Supplementary Information for. Structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction

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1 Supplementary Information for Structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction Takamasa Teramoto, Yukari Fujikawa, Yoshirou Kawaguchi, Katsuhisa Kurogi, Masayuki Soejima, Rumi Adachi, Yuichi Nakanishi, Emi Mishiro- Sato, Ming-Cheh Liu, Yoichi Sakakibara, Masahito Suiko, Makoto Kimura, and Yoshimitsu Kakuta* *To whom correspondence should be addressed. This PDF file includes: Supplementary Fig.S1 to S7 and Supplementary References

2 b1 b2 b3 5 PSB a1 b4 a2 a3 a4 S-S b5 a5 a6 b6 a7 3 PB be a8 b7 a9 a10 a11 a12 S-S a13 a14 a15 a16 Supplementary Figure S1 Sequence alignment of the secondary structures of TPST2 and TPST1. Region for crystallization of TPST2 is Gly43 Leu359 (designated TPST2DC18). The annotated secondary structures of TPST2DC18 are indicated below the alignment (arrows: β-strands, boxes: α-helices). β-strand paired with substrate peptide (C4P5Y3) is indicated as βe. Disordered regions are indicated by dashed-lines. 5 PSB motif and 3 PB motif are shown by blue bar and green bar, respectively. Two disulfide bonds (Cys96-Cys156 and Cys225-Cys233) revealed by the crystal structure of TPST2 DC18 are shown in the figure. The only free Cys (Cys210) is buried in the interior of the protein molecule and is not exposed to solvent. This observation indicates it is unlikely that the dimerization of the native enzyme could be stabilized by disulfide bonding between Cys210. Green closed-circles indicate residues involved in binding to the molecule, blue closed-squares indicate residues involved in binding to C4P5Y3 peptide, and a black closed-square indicate W133 involved in dimer formation. Red stars indicate proposed catalytic residues. All important residues for the enzymatic activity are conserved between the two TPST isoforms.

3 K300 a7 S191 Y238 R183 3 P 5 P R195 R78 P77 bs be b6 L83 T81 S285 T82 Y acceptor a1 b3 K158 E99 Supplementary Figure S2 Stereo figure of S/-binding site of TPST2DC18. The residues involved in binding to the molecule are colored slate. a-helices and b-strands are colored orange and cyan, respectively. Substrate peptide and acceptor tyrosine residues are colored green and magenta, respectively. A number of interactions are involved in positioning the molecule within the S-binding site. 5 -Phosphate forms hydrogen bonds with the side chains of residues Arg78, Thr81, and Thr82 of the 5 PSB motif and Ser285. The side chains of Arg183 and Ser191 of the 3 PB motif interact with 3 -phosphate. The side chains of Arg195 and Lys300 also interact with 3 -phosphate. Moreover, Arg78 of 5 PSB motif participates in not only 5 -phosphate binding but also 3 -phosphate binding. As for the adenosine ring, the side chain of Tyr238 interacts with the adenine nitrogens. The adenine ring is in position to form a hydrophobic interaction with Lue83 of the 5 PSB motif. These interactions are believed to help position the donor substrate, S, at a precise location for catalysis. The -binding modes of the two protomers of TPST2 DC18 in asymmetric unit are identical.

4 a 3 PB 3 P R78 5 P S285 a7 P77 be bs Y acceptor R101 Deep narrow cleft +3 C4P5Y3 A -5 a2 R105 W113 a3 5 PSB a1 b6 K158 b3 E99 R118* R122* W113* a3* a4 a2* a4* b 3 PB c 3 PB Open groove 5 PSB Hydrophobic gate 5 PSB Supplementary Figure S3 Structural comparisons of TPST2DC18 with HS3OST3 and SULT1D1. (a) S binding site (the 5 PSB and 3 PB motifs) and acceptor substrate binding site for human TPST2DC18 in protomer-a. Red asterisks indicate residues and secondary structures in protomer-b. (b) S binding site (the 5 PSB and 3 PB motifs) and acceptor substrate binding site for mouse cytosolic sulfotransferase (SULT1D1). (c) S binding site (the 5 PSB and 3 PB motifs) and acceptor substrate binding site for heparan sulfate 3O-sulfotransferase isoform 3 (HS3OST3).

5 a A (kda) b Elution Volume(ml) c C4P5Y3 B C4P5Y3 A Supplementary Figure S4 Dimer formation of TPST2. (a) Elution profiles of wild-type TPST2 (light blue) and mutant TPST2s (R78A; purple, W113A; red, and R101A; green). The Origami cells-expressed wild-type or mutants TPST2 was analyzed by gel filtration chromatography. Red arrows indicate elution positions of molecular mass standards, including chymotrypsinogen A (25 kda), ovalbumin (43 kda), BSA (67 kda), and aldolase (158 kda). The elution profiles of the R101A and W113A mutants revealed that they are unable to form a dimeric complex. The wild-type and R78A mutant were both eluted with a calculated molecular mass of ~77 kda, consistent with their being dimeric molecules. The elution profiles of the other mutants were similar to that of the R78A mutant. (b) Dimeric structure of TPST2- complex (c) Dimeric structure of the TPST2DC18--C4P5Y3 compelx

6 Relative activities (%) a b R284 R122* R118* C4P5Y3 A +3 K164 K216-5 R101 R105 K115 C4P5Y3 B c I282 R118 G171 L121-5 C4P5Y3 A +3 M109 K216 V220 V223 K115 C4P5Y3 B Supplementary Figure S5 Acceptor substrate binding site of TPST2DC18--C4P5Y3 complex. (a) Charged surface representation of TPST2DC18--C4P5Y3 complex. Blue surfaces signify the positive charge, whereas red surfaces signify the negative charge. The peptide-binding area is widely positively-charged and can accommodate negatively-charged substrate including tyrosines adjacent to several acidic residues located within -5 to +5 positions. R118* and R122* are residues in protomer-b. (b) Site-directed mutagenesis study results of TPST2. Sulfotransferase activity assay was performed using C4P5Y3 as the substrate peptide. Blue bar graphs illustrate the relative activities of different mutant TPST2s, compared with that of the wild-type TPST2 as 100%. n = 3, mean ± s.e.m. (c) Surface representation of TPST2DC18--C4P5Y3 complex. Yellow surfaces signify the conserved area between TPST1 and TPST2.

7 Y acceptor Supplementary Figure S6 Conformations of C4P5Y3 peptides. Superposition of C4P5Y3 A and C4P5Y3 B. C4P5Y3 A and C4P5Y3 B are colored green and light blue, respectively. Protomer-A and protomer-b of the TPST2DC18 were superposed using the program Coot. The conformations of C4P5Y3 A and C4P5Y3 B peptides are identical in the E -2 -D -1 -Y acceptor -E +1 region but divergent in the distal regions (E -5 -D -4 -F -3 and F +2 -D +3 ).

8 -1 +1 K m (mm) a C4P5Y3 (-5)-EDFEDYEFD-(+3) Complement C4 736-EDYEDYEYDE PSGL-1 43-ATEYEYLDYDFL CCR2 22-TFFDYDYGAP CCR8 11-TVTDYYYPDI-20 1, CCK 93-SDRDYMGWMD-102 1, CCR3 12-GTTSYYDDVG-21 3, CCR5 8-PIYDINYYTSE-18 4, Supplementary Fig. S7 Substrate peptides sequences and their K m values. Amino acid sequences of tyrosine sulfation site of previous tested peptide substrates. a The K m values are quoted from our previous published article 16. Potentially sulfatable tyrosine residues are colored in red. We predicted primary sulfation site based on previous biochemical and bioinformatics study and aligned the sequences with primary sufation site. Primarily sulfated tyrosine residues are underlined. For complement C4, the original sequence of C4P5Y3 is shown. Y16 of CCR8 was reported to be not a sulfated residue 19, 30, One of the major criteria appears to be the presence of acidic amino acid residues such as aspartic or glutamic acid (colored orange). Other than acidic residues, turn-inducing amino acids such as proline or glycine, positively charged residue, and hyrophobic residues are colored in green, blue, and gray, respectively.

9 Supplementary References 41. Danan, L.M., Yu, Z., Hoffhines, A.J., Moore, K.L. & Leary, J.A. Mass spectrometric kinetic analysis of human tyrosylprotein sulfotransferase-1 and -2. J. Am. Soc. Mass Spectrom. 19, (2008). 42. Liu, J. et al. Tyrosine sulfation is prevalent in human chemokine receptors important in lung disease. Am. J. Respir. Cell Mol. Biol. 38, (2008). 43. Preobrazhensky, A.A. et al. Monocyte chemotactic protein-1 receptor CCR2B is a glycoprotein that has tyrosine sulfation in a conserved extracellular N-terminal region. J. Immunol. 165, (2000).