Figure 1(A) Recombinant c10orf76 directly binds to PI4KB in vitro. His-pulldown assays of baculovirus/Sf9 produced 6xHis-c10orf76 (3 μM) were carried out with untagged PI4KB (2.5 μM). (B) PI4KB and c10orf76 form a stable complex. The complex of c10orf76-PI4KB eluted from a S200 superdex 10/300 GL increase gel filtration column (GE) at a volume consistent with a heterodimer (169 kDa), while c10orf76 alone eluted at a volume consistent with a monomer (79 kDa). Lines with MW values indicate elution of MW standards (158 kDa aldolase, 75 kDa conalbumin). (C) PI4KB is potently inhibited by c10orf76 in a dose-dependent manner in vitro. Kinase assays of PI4KB (20 nM) in the presence of varying concentrations of c10orf76 (1.6 nM-1 μM) were carried out on pure PI lipid vesicles (0.5 mg/L) in the presence of 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone. IC50 values were determined by one binding site, nonlinear regression (curve fit) using Graphpad. Error bars represent standard deviation of independent technical replicates (n=3). (D) PI4KB is potently inhibited by c10orf76 on pure PI vesicles and vesicles mimicking Golgi composition. Kinase assays of PI4KB and c10orf76 were carried out on lipid substrate composed of pure PI vesicles (0.5 mg/mL) with 100 μM ATP, and Golgi mimic vesicles (0.5 mg/ml, 10% PS, 20% PI, 25% PE, 45% PC) with 10 μM ATP. PI4KB was present at 20 and 300 nM in the PI and Golgi substrate assays respectively, with c10orf76 present at 500 nM in both experiments. The data is normalized to the kinase activity of PI4KB alone. Error bars represent standard deviation of independent technical replicates (n=3). (E) Changes in deuterium incorporation PI4KB in the presence of c10orf76 showed a profound ordering of the kinase domain N-lobe linker and smaller changes in the helical domain and C-lobe of the kinase domain. The sum of the difference mapped as the difference in number of deuterons incorporated for PI4KB (400 nM) in the presence and absence of c10orf76 (400 nM) over all time points (3s at 1 oC; 3s, 30s, and 300s at 23 oC). Each dot represents a peptide graphed on the x-axis according to the central residue. The red boxes highlight key regions that showed significant changes (>7% decrease in exchange, >0.5 Da difference, and unpaired two-tailed student t-test p<0.05). Error bars represent standard deviation of independent technical replicates (n=3). (F) c10orf76 binding induces differences in HDX throughout multiple domains of PI4KB. Regions of >7% difference in deuterium exchange in the presence of c10orf76 are colored onto the structure of PI4KB according to the legend (PDB: 4D0L). The N-lobe linker of the kinase domain is disordered in the structure and is represented by a dotted line. (G) Changes in the deuterium incorporation of c10orf76 in the presence of PI4KB. H/D exchange reactions displayed as the sum of the difference in HDX in the number of deuterons for c10orf76 (400 nM) in the presence of PI4KB (400 nM) at all time points (3s at 1 oC; 3s, 30s, and 300s at 23 oC) analyzed. Red boxes highlight regions that showed significant changes (>7% decrease in exchange, >0.5 Da difference, and unpaired two-tailed student t-test p<0.05). Error bars represent standard deviation of independent technical replicates (n=3). (H) The PI4KB N-lobe linker undergoes a disorder-to-order transition upon binding c10orf76. Selected peptides (including the sequence, domain information, and numbering) of both PI4KB and c10orf76 displayed as the % deuteration incorporation over time. Error bars represent standard deviation of independent technical replicates (n=3), and are typically smaller than the size of the point on the graph.