warfarin response. Here, we used multiplexed, sequencing-based assays to measure the effects 23 of 2,695 VKOR missense variants on abundance and 697 variants on activity in cultured human 24 cells. The large-scale functional data, along with an evolutionary coupling analysis, supports a 25 four transmembrane domain topology, with variants in transmembrane domains exhibiting 26 strongly deleterious effects on abundance and activity. Functionally constrained regions of the 27 protein define the active site, and we find that, of four conserved cysteines putatively critical for 28 function, only three are absolutely required. Finally, 25% of human VKOR missense variants show 29 reduced abundance or activity, possibly conferring warfarin sensitivity or causing disease. 30
The enzyme vitamin K epoxide reductase (VKOR) drives the vitamin K cycle, which 32 activates blood coagulation factors. VKOR, an endoplasmic reticulum (ER) localized 33 transmembrane protein encoded by the gene VKORC1, reduces vitamin K quinone and vitamin 34 K epoxide to vitamin K hydroquinone (Li et al., 2004; Rost et al., 2004) . Vitamin K hydroquinone 35 is required to enable gamma-glutamyl carboxylase (GGCX) to carboxylate Gla domains on 36 Topology informs basic aspects of VKOR function including where vitamin K and warfarin 51 bind, so determining the correct topology and validating the homology model is critical. In 52 particular, VKOR has four functionally important, absolutely conserved cysteines at positions 43, 53 51, 132, and 135, the orientation of which differs between the two proposed topologies. In the 54 four transmembrane domain topology, all four cysteines are located on the ER lumenal side of 55 the enzyme. In this topology, cysteines 43 and 51 are hypothesized to be "loop cysteines" that 56 line, n = 2,695). The missense variant density is colored as a gradient between the lowest 10% 132 of abundance scores (blue), the WT abundance score (white) and abundance scores above WT 133 (red). d, Heatmap showing abundance scores for each substitution at every position within VKOR. 134
Heatmap color indicates abundance scores scaled as a gradient between the lowest 10% of 135 abundance scores (blue), the WT abundance score (white), and abundance scores above WT 136 (red). Grey bars indicate missing variants. Black dots indicate WT amino acids. e, Number of 137 substitutions scored at each position for abundance. f, Scatterplot comparing VAMP-seq derived 138 abundance scores to mean eGFP:mCherry (n = 1 replicate) ratios measured individually by flow 139 cytometry. Variants were selected at random to span the abundance score range. 140 We also measured VKOR variant activity, adapting a HEK293 cell assay based on vitamin 146 K-dependent gamma-glutamyl carboxylation of a cell-surface reporter protein (Haque et al., 147 2014) . In this assay, if VKOR is active, a Factor IX domain reporter is carboxylated, secreted and 148 retained on the cell surface where it is detected with a carboxylation-specific, fluorophore-labeled 149 antibody. However, if VKOR is inactive, the reporter is not carboxylated and the antibody cannot 150 bind (Fig. 2a) . We modified the HEK293 activity reporter cell line to eliminate endogenous VKOR 151 activity by knocking out both VKORC1 and its paralog, VKORC1-like 1 (VKORC1L1) ( Recombination of WT VKORC1 into the landing pad of the HEK293 VKOR activity reporter cell 155 line yielded robust reporter activation, demonstrating that the reporter line could be used to assess 156 the activity of a library of VKOR variants (Fig. 2b) . 157
We recombined a library of VKORC1 variants into the HEK293 activity reporter cell line, 158 and sorted recombinant cells into quartile bins based on carboxylation-specific antibody binding. 159
Each bin was deeply sequenced and, as for VAMP-seq, an activity score was computed for each 160 variant. Final activity scores and confidence intervals were computed from six replicates for a total 161 of 697 missense variants, 21.5% of those possible ( 2010). We found four clearly demarcated regions where charged substitutions profoundly reduced 187 VKOR abundance, relative to aliphatic substitutions (Fig. 3b) . To exclude the possibility that the 188 eGFP tag used in our VAMP-seq assay somehow affected topology, we also analyzed the activity 189 score data. The activity data, derived using native, untagged VKOR, revealed the same four 190 minima as the abundance data (Fig. 3c ). In addition to these four minima, we also observed an 191 activity score minimum at position 57, corresponding to a conserved serine at this position. This 192 serine occurs at the end of the lumenal half-helix hypothesized to shield the active site from non-193 specific oxidation, so it is likely this signal is the result of disruption of that half helix. Together, 194 these results strongly support the hypothesis that, like its distant bacterial homolog, human VKOR 195 has four transmembrane domains. 196
To validate these findings, we performed evolutionary coupling analysis to infer the three-197 dimensional structure suggested by co-evolution. We aligned 2,770 VKOR sequences from both 198 eukaryotes and prokaryotes and identified coupled residues using the EVcouplings software 199 We identified non-local evolutionary coupling patterns characteristic of three-dimensional 205 contacts, which also strongly supported the four transmembrane domain model. Using these 206 13 contacts, we computationally folded human VKOR, yielding a modeled structure similar to the 207 bacterial structure (RMSD = 2.58 ÅA over 97/143 Calpha, Figure 3 -figure supplement 1). The 208 predicted tertiary structure hasd a four-helix topology, with antiparallel contacts between 209 transmembrane domains 1 and 2 (Fig. 3e, Fig. 3f ) and between transmembrane domains 1 and 210 4 (Fig. 3e, Fig. 3g with columns showing median abundance score, median activity score, rescaled scores, and 377 specific activity score. 378
Variation in VKOR is linked to both disease and warfarin response, but the overwhelming 380 majority of VKOR variants found in humans so far have unknown effects. Thus, we curated a total 381 of 215 variants that had either been previously reported in the literature as affecting warfarin 382 response (Supplementary Table 3 Table 4 ). All synonymous variants with the exception of two were WT-like or 393 possibly WT-like, while the three nonsense variants scored had as having low abundance (Fig.  394 6a). Missense variants spanned all abundance categories, with 129 (60%) having WT-like or 395 possibly WT-like abundance. 30 missense variants were low abundance, and 12 were high 396 abundance. The single known pathogenic variant R98W was low abundance (Fig. 6b) . We also 397 classified 54 variants according to their activity (Supplementary Table 4 , but it is unclear whether these variants have appreciable effects 405 on abundance or activity. We found that warfarin resistance variants span a range of abundances 406 and that the distribution of warfarin resistant variant abundance was not different from missense 407 variants generally (Fig. 6c , two-sided Kolmogorov-Smirnov test p= 0.438). Five warfarin-408 resistance variants had low abundance, suggesting that these variants must block drug binding 409 or increase activity to confer resistance. One variant, A26T, had high abundance, a possible 410 mechanism of warfarin resistance. The five warfarin resistance variants, R58G, W59L, V66M, 411 G71A, and N77S, whose activity we scored, were all WT-like. Thus, our abundance and activity 412 data are consistent with warfarin resistance arising largely from variants that block warfarin We conducted multiplexed assays to measure the effects of 2,695 VKOR variants on 425 abundance and 697 variants on activity. Both abundance and activity data provided evidence for 426 a four transmembrane topology, which was further supported by evolutionary couplings analysis. 427
We evaluated a VKOR homology model in the context of the patterns of variant effects on 428 abundance we measured, and found that the homology model could explain these patterns. Low positions (V112, S113). In light of the fact that substitutions at F87 and Y88 resulted in low 435 abundance, we note that the modeled vitamin K binding mode would disrupt packing of VKOR 436 core residues and require repacking of helices to maintain protein stability (Merkle et al., 2018) . 437
In addition to the active site, substitutions at the dilysine and, to a lesser extent, the diarginine ER 438 localization motifs caused abundance loss. 439
We also used our large-scale functional data to analyze 215 VKOR variants found in 440 humans. 16% of these variants affect neither activity nor abundance; we identified 54 previously 441 uncharacterized low abundance or low activity variants that could be pathogenic or alter warfarin 442 response. We found that only one warfarin resistance variant had increased abundance, 443
indicating that increased abundance is not a pervasive warfarin resistance mechanism. All five of 444 the warfarin resistance variants whose activity we scored were WT-like. Taken together these 445 data support the notion that warfarin resistance generally involves alterations to warfarin binding 446 rather than abundance or activity. We analyzed one known warfarin sensitivity variant, W5X, and 447 found that it is low abundance, suggesting that one should not exclude the possibility that any of 448 the 52 other low abundance variants, if found in a person, also confer warfarin sensitivity. 449 29 While our VKOR variant abundance and activity data illuminates various aspects of 450 VKOR's structure and function, the data have limitations. For example, neither assay captures 451 variant effects on mRNA splicing. Both assays have limited dynamic ranges, meaning that subtle 452 effects on abundance or activity cannot be discerned. In addition, both assays have inherent 453 noise, largely arising from the limited number of cells we can sample due to the bottleneck of cell 454 sorting. We account for this noise by filtering each dataset based on variant frequency and 455 presenting a confidence interval for each abundance and activity score. 456
In the future, we envision that the assays we used could be employed to better understand 457 VKOR's interaction with warfarin. Here, we could measure warfarin's effect on both variant 458 abundance and activity, mapping the warfarin binding site more finely. In addition, we could 459 identify warfarin resistance mutations that have not yet been observed in the clinic and group 460 variants by their putative resistance mechanism. Overall, our work highlights the value of 461 scores scaled as a gradient between the lowest 10% of activity scores (blue), the WT activity 524 score (white) and activity scores above WT (red). Grey indicates missing data. b, Heatmap of 525 abundance scores for cysteines. Catalytic cysteines C132 and C135 labeled in green. Color 526 legend same as described in a, applied to abundance scores. 527 (Oldenburg et al., 2004) 26 A26T (Watzka et al., 2011) 26 A26P (Bodin et al., 2008) 27 L27V (Peoc'h et al., 2009) 28 H28Q (Watzka et al., 2011) 29 V29L (Oldenburg et al., 2004) 34 A34P (Harrington et al., 2011) 36 D36Y (Loebstein et al., 2007) 36 D36G (Watzka et al., 2011) 41 A41S (Rieder et al., 2005) 45 V45A (Oldenburg et al., 2004) 52 S52W 66 V66G (Watzka et al., 2011) 68 H68Y (Osman et al., 2006) 71 G71A (Watzka et al., 2011) 77 N77Y (Watzka et al., 2011) 77 N77S (Watzka et al., 2011) 123 I123N (Watzka et al., 2011) 128 L128R (Oldenburg et al., 2004) 139 Y139H 
General reagents, DNA oligonucleotides, and plasmids. 560
Details on general reagents can be found in Supplementary Table 5. Unless otherwise  561 noted, all chemicals were obtained from Sigma and all enzymes were obtained from New 562 England Biolabs. E. coli were cultured at 37°C in Luria broth. All cell culture reagents were 563 purchased from ThermoFisher Scientific unless otherwise noted. HEK 293T cells (ATCC CRL-564 3216) and derivatives thereof were cultured in Dulbecco's modified Eagle's medium 565 supplemented with 10% fetal bovine serum, 100 U ml -1 penicillin, and 0.1 mg ml -1 streptomycin. 566
Cells were induced with 2.5 ug mL -1 doxycycline. Cells were passaged by detachment with 567 trypsin-EDTA 0.25%, and cells were prepared for sorting by detachment with versene. All cell 568 lines tested negative for mycoplasma. Because our activity assay is vitamin-K dependent, all 569 activity assays were done with the same lot of FBS to ensure similar concentrations of vitamin K 570 in each replicate. 571
All synthetic oligonucleotides were obtained from IDT and can be found in 572
Supplementary 
A gBLOCK with a codon-optimized sequence for human VKOR was ordered from IDT. It 577 was then cloned into the vector pHSG298 (Clontech). Saturation mutagenesis primers were 578 designed for each codon in VKOR from positions 2 to 163(Jain and Varadarajan, 2014) and 579 ordered resuspended from IDT. Forward and reverse primers for each position were mixed at 2.5 580 mM, and used in a PCR reaction with 125 pg of pHSG298-VKOR, 5% DMSO, and 5 uL of KAPA 581
Hifi Hotstart 2X ReadyMix. PCR products were visualized on a 0.7% agarose gel to confirm 582 amplification of the correct product. 583 584 49 PCR products were then quantified using the Quant-iT PicoGreen dsDNA Assay kit 585 (Invitrogen) using DNA control curves done in triplicate.
To pool, a total amount of DNA for 586 each reaction was calculated that maximized the volume to be drawn from the lowest 587 concentration PCR product. Pooled PCR products were cleaned and concentrated using 588
Zymogen Clean and Concentrate kit and then gel extracted. The pooled library was 589 phosphorylated with T4 PNK (NEB), incubated at 37°C for 30 minutes, 65°C for 20 minutes, and 590 then 4° indefinitely. 8.5 uL of this phosphorylated product was combined with 1 uL of 10X T4 591 ligase buffer (NEB) and 0.5 uL of T4 DNA ligase (NEB) to make a 10 uL overnight ligation reaction. 592
This reaction was incubated at 16°C overnight. 593
The overnight ligation was then cleaned and concentrated (Zymogen) and eluted in 6 uL 594 of ddH2O. 1 uL of this ligation was then transformed into high efficiency E. coli using 595 electroporation at 2 kV. Each reaction contained 1 uL of ligation (or ligation control or pUC19 10 596 pg/uL) and 25 uL of E. coli. 975 uL of pre-warmed SOC media was added to each cuvette after 597 electroporation, transferred to a culture tube, and recovered at 37°C, shaking for 1 hour. At 1 598 hour, 1 and 10 uL samples from all cultures were taken and plated on appropriate media (LB + 599 kanamycin for ligation and ligation control; LB + ampicillin for pUC19), the remaining 989 uL was 600 used to inoculate a 50 mL culture (+ kanamycin). Plates and 50 mL culture were incubated at 601 37°C overnight (shaking for 50 mL culture). Colonies on plates were then counted, and counts 602 were used to calculate how many unique molecules were transformed to gauge coverage of the 603 library. 50 mL culture was spun down and midiprepped. 604
To transfer the library from pKan to the recombination vector, the pKan library and 605 recombination vector were digested with XbaI and AflII for 1 hour at 65°C. The library and cut 606 vector were then gel extracted. The library was then ligated with the cut vector at 5:1 using T4 607 ligase, overnight at 16°C. The ligation was heat inactivated the next morning, clean and 608 concentrated. Another high efficiency transformation was performed the same as described 609 above, except this ligation was plated on LB + ampicillin (antibiotic switching strategy). Plates and 610 50 50 mL culture were incubated at 37°C overnight (shaking for 50 mL culture). Colonies on plates 611 were then counted, and counts were used to calculate how many unique molecules had been 612 transformed to gauge coverage of the library. A 50 mL culture was spun down and midiprepped. 613
To barcode individual variants, plasmid library harvested from midiprep was digested with 614
EcoRI-HF and NdeI at 37°C for 1 hour, 65°C for 20 minutes. Barcode oligos were ordered from 615 IDT, resuspended at 100 uM, and then annealed by combining 1 uL each of primer with 4 uL 616 CutSmart Buffer and 34 uL ddH2O and running at 98°C for 3 minutes followed by ramping down 617 to 25°C at -0.1°C/second. After annealing, 0.8 uL of Klenow polymerase (exonuclease negative, 618 NEB) and 1.35 uL of 1 mM dNTPS was then combined with the 40 uL of product to fill in the 619 barcode oligo (cycling conditions: 25°C for 15:00, 70°C for 20:00, ramp down to 37°C at -0.1°C/s). 620
Digested vector and barcode oligo were then ligated overnight at 16°C. 621
The overnight ligation was then cleaned and concentrated and eluted in 6 uL of ddH2O. 1 622 uL of this ligation was then transformed into high efficiency E. coli using electroporation at 2 kV. 623
Each reaction contained 1 uL of ligation (or ligation control or pUC19 10 pg/uL) and 25 uL of E. 624 coli. 975 uL of pre-warmed SOC media was added to each cuvette after electroporation, 625 transferred to a culture tube, and recovered at 37°C, shaking for 1 hour. At 1 hour, 1 and 10 uL 626 samples from water and pUC19 cultures were taken and plated on LB supplemented with 627 ampicillin. For ligation and ligation control, four flasks were prepared with 50 mLs of LB and 628 ampicillin, and then 500 uL, 250 uL, 125 uL, 62.5 uL was sample from the 1 mL of recovery and 629 transferred into a corresponding flask. From those flasks, 1 uL, 10 uL, and 100 uL, were sampled 630 and plated onto LB ampicillin plates. Plates and 50 mL culture were incubated at 37°C overnight. 631
Colonies on plates were then counted, and counts were used to calculate how many unique 632 molecules were transformed to gauge number of barcodes. Flask with the target number of 633 barcodes was then spun down and midiprepped. 634 635 Cell line description 636