mutants fks1 v595i  (ATCC)

Journal: Science Advances

Article Title: Structure of a fungal 1,3-β-glucan synthase

doi: 10.1126/sciadv.adh7820

Figure Lengend Snippet: ( A ) Synthesis and transport scheme of 1,3-β-glucan by Fks1. The activity was found to be regulated by Rho1. ( B ) In vitro UDP-Glo GT assay with purified Fks1 with or without purified Rho1 and GTP-γ-S. The assay under condition with Rho1 and GTP-γ-S was also performed with glucose, Mg 2+ , caspofungin, or enfumafungin. The reaction without Fks1 was used as the control. Data points represent the means ± SD in triplicate. ( C ) Cryo-EM map and atomic model of Fks1. The polypeptide is shown in rainbow from N to C terminus. ( D ) Fks1 domain map. Major domains and motifs are labeled. The regions not observed are in hollow.

Article Snippet: During preparation of this manuscript, another group reported the cryo-EM structures of S. cerevisiae FKS1 and the echinocandin-resistant mutant FKS1 (S643P) (fig. S12) ( ).

Techniques: Activity Assay, In Vitro, Purification, Cryo-EM Sample Prep, Labeling

Journal: Science Advances

Article Title: Structure of a fungal 1,3-β-glucan synthase

doi: 10.1126/sciadv.adh7820

Figure Lengend Snippet: ( A ) Overall structure of Fks1 in cartoon representation. The central catalytic region (green) is sandwiched by N-terminal (gray) and C-terminal (black) regulatory domains. The putative active site is highlighted by a circle (salmon). Flexible IF2 and IF3 are highlighted by two open black cylinders. ( B ) A putative active site in Fks1, with residues forming the pocket shown in stick representation. ( C ) Structural comparison between the soluble GT domains of Fks1 (green) and BcsA (magenta) in complex with cellulose (orange), UDP (red), and Mg 2+ (lemon). ( D ) UDP-Glc hydrolysis activity of wild-type (WT) Fks1 and Fks1 mutant. Data points represent the means ± SD in triplicate. ( E ) Growth complementation of fks1 Δ cells with empty plasmid ( fks1 Δ) or plasmid carrying either WT FKS1 (FKS1) or mutants. Either 2 μl (top box) or 5 μl (bottom box) of cells was spotted onto SD-His plates and SD-His plates with FK506 (0.1 μg/ml). Plates were incubated at 30°C for 2 days.

Article Snippet: During preparation of this manuscript, another group reported the cryo-EM structures of S. cerevisiae FKS1 and the echinocandin-resistant mutant FKS1 (S643P) (fig. S12) ( ).

Techniques: Comparison, Activity Assay, Mutagenesis, Plasmid Preparation, Incubation

Journal: Science Advances

Article Title: Structure of a fungal 1,3-β-glucan synthase

doi: 10.1126/sciadv.adh7820

Figure Lengend Snippet: ( A ) Structural comparison between the central catalytic region of Fks1 (green) and BcsA (Protein Data Bank ID: 5EJZ, magenta) in complex with cellulose (orange), UDP (red), and Mg 2+ (lemon) by aligning their respective TMs. The cyan and red rhomboids mark the positions for cut-in views shown in (C) and (D). ( B ) A close-up view of the cytosolic region in (A). Substrates in cyan are modeled onto Fks1’s active site basing on structural alignment of GT domains of Fks1 and BcsA in . Movements of the GT domain and substrates are highlighted by yellow and red arrows, respectively. The glucan shifts downward by ~3 Å. ( C and D ) Cut-in views from the cytosolic side of the superposition of Fks1 and BcsA in (A). TM8 and IF1 of Fks1 form part of the putative glucan transport path. Movements of the TM8 and IF1 are highlighted by red and yellow arrows, respectively. Compared with BcsA, TM8 of Fks1 is shifted inward by ~9 Å and occupy the glucan transporting path, resulting in a closed channel. Corresponding sequences of IF2 and IF3 and gating loop are disordered in Fks1. ( E ) Mapping of the echinocandins resistance mutant hotspots (HS1 to HS3) onto the structure of Fks1. ( F ) Growth complementation of fks1 Δ cells with empty plasmid ( fks1 Δ) or plasmid carrying either WT Fks1 (FKS1) or mutants (S643P and S643Y in HS2). Cells were serially diluted, spotted onto SD-His plates and SD-His plates with FK506 (0.1 μg/ml), and incubated at 30°C for 2 days.

Article Snippet: During preparation of this manuscript, another group reported the cryo-EM structures of S. cerevisiae FKS1 and the echinocandin-resistant mutant FKS1 (S643P) (fig. S12) ( ).

Techniques: Comparison, Mutagenesis, Plasmid Preparation, Incubation

Journal: Science Advances

Article Title: Structure of a fungal 1,3-β-glucan synthase

doi: 10.1126/sciadv.adh7820

Figure Lengend Snippet: ( A ) Two disulfide bonds (Cys 658 with Cys 669 and Cys 1328 with Cys 1345 ) are located in the exoplasmic loops between TM5 and TM6 (EL3) and TM7 and TM8 (EL4) of Fks1, respectively. ( B ) UDP-Glc hydrolysis activity of WT Fks1 (WT), Fks1 in a buffer containing DTT, and Fks1 C658A/C1328A mutant. The reaction without Fks1 was used as the control. Data points represent the means ± SD in triplicate. ( C ) Growth complementation of fks1 Δ cells with a plasmid carrying Fks1 mutant (C658A/C1328A or C669A/C1345A). The empty plasmid ( fks1 Δ) and a plasmid carrying WT FKS1 (FKS1) were used as controls.

Article Snippet: During preparation of this manuscript, another group reported the cryo-EM structures of S. cerevisiae FKS1 and the echinocandin-resistant mutant FKS1 (S643P) (fig. S12) ( ).

Techniques: Activity Assay, Mutagenesis, Plasmid Preparation

Journal: Science Advances

Article Title: Structure of a fungal 1,3-β-glucan synthase

doi: 10.1126/sciadv.adh7820

Figure Lengend Snippet: The cryo-EM structure reflects the apo state (left) with flexible IF2 and IF3 and an empty open, active site for substrate binding. The glucan transporting channel is closed and engaged by TM8. Substrate binding and subsequent reaction may trigger the downward movement of the GT domain, which pushes the glucan product out of the cell. IF2 and IF3 may stabilize the glucan during glucan elongation, and TM8 is shifted outward, resulting in an open channel. The conformation of Fks1 in complex with substrates (right) is modeled according to the structure of cellulose synthases in complex with UDP and cellulose. More details are provided in the main text.

Article Snippet: During preparation of this manuscript, another group reported the cryo-EM structures of S. cerevisiae FKS1 and the echinocandin-resistant mutant FKS1 (S643P) (fig. S12) ( ).

Techniques: Cryo-EM Sample Prep, Binding Assay