Genetic Analysis of Allosteric Signaling in RhaR from Escherichia coli and Characterization of the VirF Protein from Shigella flexneri
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1 Genetic Analysis of Allosteric Signaling in RhaR from Escherichia coli and Characterization of the VirF Protein from Shigella flexneri By Bria Collette Kettle Submitted to the graduate degree program in Molecular Biosciences and the Graduate Faculty of the University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Chairperson Dr. Susan M. Egan Dr. P. Scott Hefty Dr. Steve Benedict Dr. David Davido Dr. Liskin Swint-Kruse Date Defended: November 14, 2013 Dr. Edith L. Taylor
2 The Dissertation Committee for Bria Collette Kettle certifies that this is the approved version of the following dissertation: Genetic Analysis of Allosteric Signaling in RhaR from Escherichia coli and Characterization of the VirF Protein from Shigella flexneri Chairperson Dr. Susan M. Egan Date approved: December 20, 2013
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28 GTGAAGCTTTTAATCTTTCTGCGAATTGAG GATGAATTCGGTCACCGCGTGATATTCGCC CGATGACTCTTCAGGTGTAACGATGGCGATTC CGATGACTCTTCAACCAGTGATTCGTTGCCG CACCTCTTCCTCTGACCTGTCGCAGATATT GCGCTCTTCCATGACACACTCTGACCTGTC ATCCTCTTCTCTCCAGNNNAGCCGCCT GGTCTCTTCGGAGAAGATATTGCGCAT GTCCTCTTCCAGAGTGTGTNNNGCGCAATATCTTCTCCAGCATA Construct rhar H262X (middle G downstream) GTGCTCTTCTCATGCGCAANNNCTTCTCCAGCATAGCCG ATCCTCTTCTCTCCAGNNNAGCCGCCTGTTAATCAGTGATAT ATCCTCTTCTCTCCAGCATNNNCGCCTGTTAATCAGTGATAT GTGCTCTTCTCATGCGCAATATNNNCTCCAGCATAGCCG TTACTCTTCACTGATGCGCCACCGTGGC GCACTCTTCTCAGTTAAAANNNCTCAAAGATGATTTTTTT GGCCTCTTCTATGCTGGAGAAGATATTGCGCATGACAC CCACTCTTCGCATAGCNNNCTGTTAATCAGTGATATTTGCACCG CCACTCTTCGCATAGCCGCNNNTTAATCAGTGATATTTGCACCG CCA CTC TTCGCATAGCCGCCTGNNNATCAGTGATATTTGC GATGAATTCTAAATATAGTTTGGTTATTCTGTTGAATTTATG Clone wild type virf into phg165 (upstream) GTGGAATTCTATTCTGTTGAATTTATGGATCAGATAAGGAAGAT TGTTGAAAAAAACATC Construct virf (upstream) GTGGAATTCGCCAGGATGTTAAAACTTCTCAAAGATGATTTTTT TGCC TCTCTCTTCGCGCATCAGNNNAAACTTCTCAAAGATGAT GAACTCTTCTGCGCCACCGTGGCTACCTCGGCCAGAG
29 GTGCTCTTCATATCTTCTCNNNCATAGCCGCCTGTTAATCAGTG ATAT GTGGAATTCCGCGTGATATTCGCCAGGGACGGGATGTTAAAACT TCTCAAAGATGATTTTTTGCC GTGGAATTCCGCGTGATATTCGCCAGGGACGGGATGGATTTTTT TGCCAGCGACCAGCAG GATGAATTCCGCGTGATATTCGCCAGGGACGGGATG GTGCTCTTCGATATTGCGCATGACACACTCTGAC Clone wild type virf into phg165 (downstream) GTGCTGCAGTTAAAATTTTTTATGATATAAGTAAAATTTCTTTG 3042 GAG GTGAAGCTTTTAACGTTTCTGCGAATTGAG GTGAAGCTTTTACTGCGAATTGAGATGACG GTGAAGCTTTTAACGCAGCAGATCTTTCTGCGAATTGAG GTGAAGCTTTTAACGNNNNNNNNNATCTTTCTGCGAATTGAG GCGCATATGATGGATATGGGACATAAAAACAAAATAGATATAAA GG Clone wild type virf into pdz1 and pdz3 (upstream) 3049 GCGGGATCCTTAAAATTTTTTATGATATAAGTAAAATTTCTTTG GAG Clone wild type virf into pdz1 (downstream) Clone wild type virf into pdz3 GCGGGATCCAAATTTTTTATGATATAAGTAAAATTTCTTTGGAG 3050 (downstream) GTGAAGCTTTTAACGNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNATCTTTCTGCGAATTGAGATGACG GTGAAGCTTTTAATCTTCCTGCGAATTGAGATGACG [IRD700]CGCTGTATCTTGAAAAATCGACGTTTTTTACGTGGT TTTCCGTCGAAAATTTAAGGTAAGAAC [IRD700]GTTCTTACCTTAAATTTTCGACGGAAAACCACGTAA AAAACGTCGATTTTTCAAGATACAGCG Clone wild type virf-dbd into GCGCATATGGATCAGATAAGGAAGATTGTTGAAAAAAACATCGA 3068 G pdz1 and pdz3 (upstream) GCGAAGCTTGCTCTTCACTGATCTTTCTGCGAATTGAGATGACG CCACTG GCGGAATTCATATTCGCCAGGGACGGGATGAGTGATTCGTTGCC GCC GCGGCTCTTCTCAGTTAAAACTTCTCAAAGATGATTTTTTTTAA AAGCTTCGC GCGAAGCTTTTAAAAAAAATCATCTTTGAGAAGTTTTAACTGAG AAGAGCCGC GTCCTCTTCCAGAGTGTGTCATGCGNNNTATCTTCTCCAGCATA G GTGCTCTTCTCATGCGCAATATCTTNNNCAGCATAGCCGCCTG GTGAAGCTTTTAATCTTTCTGCGAATTGAGATGACGCCACTGGC GTGAAGCTTTTAATCTTTCTGCGAATTGAGATGACGCCACTGGC TGGGCGTCATCCCGGTTTCNNNGGTAAACACCACCGA TGGGCGTCATCCCGGTNNNCCGGGTAAACACCACCGA
30 GTGAAGCTTTTAATCTTTCTGCGAATTGAGATGACGCCACTGGC TGGGCGTCATCCCNNNTTCCCGGGTAAACACCACCGA GTGAAGCTTTTAATCTTTCTGCGAATTGAGATGACGCCACTGGC TGGGCGTCATNNNGGTTTCCCGGGTAAACACCACCGA GTGAAGCTTTTAATCTTTCTGCGAATTGAGATGACGCCACTGGC TGGGCGTNNNCCCGGTTTCCCGGGTAAACACCACCGA GATGAATTCGGTCACCGCGTGATATTCGCCAGGGACGGG Clone wild type virf-ntd into GCGGGATCCTTAAGAAAAACTAAGAGAAGAAGCTATCGATATCG 3117 pdz1 (downstream) Clone wild type virf-ntd into GCGGGATCCAGAAAAACTAAGAGAAGAAGCTATCGATATCGA 3118 pdz3 (downstream) 3159 [AminoC6+DY682]TTGCATTGTCTTGTTTTTTTATCTCATTA ATC [AminoC6+DY682]GATTAATGAGATAAAAAAACAAGACAATG CAA 3160 GTCCTCTTCAACGTTGCTGNNNAAGCTGATTACC GTCCTCTTCACGTTTCGCTGGATGTTGGCGG GTCCTCTTCGGATAAGCTGATTNNNCGGCTGGCG GTCCTCTTCTATCCAGCAACGTTTCGCTGGATGTTGG GTCCTCTTCCCGGCTGGCGNNNAGCCTGAAAAG GTGCTCTTCATATCTTCTCNNNTACAGCCGCCTGTTAATC GTCCTCTTCCAGAGTGTGTNNNGCGCAATATCTTC GTGCTCTTCTCATGCGCAANNNCTTCTCCAGTACAGCCG GTGCTCTTCATATCTTCTCTGCTACAGCCGCCTGTTAATC GTGCTCTTCATATCTTCTCCGTTACAGCCGCCTGTTAATC GTCCTCTTCCAGAGTGTGTTCTGCGCAATATCTTC GTGCTCTTCTCATGCGCAAAAGCTTCTCCAG GTGCTCTTCGCTGACGCAAAACGCGCTCAC GTCCTCTTCTCAGCAANNNCGCCAGCAGACTGGAATGACC GTCCTCTTCTCAGCAATTTCGCNNNCAGACTGGAATGACC GTCCTCTTCTCAGCAATTTCGCCAGNNNACTGGAATGACC GTCCTCTTCTCAGCAATTTCGCCAGCAGNNNGGAATGACC GTCCTCTTCGATATTGATTGATGGTCATTCCAGTCTGCTGG GTCCTCTTCATATCTGCGANNNGTCAGAGTGTGTCATGCG
31 GTCCTCTTCATATCTGCGACAGGTCAGAGTGNNNCATGCGC GGTCTCTTCGGAGAAGATATTGCGCAT GCGCATATGATGGACATGGGTCACAAAAACAAAATCGACATTAA A GCGGGATCCTTAGAATTTTTTGTGGTACAGGTAGAATTTTTTCG GGG GCGGGATCCGAATTTTTTGTGGTACAGGTAGAATTTTTTCGGGG GTGAAGCTTTTAGAATTTTTTGTGGTACAGGTAGAATTTTTTCG GGG GTGGAATTCTATTCTGTTGAATTTATGGACCAGATTCGTAAAAT CGTGG GCGGGATCCATGATGGACATGGGTCACAAAAACAAAATCGACAT TAAAG GCACATATGAAACTGAAACAGAACATCG ACGGGATCCTTATTGCGATTTGAAGTAGGTCAGG ACGGGATCCTTGCGATTTGAAGTAGGTCAGG GATGAATTCGATAAAGACATTTTTTCAAGGAGGAAAGATATG GTGAAGCTTTTATTGCGATTTGAAGTAGGTCAGG GCGGGATCCATGAAACTGAAACAGAACATCG GTCCTCTTCGCCGGGTAATCAGCTTATCCAGCAACG GTGGAATTCCGCGTGATATTCGCCAGGGACGGGATGTTAAAACG CCTCAAAGATGATTTTTTTGCC GTTCTTACCTTAAATTTTCGACGGAAAACCACGTAAAAAACGTC GATTTTTCAAGATACAGCG GCGGAATTCATGATGGACATGGGTCACAAAAACAAAATCGACAT TAAAG GCGGGATCCATGATGGATATGGGACATAAAAACAAAATAGATAT AAAGG GCGCTCGAGAAATTTTTTATGATATAAGTAAAATTTCTTTGGAG GCGGAATTCCGCGTGATATTCGCCAGGGACGGGATGNNNAAACT TCTCAAAGATGATTTTTTTGCC TTGCATTGTCTTGTTTTTTTATCTCATTAATC GTGGAATTCCGCGTGATATTCGCCAGGGACGGGATGACGGTGGC GCATCAGTTAAAACTTCTC [AminoC6+DY682]GTGCCCTGGTCTGG AGAATATTATTCTTTTATCCAATAAAGATAAATTGCATCAATCC AGCTATTAAAATAGTA Clone codon optimized virf into pdz1 and pdz3 (upstream) Clone codon optimized virf into pdz1 (downstream) Clone codon optimized virf into pdz3 (downstream) Clone codon optomized virf into phg165 (downstream) Clone codon optimized virf-dbd into phg165 (upstream) Clone codon optimized virf into pse264 (upstream) Clone codon optimized aggr into pdz1 and pdz3 (upstream) Clone codon optimized aggr into pdz1 (downstream) Clone codon optimized aggr into pdz3 (downstream) Clone codon optimized aggr into phg165 (upstream) Clone codon optimized aggr into phg165 (downstream) Clone codon optimized aggr into pse264 (upstream) Clone codon optimized virf into pmal-c2 (upstream) Clone wild type virf into pet24b (upstream) Clone wild type virf into pet24b (downstream) Labeling oligo used for LUEGO protocol
32 TACTATTTTAATAGCTGGATTGATGCAATTTATCTTTATTGGAT AAAAGAATAATATTCTCCAGACCAGGGCAC AGAATATTATTCTTTTATCCAATAAAGA TCTTTATTGGATAAAAGAATAATATTCTCCAGACCAGGGCAC CGATCACTCTTCTCTGTTCGGGCAGTTGG CGATCACTCTTCTCAGCAACTCAGCCATTTC GTCCTCTTCGGATAAGCTGATTACCCGGCTGGCG GTGCTCTTCGTTGCTGAATCGCCATCGTTACACCAGTGATTCG GTGCTCTTCGCAACATCACCTCCTGCCCGAACTCCAACTCAGC GTGCTCTTCGCAACATCACCTCCTGCCCGAACAGCAACTCAGC Downstream middle oligo to make RhaR L185E and L181E/L185E. Upstream middle oligo to make RhaR L181E/L185E Upstream middle oligo to make RhaR L185E
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34 E. coli strains Genotype SME1049 ECL116 [F- lacu169 enda hsdr thi] SME1541 CSH141 Source or Reference SME BS536 Shigella flexneri virb-lacz T. Maurelli SME2503 ECL116 (rhas-lacz) 128 reca::cat Laboratory collection SME2525 ECL116 (rhas-lacz)128 rhasr-kan reca::cat Laboratory collection SME3053 ECL116 (rhas-lacz) 85 reca::cat Laboratory collection SME3160 ECL116 rhas-lacz 85rhaSR::kan reca::cat Laboratory collection SME3564 SME3160+ phg165/rhar This study SME3565 SME2525+pHG165/rhaR This study SME3613 AP76 [[MC4100 (nlpa-lacz) 391] G.P. Munson SME3629 SME3792+pATM323/virF T. Maurelli SME3653 SME3160+pHG165/rhaR Y265H This study SME3654 SME3160+pHG165/rhaR Y265T This study SME3655 SME3160+pHG165/rhaR Y265G This study SME3656 SME3160+pHG165/rhaR Y265L This study SME3734 SME3160+pHG165/rhaR S270R This study SME3735 SME3160+pHG165/rhaR S270T This study SME3736 SME3160+pHG165/rhaR S270D This study SME3737 SME3160+pHG165/rhaR S270V This study SME 3738 SME3160+pHG165/rhaR S270I This study SME3739 SME3160+pHG165/rhaR S270G This study SME3750 BL21(DE3) DnaY R. DeGuzman SME3754 AF51 [MC4100 (cfaa-lacz) 469] G.P. Munson SME3773 SME3160+pHG165/rhaR H262D This study SME3774 SME3160+pHG165/rhaR H262R This study SME3775 SME3160+pHG165/rhaR H262V This study SME3776 SME3160+pHG165/rhaR wt (for N-terminal deletions) This study SME3777 SME3160+pHG165/rhaR 2-34 This study SME3778 SME3160+pHG165/rhaR 2-40 This study SME3792 AB097 [MC4100 (virb-lacz) 259] G.P. Munson SME3794 SME3792+pHG165/virF-DBD (2-160) This study SME3797 SME3160+pHG165/rhaR Y265M This study SME3798 SME3160+pHG165/rhaR Y265E This study SME3799 SME3160+pHG165/rhaR Y265K This study
35 SME3805 Rosetta 2(DE3) Novagen SME3821 SME3160+pHG165/rhaR-DBD This study SME3822 SME3160+pHG165 SapI-/rhaR This study SME3823 SME3160+pHG165 SapI-/rhaR-DBD This study SME3824 SME3792+pDZ1/virF This study SME3825 SME3160+pHG165/rhaR Q268C This study SME3826 SME3160+pHG165/rhaR Q268V This study SME3827 SME3160+pHG165/rhaR Q268G This study SME3828 SME3160+pHG165/rhaR Q268M This study SME3829 SME2525+pHG165/rhaR 2-34 This study SME3830 SME2525+pHG165/rhaR 2-40 This study SME3841 SME3792+pDZ3/virF-DBD This study SME3842 SME3792+pDZ3/virF This study SME3843 SME3792+pDZ1/virF-DBD This study SME3850 BL21(DE3)pLysS Promega SME3857 SME3160+pHG165/rhaR L266V This study SME3858 SME2525+pHG165/rhaR WT (for N-term deletions) This study SME3866 KS1000 New England Biolabs SME3873 SME3160+pHG165/rhaR R295L This study SME3874 SME3160+pHG165/rhaR R295Q This study SME3875 SME3160+pHG165/rhaR E296T This study SME3876 SME3160+pHG165/rhaR E296L This study SME3877 SME3160+pHG165/rhaR E296Q This study SME3878 SME3160+pHG165/rhaR E296Y This study SME3879 SME3160+pHG165/rhaR E296H This study SME3880 SME3160+pHG165/rhaR T297N This study SME3881 SME3160+pHG165/rhaR T297A This study SME3882 SME3160+pHG165/rhaR T297I This study SME3883 SME3160+pHG165/rhaR T297S This study SME3884 SME3160+pHG165/rhaR G298L This study SME3885 SME3160+pHG165/rhaR G298T This study SME3886 SME3160+pHG165/rhaR G298S This study SME3887 SME3160+pHG165/rhaR G298K This study SME3888 SME3160+pHG165/rhaR M299T This study SME3889 SME3792+pHG165/virF This study SME3889 SME3160+pHG165/rhaR M299E This study SME3890 SME3160+pHG165/rhaR M299L This study
36 SME3897 SME3862 (SME2999[2985 (S-Z 85) rhasr::kan] malp::laciq reca::cat) + phg165rhar This study SME3909 SME3160+pHG165/rhaR Q264V This study SME3910 SME3160+pHG165/rhaR Q264L This study SME3910 SME3160+pHG165/rhaR R295K This study SME3911 SME3160+pHG165/rhaR L267F This study SME3912 SME3160+pHG165/rhaR L267S This study SME3913 SME3160+pHG165/rhaR L267A This study SME3914 SME3160+pHG165/rhaR L267M This study SME3920 SME3160+pHG165/rhaR L267I This study SME3921 SME3160+pHG165/rhaR L267T This study SME3922 SME3160+pHG165/rhaR R295I This study SME3923 SME3160+pHG165/rhaR M299V This study SME3924 SME3160+pHG165/rhaR H262I This study SME3925 SME3160+pHG165/rhaR H262S This study SME3926 SME3160+pHG165/rhaR H262F This study SME3927 SME3160+pHG165/rhaR H269P This study SME3928 SME3160+pHG165/rhaR Y265F This study SME3930 SME3160+pHG165/rhaR H262Y This study SME3931 SME3160+pHG165/rhaR H262L This study SME3932 SME3160+pHG165/rhaR Y265P This study SME3933 SME3160+pHG165/rhaR R271V This study SME3934 SME3160+pHG165/rhaR L266C This study SME3935 SME3160+pHG165/rhaR L266E This study SME3936 SME3160+pHG165/rhaR L266A This study SME3937 SME3160+pHG165/rhaR H269I This study SME3938 SME3160+pHG165/rhaR H269L This study SME3939 SME3160+pHG165/rhaR H269K This study SME3940 SME3160+pHG165/rhaR H269Y This study SME3941 SME3160+pHG165/rhaR H269S This study SME4117 Arctic Express +pse290/rhar Stratagene SME4158 SME3754+pHG165/aggR co This study SME4243 SME2525+ phg165/rhar 34 Q264L This study SME4244 SME2525+ phg165/rhar 34 Y265M This study SME4245 SME2525+ phg165/rhar 34 L266A This study SME4246 SME2525+ phg165/rhar 34 L266E This study SME4247 SME2525+ phg165/rhar 34 Q268V This study SME4248 SME2525+ phg165/rhar 34 H269Y This study
37 SME4249 SME2525+ phg165/rhar 34 H269S This study SME4250 SME2525+ phg165/rhar 34 S270G This study SME4251 SME2525+ phg165/rhar 34 E296T This study SME4252 SME2525+ phg165/rhar 34 T297S This study SME4253 SME2525+ phg165/rhar 34 G298S This study SME4254 SME2525+pHG165/rhaR L37R-H269S This study SME4255 SME3160+pHG165/rhaR D209A This study SME4258 SME3792+pHG165/aggR co This study SME4272 SME2525+ phg165/rhar 34 H262V This study SME4273 SME2525+ phg165/rhar 34 L267M This study SME4274 SME2525+ phg165/rhar 34 R271V This study SME4275 SME2525+ phg165/rhar 34 R295Q This study SME4276 SME3160+pHG165/rhaR Q264K This study SME4277 SME3160+pHG165/rhaR Q264T This study SME4278 SME2525+ phg165/rhar 34 H269T This study SME4279 SME2525+ phg165/rhar 34 H269V This study SME4280 SME3160+pHG165/rhaR Q245C This study SME4281 SME3160+pHG165/rhaR Q245A This study SME4282 SME3160+pHG165/rhaR Q245V This study SME4283 SME3160+pHG165/rhaR Q245G This study SME4284 SME3160+pHG165/rhaR Q246L This study SME4285 SME3160+pHG165/rhaR Q246S This study SME4286 SME3160+pHG165/rhaR F243L This study SME4287 SME3160+pHG165/rhaR F243M This study SME4288 SME3160+pHG165/rhaR F243I This study SME4289 SME3160+pHG165/rhaR Q257I This study SME4290 SME3160+pHG165/rhaR Q257R This study SME4291 SME3160+pHG165/rhaR Q257L This study SME4292 SME3160+pHG165/rhaR Q257V This study SME4292 SME3160+pHG165/rhaR L35K-H269S This study SME4293 SME3160+pHG165/rhaR C261T This study SME4293 SME3160+pHG165/rhaR L35K-H269Y This study SME4294 SME3160+pHG165/rhaR C261V This study SME4294 SME3160+pHG165/rhaR L35K-H269V This study SME4295 SME3160+pHG165/rhaR C261I This study SME4296 SME3160+pHG165/rhaR Y265Q-H269Y This study SME4297 SME3160+pHG165/rhaR Y265A-H269Y This study SME4298 SME3160+pHG165/rhaR Y265G-H269Y This study
38 SME4299 SME3160+pHG165/rhaR Y265R-H269Y This study SME4300 SME3160+pHG165/rhaR A217G This study SME4301 SME3160+pHG165/rhaR A217P This study SME4302 SME3160+pHG165/rhaR A217V This study SME4303 SME3160+pHG165/rhaR T247I This study SME4304 SME3160+pHG165/rhaR T247G This study SME4304 SME2525+ phg165/rhar 34 Q268M This study SME4305 SME3160+pHG165/rhaR T247W This study SME4306 SME3160+pHG165/rhaR D209E This study SME4307 SME3160+pHG165/rhaR T213A This study SME4308 SME2525+ phg165/rhar 34 H269C This study SME4309 SME2525+pHG165/rhaR 34 Q268M This study SME4310 SME3160+pHG165/rhaR Q246R This study SME4311 SME3160+pHG165/rhaR Q246W This study SME4312 SME3160+pHG165/rhaR T213L This study SME4313 SME3160+pHG165/rhaR H262Y-H269Y This study SME4314 SME3160+pHG165/rhaR H262A-H269Y This study SME4315 SME3160+pHG165/rhaR T247A This study SME4319 KS1000+pMAL-C2x/virF co This study SME4320 SME3160+pHG165/rhaR C261L This study SME4322 SME2525+pHG165/rhaR 34 L35C-H269S This study SME4323 SME2525+pHG165/rhaR 34 L35H-H269S This study SME4381 SME3160+pHG165/rhaR L37R-H269S This study SME4391 SME3160+pHG165/rhaR 2-29 This study SME4413 SME2525+pHG165/rhaR 2-29 This study SME4419 SME3160+pHG165/rhaR W75R-H269K This study SME4420 SME3160+pHG165/rhaR W75R-H269S This study SME4421 SME3160+pHG165/rhaR D197G-H269K This study SME4422 SME3160+pHG165/rhaR L35K-H269K This study SME4423 SME3160+pHG165/rhaR L37R-H269K This study SME4428 SME4429 SME4430 SME4431 SME2503 Tn-10 linked to pcnb mutant (from P1 on SME2595) SME2525 Tn-10 linked to pcnb mutant (from P1 on SME2595) SME3053 Tn-10 linked to pcnb mutant (from P1 on SME2595) SME3160 Tn-10 linked to pcnb mutant (from P1 on SME2595) This study This study This study This study SME4469 SME3160+pHG165/rhaR D41A-T279A This study SME4470 SME3160+pHG165/rhaR D41S-T279S This study
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56 A AS2 AS1 C-HTH1 C-HTH2 B AS2 C-HTH2 AS1 C-HTH1
57 AS1 C-HTH1 AraCxx0 REACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGISVLSWRED 229 RhaRxx1 LDKLITRLAASLKSPFALDKFCDEASCSERVLRQQFRQQTGMTINQYLRQ 257 ToxTXx2 MEKISCLVKSDITRNWRWADICGELRTNRMILKKELESR-GVKFRELINS 219 : :..... * : :..: *:..... AS2 C-HTH2 AraCxx0 QRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAG 279 RhaRxx1 VRVCHAQYLLQHSRLLISDISTECGFEDSNYFSVVFTRETGMTPSQWRHL 307 ToxTXx2 IRISYSISLMKTGEFKIKQIAYQSGFASVSYFSTVFKSTMNVAPSEYLFM 269 *:. : *:..: * :. : **. *** **.. :**:: AraCxx0 CEEKVNDVAVKLS 292 RhaRxx1 NSQKD ToxTXx2 LTGVAEK
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61 Mutation (-) Rha (+) Rha Category c % WT Activation a % WT Protein Levels b % WT Activation a % WT Protein Levels b WT 100± ± C261I 22±4 110±39 113±5 93±10 Down (-) C261V 29±3 77±2 Down (-) C261L 29±2 86±9 Down (-) C261T 40±3 95±3 Down (-) H262D 8±1 57±8 15±1 68±14 Down H262V 8±1 25±5 Down H262I 8±1 5±0.2 Down H262Y 13±3 47±11 Down H262L 10±0.3 56±4 153±50 51±29 Down (-) H262S 11±2 117±14 Down (-) H262F 13±0.2 88±10 Down (-) H262R 86±7 139±14 ~WT Q264T 21±2 104±8 Down (-) Q264L 27±4 73±14 93±2 84±5 Down (-) Q264K 35±3 102±17 Down (-) Q264V 36±5 111±5 Down (-) Y265T 9±1 85±3 Down (-) Y265G 11±1 95±9 Down (-) Y265K 19±1 51±11 102±21 79±9 Down (-) Y265E 30±13 119±4 Down (-) Y265M 32±3 121±20 Down (-) Y265L 9± ±6 Down (-) Up (+) Y265F 81±6 110±17 ~WT Y265H 124±4 120±6 ~WT L266E 9±1 27±5 Down L266A 8±0.1 82±3 Down (-) L266C 9±0.4 45±1 86±4 70±11 Down (-) L266V 11±3 93±10 Down (-) L267S 8±1 2±0.1 Down L267F 9±1 9±1 Down L267A 9±0.5 52±23 11±1 71±9 Down L267T 10±1 5±0.3 Down L267M 11±2 53±13 56±2 79±5 Down L267I 13±4 58±4 Down Q268V 71±11 77±1 ~WT Q268G 86±10 76±18 70±5 164±97 ~WT Q268C 99±3 95±12 ~WT Q268M 106±21 77±4 ~WT
62 H269P 11±0.4 2±0.1 Down H269L 12±2 78±2 Down (-) H269I 17±0.5 57±25 94±20 79±2 Down (-) H269Y 25±2 102±12 Down (-) H269S 184±34 78±30 94±5 162±85 Up (-) H269K 185±8 75±24 111±2 150±76 Up (-) S270V 10±1 15±1 Down S270I 10±0.2 3±0.2 Down S270D 14±1 86±4 Down (-) S270R 15±1 101±2 Down (-) S270G 20±2 38±23 106±4 85±28 Down (-) S270T 116±8 106±6 ~WT b. Quantification of variant protein levels compared c.
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64 A B (-)Rha C 118±69 1±1 9±6 29±23 (+)Rha 100±0 2±1 18±8 45±21
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68 Mutation (-) Rha (+) Rha Category c % WT Activation a % WT Protein Levels b % WT Activation a % WT Protein Levels b WT 100± ±6 100 R295L 9±1 128±3 160±12 176±70 Down (-) Up (+) R295I 10±1 169±22 Down (-) Up (+) R295Q 25±2 238±76 262±41 94±16 Down (-) Up (+) R295K 118±4 204±91 111±15 153±21 ~WT E296Y 14±7 113±33 4±1 117±75 Down E296H 11±5 134±46 4±1 157±17 Down E296L 14±2 91±11 Down (-) E296Q 15±1 110±41 Down (-) E296T 58±8 149±60 125±10 163±0.8 ~WT T297N 9±1 91±8 Down (-) T297A 30±2 69±2 89±30 161±30 Down (-) T297S 30±2 96±77 141±20 119±30 Down (-) T297I 102±6 136±49 109±4 148±44 ~WT G298T 7±1 42±29 83±8 54±7 Down (-) G298L 13±4 70±44 81±8 79±16 Down (-) G298S 13±2 86±10 Down (-) G298K 14±1 80±2 Down (-) M299E 8±0.5 38±6 7±1 10±5 Down M299T 30±3 96±9 Down (-) M299L 50±4 111±22 Down (-) M299V 51±2 231± ±6 158±0.7 Down (-) b. Quantification of variant protein levels compared c.
69
70
71 Mutation % WT Activation (-) Rha (+) Rha WT Category b F243I Down F243L Down (-) F243M Down (-) Q245G Down (-) Q245V Down (-) Q245C Down (-) Q245A ~WT Q246L Down (-) Q246R Down (-) Up (+) Q246S Up (+) Q246W ~WT T247G 13 3 Down T247W 17 5 Down T247A Down (-) T247I Down (-) Q257I Down Q257L Down (-) Q257R Down (-) Q257V ~WT b.
72 (-) Rha (+) Rha Mutation % WT Activation % WT Protein b % WT Activation % WT Protein b Category c WT 100± ± Q246R 36±9 159±29 658±15 108±25 Down (-) Up (+) Q246S 77±7 93±15 149±10 61±17 Up (+) b. Quantification of variant protein levels compared c.
73 Mutation % WT Activation (-) Rha (+) Rha WT Category b D209A Down (-) D209G Down (-) Up (+) D209E Down (-) T213L * Down (-) T213A * ~WT A217P Down (-) A217V ~WT A217G * ~WT b.
74
75 L35 H269
76
77 Mutation % WT Activation (-) Rha (+) Rha WT 100±4 100±10 L35K 190±4 117±9 H269V 96±4 117±10 L35K-H269V 115±13 122±2 a. b. Quantification of variant protein levels compared
78
79 (-) Rha (+) Rha Mutation % WT Activation % WT Protein b % WT Activation % WT Protein b ±16 ±12 ±22 ±4 ±2 ±4 ±37 ±3 ±5 ±28 ±28 ±12 ±3 ±35 ±7 ±2 ±5 ±6 ±12 ±5 ±4 ±8 ±17 ±7 ±12 ±5 ±27 ±1 ±1 ±32 ±29 ±7 ±1 ±17 ±21 ±3 ±7 ±20 ±16 ±1 ±4 ±18 b. Quantification of variant protein levels compared
80 A W75 B D197 H269
81
82 Mutation % WT Activation (-) Rha (+) Rha WT D41A D41S T279A T279S D41A-T279A D41S-T279S
83 D41 T279
84
85
86
87 Quantification of variant protein levels compared E
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96 A C B
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109 kda GB1-VirF pdz1/virf (38kDa) VirF-GB1 pdz3/virf (38kDa) GB1-VirF DBD pdz1/virf-dbd (20kDa) VirF DBD-GB1 pdz3/virf-dbd (20kDa)
110 kda
111 1 2 3 kda
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128 Effector Binding Pocket L35 H contacts T279
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140 C183 Q218
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142 RhaR MAFCNNANLLNVFVRHANNQLRSLAEVATVAHQLKLLKDDFFASDQQA--VAVADRYPQDV 60 RhaS MTVLHSVDFFPSGNAS--VAIEPRLPQAD 27
143
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146 ToxTxx0 MIGKKSFQTNVYRMSKFDTYIFNNLYINDYKMFWIDSGIAKLIDKNCLVSYEINSSSIIL 60 VirFxx1 -MMDMGHKNKIDIKVRLHNYIILYAKRCSMETTVSSGNETLTIDEGQIAFIERNIQINVS 59 AraCxx2 --MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGYILNLTIRGQGVVK 58. : : :. ::.. : :. :.. : ToxTxx0 LKK NSIQRFSLTSLSDENINVSVITISDSFIRSLKSYILGDLMIRNLYSENKD 113 VirFxx1 IKKS------DSINPFEIISL-DRNLLLSIIRIMET--EP--IYSFQHSYSEEKRGLNKK 108 AraCxx2 NQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYWHEWLNWPSIFANTG 118 :..* *.:..... *. Dimerization ToxTxx0 LLLWNCEHND--IAVLSEVVNGFREINYSDEFLKVFFSG--FFSKVEKKYN 160 VirFxx1 IFLLSEEEVS--IDLFKSIKEMETPFGKRKIYSLACLLS--AVSDEEALYT 155 AraCxx2 FFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRRMEAINE 169 ::.. ::.: :.. :. *
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To test the possible source of the HBV infection outside the study family, we searched the Genbank
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