Data collection and structure determination
X-ray diffraction data were collected at the synchrotron beamline BL19U1
in SSRF, Shanghai, China. The crystals were maintained at 100 K during
data collection. Data were indexed, integrated and scaled using HKL3000
(HKL Research, Charlottesville, VA). The structure of the complex was
determined by molecular replacement
using
molrep (CCP4) and PDB entry 4JTU as an initial model. The resulting
model was re-builded and refined in
Coot. The images were generated using the software PyMOL (Molecular
Graphics System, Version 2.2.3; Schrödinger, LLC). A summary of the data
collection and processing statistics was given in Table S1. The
coordinates and structure factors of the final model of DHODH-piprine
were deposited in the Protein Data Bank (PDB, www.rcsb.org, PDB ID:
6IWU).
Mouse spleen cell
proliferation
Single-cell suspensions of spleens from individual C57BL/6 mice were
harvested as described previously(Wanke et al., 2017). Briefly, the
spleen was aseptically taken from mice, cut into small pieces and crush
gently through the cell strainers in 5 ml 1×HBSS. Cells were suspended
in Ammonium-Chloride-Potassium (ACK) lysing buffer to remove the
erythrocytes and then collected by centrifugation at 2000 rpm for 10
min. After washing twice with 1×HBSS, cells were re-suspended in
RPMI-1640 with 10% FBS for culture. Spleen cells were seeded into
96-wells cell culture plates at a density of 5×105cells/well in RPMI-1640 with 10% FBS and subsequently incubated with
various concentrations of PIP in the presence or absence of uridine (100
μM). Cell proliferation was stimulated by adding 10 μg/ml of Con
A. After 72 h treatment, cells
growth was measured by MTT assay with EnVision Multimode Plate Reader
(Perkin Elmer, Waltham, MA).