Structural And Functional Studies Of The Complex Of Yeast Chitin Synthase Chs1 With An Inhibitor

Background:Fungal infections are seriously affecting human health worldwide.Chitin is essential for fungal cell walls,which,together withβ-1,3-glucan,form the inner core skeleton of the fungal cell wall.Chitin is a linear N-acetylglucosamine（Glc NAc）polymer withβ-1,4-linkage,which are synthesized by chitin synthase（Chs）and transported across the membrane to participate in cell wall formation.Loss or inactivation mutations of chitin synthase can result in abnormal structure and function of the fungal cell wall.Nikkomycin Z（Nik Z）,a nucleoside-peptide antifungal drug targeting chitin synthase,has demonstrated excellent therapeutic efficacy in clinical trials against various fungal infections.Although the study of chitin synthase has important clinical value,the protein is a highly complex membrane protein,and there are still many unknown and unresolved issues regarding its structure and function.Therefore,a deeper understanding of the synthesis mechanism of chitin synthase and the inhibitory mechanism of Nik Z is still lacking.That severely restricts the development of clinical antifungal drug research and related fields.Objective:This study aims to explore the catalytic synthesis and transmembrane transport molecular mechanisms of fungal chitin by analyzing the structure of chitin synthase Chs1in Saccharomyces cerevisiae and its complex with the inhibitor Nik Z.The study further aims to reveal the molecular mechanism of Nik Z inhibition of fungal chitin synthase and to provide critical structural information for the development of novel antifungal drugs.Methods:Chitin synthase Chs1 from Saccharomyces cerevisiae was heterologously expressed in HEK 293F cells and purified using affinity chromatography and size exclusion chromatography.The enzyme activity of Chs1 was explored by detecting UDP production and calcofluor-specific staining of chitin products.The structure of Chs1（Chs1-apo）and its complex with the inhibitor Nikkomycin Z（Chs1-Nik Z）was analyzed using single-particle cryo-electron microscopy data.The structural basis of Chs1 activity and the mechanism of Nik Z inhibition of chitin synthase were investigated through structural and functional experiments.Results:1.Two convenient and rapid methods for detecting chitin synthase activity in vitro were established.UDP-Glc NAc was identified as the specific substrate of Chs1,and its catalytic activity was dependent on Mg²⁺.The free Glc NAc molecule could enhance the reaction rate.2.Chs1 forms a homodimer with C2 symmetry,and destabilization of the dimer leads to loss of activity.3.Each Chs1 monomer contains a transmembrane domain and a N-terminal glycosyltransferase（GT）domain.The GT domain and three interface helices（IF1-3） form the catalytic center of chitin synthase,where D717,R759,and W760 are critical amino acids,and Y455,E457,K578,and R759 participate in the binding of UDP- Glc NAc substrate.4.TM1,TM3,TM4,and TM6 in the transmembrane domain of Chs1 form a hydrophobic transmembrane transport channel for chitin.5.Nik Z binds to the catalytic pocket of Chs1,exerting a competitive inhibition effect on Chs1 by blocking the channel leading to the transmembrane cavity through the pyridine-3-ol group.The binding of Nik Z causes significant changes in the amino acids near the catalytic site and the"plug loop".The"plug loop"functions as a gating system, controlling the switch of the catalytic pocket to the transmembrane channel.Conclusions:This study provides a comprehensive analysis of the structure and function of Chs1and its complex with the inhibitor Nik Z,revealing the key residues required for substrate binding and catalysis as well as the potential chitin transmembrane transport channel.We also elucidated the mechanism by which Nik Z competitively inhibits chitin synthesis by binding to chitin synthase and blocking the transport channel for chitin.Our findings provide important theoretical foundations for the development of antifungal drugs targeting chitin synthesis.