调节性CyaY蛋白，促进硫从IscS传递到其他重要的细胞过程，并增加SufSE-CyaY相互作用的可能性。
