National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01GM137421
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
U24GM129547
United States
American Heart Association
20PRE35210058
United States
Citation
Journal: Nature / Year: 2022 Title: Methotrexate recognition by the human reduced folate carrier SLC19A1. Authors: Nicholas J Wright / Justin G Fedor / Han Zhang / Pyeonghwa Jeong / Yang Suo / Jiho Yoo / Jiyong Hong / Wonpil Im / Seok-Yong Lee / Abstract: Folates are essential nutrients with important roles as cofactors in one-carbon transfer reactions, being heavily utilized in the synthesis of nucleic acids and the metabolism of amino acids during ...Folates are essential nutrients with important roles as cofactors in one-carbon transfer reactions, being heavily utilized in the synthesis of nucleic acids and the metabolism of amino acids during cell division. Mammals lack de novo folate synthesis pathways and thus rely on folate uptake from the extracellular milieu. The human reduced folate carrier (hRFC, also known as SLC19A1) is the major importer of folates into the cell, as well as chemotherapeutic agents such as methotrexate. As an anion exchanger, RFC couples the import of folates and antifolates to anion export across the cell membrane and it is a major determinant in methotrexate (antifolate) sensitivity, as genetic variants and its depletion result in drug resistance. Despite its importance, the molecular basis of substrate specificity by hRFC remains unclear. Here we present cryo-electron microscopy structures of hRFC in the apo state and captured in complex with methotrexate. Combined with molecular dynamics simulations and functional experiments, our study uncovers key determinants of hRFC transport selectivity among folates and antifolate drugs while shedding light on important features of anion recognition by hRFC.
Model: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: GOLD / Support film - #0 - topology: HOLEY ARRAY / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: CARBON / Support film - #1 - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 200 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.00039000000000000005 kPa
Vitrification
Cryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 277 K / Instrument: LEICA EM GP
-
Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recording
Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 4 / Number real images: 21002 / Average exposure time: 4.6 sec. / Average electron dose: 60.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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