Structural And Functional Studies Of Chitin Synthase Chs1 And β-(1,3)-Glucan Synthase Fks1 From Saccharomyces Cerevisiae

Fungal cell walls are dynamic structures essential for cell viability,morphogenesis,and pathogenesis.Chitin and β-(1,3)-glucan are representative key components of typical fungal cell walls.Chitin and β-(1,3)-glucan are synthesized and transported by the membrane integrated proteins chitin synthase and β-(1,3)-glucan synthase.Due to the absence of highresolution three-dimensional structural studies,the molecular mechanisms of catalysis and substrate transport of these enzymes remain elusive,which hinders the understanding of the mechanism of fungal cell wall biogenesis as well as chitin and β-(1,3)-glucan synthesis.According to their important biological functions,these enzymes have been considered important targets for developing antifungal drugs.Clinical antifungal agents targeting β-(1,3)-glucan synthase(e.g.,echinocandins)and small molecule inhibitors targeting chitin synthase(e.g.,Nikkomycin Z)have been developed.However,the molecular mechanism of their inhibitory effect remains unclear.In addition,a variety of β-(1,3)-glucan synthetase mutant strains resistant to echinocelins have appeared in clinical,and the resistance mechanism remains to be studied.Therefore,in this study,in order to explore the synthesis and transport mechanism of chitin and β-(1,3)-glucan and answer the above scientific problems related to antifungal drug,the chitin synthase Chs1 and β-(1,3)-glucan synthase Fks1 of model organism S.cerevisiae were investigated by structural biology/cryo-EM single-particle analysis combined with molecular and cellular functional studies as follows:(1)We for the fisrt time determind the cryo-EM structures of chitin synthase Chs1 in multiple states,including the apo-Chs1 structure(3.3 (?))and the glycosyl donor UDPGlc NAC-bound(3.6 (?))and the product UDP-bound(3.9 (?))complex structures of Chs1.These structures show that Chs1 exists as a homodimer.The two subunits of the dimer are closely associated with each other with sub-domains swapped between them.Each Chs1 subunit consists of a cytoplasmic GT domain and a transmembrane domain.A switching loop located at the entrance of the product channel within the transmembrane domain may be involved in glycosyl transfer,the channel switch and the trans-membrane transportation of the product chitin.For exploring the inhibition mechanism of Nikkomycin Z,we also determind the cryo-EM structures of Nikkomycin Z-bound(3.2 (?))complex structures of Chs1.We found that the sugar moiety of the compound plays the major role in stable binding with Chs1,and its long scaffold occupies both the catalytic pocket and the entrance of the transmembrane channel,preventing binding of the substratesto exert inhibition function.(2)We for the fisrt time determined the high-resolution cryo-EM structure of β-(1,3)-glucan synthase,Fks1(3.0 (?)),which is also the first structure of the glycosyltransferase subfamily GT48.The structure reveals that Fks1 consists of a central catalytic region flanked by the N-and C-terminal regulatory regions.The central catalytic region adopts a cellulose synthase fold with a cytosolic conserved GT-A type glycosyltransferase domain and a transmembrane glucan-transporting channel adopting a closed conformation in our structure.We observed two conserved disulfide bonds in the extracellular part of Fks1 which are found to be crucial for the enzym activity.We proposed a working model of Fks1 based on the analysis of our structure and the comparison with cellulose synthases.In addition,our strcutual studies indicate that echinocandins inhibit Fks1 activity likely by affecting glucan transportation rather than synthesis.In summary,our study determined the cryo-EM structures of Chs1 and Fks1,responsible for the synthesis of key polysaccharide components from S.cerevisiae cell wall,and analyzed the molecular mechanisms of their synthesis and transport based on structural-functional studies.These findings shed light on the mechanisms underlying the biosynthesis of chitin andβ-(1,3)-glucan in fungal cell walls.The primary sequences of these two synthases are highly similar in different fungal species and S.cerevisiae has been widely used as an experimental model for exploring the mechanism of pathogenicity and drug resistance.Therefore our studies will also provide insights into the development of novel antifungal drugs targeting these two targets in pathogenic fungi.