Human Viperin Causes Radical SAM Dependent Elongation of E. coli Hinting at its Physiological Role

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1 Supporting Information Human Viperin Causes Radical SAM Dependent Elongation of E. coli Hinting at its Physiological Role Micah T. Nelp, Anthony P. Young, Branden M. Stepanski, Vahe Bandarian* Department of Chemistry, University of Utah, 314 S 1400 E., Salt Lake City, UT 84112, USA Table of Contents S1. Supporting Figures Figure S1. Additional Images of E. coli Expressing (A) modified pmal-c2x lacking viperin gene insert, (B) WT Viperin, and (C) C83A Viperin. Figure S2. Nucleotide sequence of codon-optimized viperin Figure S3. Protein sequence of viperin with MBP tag S2. Supporting Methods Cloning and Expression of Viperin. Purification of Viperin. Microscope imaging of E.coli. 5 -Deoxyadenosine production from SAM and Viperin S1

2 Figure S1. Additional images of E. coli transformed with vector alone, vector containing viperin, and vector containing inactive viperin variant. S2

3 Figure S2. The DNA sequence of the codon optimized viperin gene. The region of viperin used in this study is shown in bold. The NdeI and XhoI sites used to put the plasmid into the puc57 are shown in italics. CATATGTGGGTGCTGACCCCGGCTGCTTTTGCTGGTAAACTGCTGAGCGTGTTCCGTCAA CCGCTGTCTTCTCTGTGGCGTTCACTGGTGCCGCTGTTTTGCTGGCTGCGTGCAACCTTC TGGTTGCTAGCACGAAACGTCGCAAGCAGCAACTGGTGCTGCGCGGTCCGGATGAAACCA AAGAAGAAGAAGAAGATCCGCCGCTGCCGACCACGCCGACCAGCGTTAACTATCATTTTA CGCGTCAGTGTAATTACAAGTGCGGCTTTTGTTTCCACACCGCAAAAACGTCTTTCGTGC TGCCGCTGGAAGAAGCGAAACGTGGTCTGCTGCTGCTGAAGGAAGCCGGCATGGAAAAAA TTAACTTTAGCGGCGGTGAACCGTTCCTGCAGGATCGCGGTGAATATCTGGGCAAGCTGG TTCGTTTTTGCAAAGTCGAACTGCGCCTGCCGAGCGTTTCTATTGTCTCAAACGGTTCGC TGATCCGTGAACGCTGGTTTCAAAATTATGGCGAATACCTGGATATTCTGGCGATCAGCT GCGATTCTTTCGACGAAGAAGTGAACGTTCTGATCGGCCGCGGTCAGGGCAAAAAGAACC ATGTCGAAAATCTGCAAAAACTGCGTCGCTGGTGTCGTGATTACCGCGTTGCATTCAAGA TCAACTCAGTGATCAACCGTTTCAATGTTGAAGAAGACATGACCGAACAGATTAAGGCTC TGAACCCGGTGCGCTGGAAAGTTTTTCAATGCCTGCTGATCGAAGGTGAAAATTGTGGCG AAGATGCGCTGCGTGAAGCCGAACGCTTCGTGATTGGTGACGAAGAATTTGAACGTTTCC TGGAACGCCACAAAGAAGTCAGTTGCCTGGTGCCGGAATCCAACCAGAAAATGAAGGATT CCTATCTGATCCTGGACGAATACATGCGTTTTCTGAATTGTCGTAAAGGCCGCAAGGACC CGAGTAAATCCATTCTGGACGTCGGTGTGGAAGAAGCGATCAAGTTCTCAGGCTTCGATG AAAAGATGTTCCTGAAGCGCGGCGGTAAATATATTTGGTCGAAGGCCGATCTGAAACTGG ACTGGTAATAACTCGAG S3

4 Figure S3. The protein sequence of the viperin construct used in this study with an N-terminal MBP tag. The region corresponding to viperin is shown in bold. MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYA QSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKG KSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEA AFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTD EGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDE ALKDAQTNSSSNNNNNNNNNNLGIEGRISHHHHHHSSASENLYFQGHGTEFASTKRRKQQLVLRGPDETKEE EEDPPLPTTPTSVNYHFTRQCNYKCGFCFHTAKTSFVLPLEEAKRGLLLLKEAGMEKINFSGGEPFLQDRGE YLGKLVRFCKVELRLPSVSIVSNGSLIRERWFQNYGEYLDILAISCDSFDEEVNVLIGRGQGKKNHVENLQK LRRWCRDYRVAFKINSVINRFNVEEDMTEQIKALNPVRWKVFQCLLIEGENCGEDALREAERFVIGDEEFER FLERHKEVSCLVPESNQKMKDSYLILDEYMRFLNCRKGRKDPSKSILDVGVEEAIKFSGFDEKMFLKRGGKY IWSKADLKLDW S4

5 Cloning and Expression of Viperin The pmal-c2x vector was modified to encode an N-terminal maltose binding protein with a tobacco etch virus protease cut site and asparagine-rich linker preceding the inserted sequence. Two cycles of site directed mutagenesis, following the Stratagene QuikChange protocol, was used to insert the tobacco etch virus protease site and linker. The first set of mutagenesis primers, 5 - GGATCGAGGGAAGGATTTCACATCATCATCATCATCACAGCAGCGCTAGCGA ATTCGGATCCTCTAGATCG-3 and the reverse complement, contained an NheI cut site which was used to check for successful insertion. The second set of mutagenesis primes, 5 - CATCATCACAGCAGCGCTAGCGAGAACCTGTACTTCCAGGGCCATGGTACCG AATTCGGATCCTCTAGAGTCG-3 and the reverse complement, contained a KpnI cut site which was used to check for successful insertion. The resulting vector (pay581) sequence was then confirmed by sequencing at the DNA Sequencing Core of the University of Michigan. The codon optimized gene for viperin was obtained from Genscript. A fragment of viperin, which did not include the N-terminal membrane domain (corresponding to amino acids ), was amplified via PCR using the following two primers 5 - AAAAAAAAGAATTCGCTAGCACGAAACGTCGCAAGC-3 and 5 - AAAAAAAAAGCTTCTCGAGTTATTACCAGTCCAGTTTCAG-3. The forward primer introduced an EcoRI cut site, while the reverse primer introduced an HindIII cut site. The amplified PCR insert was ligated into pay581 using EcoR1 and HindIII restriction sites. The DNA sequence of the codon optimized viperin gene is shown in figure S.1, and the protein sequence of the viperin construct, with N-terminal MBP tag, used in this study is shown in figure S.2. The C83A variant (using WT full length viperin numbering) was constructed using site directed mutagenesis following the Stratagene QuickChange protocol using the following primer 5 - CATTTTACGCGTCAGGCGAATTACAAGTGCGGC-3 and the reverse complement. Both constructs were confirmed with sequencing at the DNA Sequencing Core of the University of Michigan. E. coli BL21 (DE3) (NEB catalog # C2527 resistant to phage T1) were transformed with the appropriate plasmid (pmn697 for viperin and pmn699 for C83A variant and pay581 for empty plasmid) through electroporation and plated onto lysogeny broth (LB) agar containing 100 µg/ml ampicillin. A single colony was used to inoculate 0.05 L of LB, and the culture was allowed to grow overnight at 37 C. The overnight culture was used to initiate six 2 L baffled growth flasks containing 1.5 L LB, 0.2% glucose, and 100 µg/ml ampicillin. These were grown at 37 C until OD 600 nm = 0.4 at which point the cultures were brought to 0.05 mm ferric iron chloride and overexpression was initiated with 0.1 mm isopropyl β-d-1-thiogalactopyranoside. After 4 h growth at 37 C cells were collected by centrifugation at 5,000 xg at 4 C, and the cell paste was frozen in liquid nitrogen and stored at -80 C. The yield of cells for viperin was 1.5 g per liter, and for the C83A variant it was 1.7 g per liter. Purification of Viperin S5

6 Viperin was purified with all steps taking place in a Coy anaerobic chamber using 2% H 2 98% N 2 atmosphere and O 2 no greater than 50 ppm. Cell paste was resuspended in 0.05 M Tris HCl (ph 8.0) containing 10% glycerol, 0.05 M KCl, and 1 mm phenylmethylsulfonyl fluoride. The cells were disrupted with sonication (Branson digital sonifier) at 50% amplitude with 15 s bursts and 45 s rests while stirring in an ice bath. Cell debris was removed by centrifugation in sealed vials at 18,500 xg for 30 min at 4 C. The clarified lysate was then loaded onto a 30 ml Q-sepharose FF column (GE healthcare), equilibrated in 0.05 M Tris HCl (ph 8.0) containing 10% glycerol and 0.05 M KCl. The column was washed with the equilibration buffer, and protein was eluted with a step gradient to 0.5 M KCl in the equilibration buffer. The eluted fractions were pooled and loaded onto a 2 x 5 ml amylose columns, connected in series, (GE healthcare) equilibrated in 0.05 M Tris HCl (ph 8.0) containing 10% glycerol and 0.05 M KCl, washed with equilibration buffer, and eluted with the same buffer supplemented with 10 mm maltose. Fractions containing viperin were pooled and again loaded onto a 30 ml Q-sepharose column as before but eluted with a gradient from M KCl over 10 column volumes (this step separates the impurities present following the amylose columns, while the first Q-sepharose column was utilized to remove amylases that could degrade the amylose column). Fractions containing viperin, identified via color and SDS- PAGE analysis, were pooled and exchanged into 0.05 M Tris HCl (ph 8.0) containing 10% glycerol and 0.05 M KCl with a desalting column (Biorad 10-DG). The enzyme was then reconstituted by adding to the solution four equivalents of FeCl 3 and Na 2 S and stirring the resulting solution for 2 h at room temperature. The reconstituted protein was exchanged into 0.05 M Tris HCl (ph 8.0) containing 10% glycerol and 0.05 M KCl using a desalting column (Biorad 10-DG). The protein solution was concentrated using 0.5 ml centrifugal concentrators (polyethersulfone membrane 10 kda cutoff) to approximately 300 µl. The concentration of viperin purified in this manner from 13 g of cells was 27 µm. The C83A variant was purified in a similar manner with the following differences. When the protein was run over the Q sepharose column for the second time, 3 x 1 ml Q sepharose FF, connected in series, were used instead of the 30 ml Q sepharose FF column as there was a lower amount of protein present. The concentration of C83A variant purified from 17 g of cells was 3 µm. Microscope Imaging of E.coli E. coli harboring the wild type MBP-viperin, MBP-C83A viperin, or MBP lacking viperin insert, were grown in 0.1 L LB cultures containing 100 µg/ml ampicillin at 37 C until OD 600 nm ~ 0.4, at which point expression was induced by bringing the cultures to 0.1 mm isopropyl β-d-1-thiogalactopyranoside and 0.05 mm ferric chloride. These were grown for an additional 12 hours at 37 C. The cultures were diluted 10-fold into 50 mm potassium phosphate ph 7.0 buffer from which µl were used to place onto microscope slides. All images were acquired using a NikonTi with a 60xPLANAPO NA 1.42 microscope and an Andor Clara CCD. 5 -deoxyadenosine production by viperin and C83A variant S6

7 Assays to determine if viperin reductively cleaved S-adenosyl-L-methionine to form 5 -deoxydenosine were performed in a Coy anaerobic chamber at room temperature using anaerobic solutions throughout. Experiments were performed in 100 mm Tris HCl (ph 8.0) containing 4 mm DTT, 10 mm sodium dithionite, 2 mm S-adenosyl-Lmethionine, and 2 µm protein. Aliquots (45 µl) were removed at 30, 60, 120, and 240 minutes and were quenched by combining with 4.5 µl of 30% (w/v) trichloroacetic acid. Precipitated protein was removed by centrifugation, and 45 µl were analyzed on an Agilent 1100 HPLC. Analytes were separated using an Eclipse XDB-C18 reverse phase column (250 x 2.6 mm) with a linear gradient from 0 to 10% acetonitrile with 0.1% (v/v) triflouroacetic acid over 20 minutes at a flow rate of 0.75 ml/min. The peak corresponding to 5 -deoxydenosine was integrated and quantified using a standard curve created from authentic 5 -deoxydenosine of varying concentrations 1. S7

8 References (1) Bruender, N. A., Young, A. P., and Bandarian, Vahe. (2015) Chemical and Biological Reduction of the Radical SAM Enzyme Carboxy-7-deazaguanine [corrected] Synthase. Biochemistry 54, S8