Supporting Information for Initial Biochemical and Functional Evaluation of Murine Calprotectin Reveals Ca(II)- Dependence and Its Ability to Chelate Multiple Nutrient Transition Metal Ions Rose C. Hadley, Yu Gu, and Elizabeth M. Nolan* Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA *Corresponding author: lnolan@mit.edu Phone: 617-452-2495 This Supporting Information includes: Design of the Synthetic Genes S2 Supporting Tables... S4 Table S1. Mass spectrometry of mcp subunits..... S4 Table S2. Metal content of representative protein preparations S4 Table S3. Analytical SEC elution volumes... S5 Table S4. Metal content of Tris:TSB from metal-depletion studies.. S6 Table S5. Bacterial strains used in this work S7 Supporting Figures S7 Figure S1. SDS-PAGE of purified proteins... S7 Figure S2. Circular dichroism spectra of.... S8 Figure S3. Thermal denaturation of mcp and.... S8 Figure S4. Antimicrobial activity of mcp, 20-h time point S9 S1
Design of the Synthetic Genes All synthetic genes were optimized for E. coli codon usage and ordered from DNA 2.0 in the pet41a vector. An NdeI restriction site was placed at the 5 end and a stop codon and an XhoI restriction site was placed at the 3 end. 1. Synthetic Gene for ms100a8 E. coli optimized nucleotide sequence for NdeI-mS100A8-Stop-XhoI: CAT ATG CCG AGC GAA CTG GAG AAA GCA CTG AGC AAC CTG ATC GAC GTC TAC CAC AAC TAC AGC AAT ATT CAA GGT AAT CAT CAC GCT CTG TAC AAA AAT GAT TTC AAG AAG ATG GTT ACC ACG GAG TGC CCG CAG TTC GTG CAG AAT ATC AAC ATT GAA AAC CTG TTC CGT GAG CTG GAC ATC AAC TCC GAT AAT GCC ATT AAC TTT GAA GAG TTT TTG GCG ATG GTT ATC AAA GTG GGC GTC GCG AGC CAC AAG GAC TCT CAT AAA GAG TAA CTC GAG Translated sequence for NdeI-mS100A8-Stop-XhoI: M P S E L E K A L S N L I D V Y H N Y S N I Q G N H H A L Y K N D F K K M V T T E C P Q F V Q N I N I E N L F R E L D I N S D N A I N F E E F L A M V I K V G V A S H K D S H K E Stop L E 2. Synthetic Gene for ms100a9 E. coli optimized nucleotide sequence for NdeI-mS100A9-Stop-XhoI: CAT ATG GCG AAC AAA GCA CCT AGC CAA ATG GAA CGC AGC ATC ACT ACT ATC ATC GAC ACT TTT CAT CAA TAC TCT CGT AAA GAG GGC CAC CCG GAT ACG CTG TCC AAG AAA GAG TTC CGC CAG ATG GTT GAG GCC CAG CTG GCG ACC TTT ATG AAG AAA GAA AAA CGT AAC GAG GCA CTG ATT AAC GAC ATT ATG GAA GAT CTG GAC ACC AAT CAA GAT AAT CAG CTG AGC TTC GAA GAG TGC ATG ATG CTG ATG GCG AAG TTG ATT TTC GCT TGC CAC GAG AAG CTG CAT GAA AAC AAT CCG CGT GGT CAT GGT CAC AGC CAC GGT AAG GGT TGT GGC AAA TAA CTC GAG Translated sequence for NdeI-mS100A9-Stop-XhoI: M A N K A P S Q M E R S I T T I I D T F H Q Y S R K E G H P D T L S K K E F R Q M V E A Q L A T F M K K E K R N E A L I N D I M E D L D T N Q D N Q L S F E E C M M L M A K L I F A C H E K L H E N N P R G H G H S H G K G C G K Stop L E S2
3. Synthetic Gene for ms100a9(c80s)(c91s)(c111s) E. coli optimized nucleotide sequence for NdeI-mS100A9(C80S)(C91S)(C111S)-Stop-XhoI: CAT ATG GCG AAC AAA GCA CCT AGC CAA ATG GAA CGC AGC ATC ACT ACT ATC ATC GAC ACT TTT CAT CAA TAC TCT CGT AAA GAG GGC CAC CCG GAT ACG CTG TCC AAG AAA GAG TTC CGC CAG ATG GTT GAG GCC CAG CTG GCG ACC TTT ATG AAG AAA GAA AAA CGT AAC GAG GCA CTG ATT AAC GAC ATT ATG GAA GAT CTG GAC ACC AAT CAA GAT AAT CAG CTG AGC TTC GAA GAG AGC ATG ATG CTG ATG GCG AAG TTG ATT TTC GCT AGC CAC GAG AAG CTG CAT GAA AAC AAT CCG CGT GGT CAT GGT CAC AGC CAC GGT AAG GGT AGT GGC AAA TAA CTC GAG Translated sequence for NdeI-mS100A9(C80S)(C91S)(C111S)-Stop-XhoI: M A N K A P S Q M E R S I T T I I D T F H Q Y S R K E G H P D T L S K K E F R Q M V E A Q L A T F M K K E K R N E A L I N D I M E D L D T N Q D N Q L S F E E S M M L M A K L I F A S H E K L H E N N P R G H G H S H G K G S G K Stop L E Mutations are highlighted in yellow. S3
Supporting Tables Table S1. Mass spectrometry of mcp subunits. Protein ms100a8 Calculated Mass ± N Met (g/mol) b ms100a8 Observed Mass (g/mol) c ms100a9 Calculated Mass ± N Met (g/mol) d ms100a9 Observed Mass (g/mol) mcp 10294.59 10163.40 10294.71 10163.52 13048.86 12917.67-12917.78 10278.53 10147.34 10278.82 10147.60 13008 12869.49-12869.50 Table S2. Metal content of representative purified murine proteins. a Metal mcp (24 µm) b (7.7 µm) b [Mn] (µm) 025 0079 equivalents 001 001 [Fe] (µm) 98 36 equivalents 041 046 [Co] (µm) 0017 00 equivalents 00 00 [Ni] (µm) 19 094 equivalents 008 012 [Cu] (µm) 18 089 equivalents 0075 012 [Zn] (µm) 0 64 equivalents 042 084 a Metal content was determined by ICP-MS. b Concentration of protein in the sample. S4
Table S3. Analytical SEC elution volume and calculated molecular weight of proteins. Protein a [Ca(II)] (mm) V e (ml) b MW (kda) mcp 0 12.2 27.2 1 11.9 3 2 11.8 32.3 5 11.6 34.6 10 11.5 36.1 25 11.4 37.6 0 12.2 27.8 1 11.7 33.4 2 11.6 34.5 5 11.5 35.8 10 11.4 37.6 25 11.3 38.1 a The protein concentration was 100 µm. The running buffer was 75 mm HEPES, 100 mm NaCl, ph 7.0. The elution volume (V e) corresponds to the maximum peak absorbance at 280 nm. S5
Table S4. Metal content (µm) of Tris:TSB medium with or without treatment with 250 µg/ml mcp,, or hcp-ser. a Metal Protein n.s. b SEM Ca(II) SEM BME SEM Ca(II), SEM BME Mn untreated 22 05 26 06 25 05 28 06 mcp 02 00 01 00 01 00 00 00 02 01 01 00 00 00 01 00 hcp-ser 01 00 03 02 02 01 01 01 Fe untreated 1.717 06 1.824 79 1.712 97 1.768 82 mcp 1.293 05 1.119 03 1.154 88 96 78 1.586 84 1.260 64 0.990 25 71 82 hcp-ser 1.392 38 1.179 57 0.830 95 44 51 Co untreated 32 01 32 01 33 01 32 01 mcp 30 01 31 02 33 02 31 01 32 02 30 02 29 02 29 02 hcp-ser 31 02 31 01 30 01 30 01 Ni untreated 70 59 15 51 50 61 88 35 mcp 78 15 66 11 26 30 81 13 05 28 80 10 92 18 79 13 hcp-ser 96 20 60 16 73 20 91 35 Cu untreated 74 04 73 04 70 03 68 03 mcp 53 01 52 03 45 03 36 02 62 05 54 05 49 05 42 07 hcp-ser 50 02 70 23 44 05 34 02 Zn untreated 3.292 26 3.351 23 3.629 34 3.548 30 mcp 89 23 79 18 00 22 69 12 24 70 55 14 65 16 52 18 hcp-ser 64 11 67 15 75 18 48 14 a Metal content was determined by ICP-MS (mean ± SEM, n ³ 4). b No supplement was added to the Tris:TSB medium. S6
Table S5. Bacterial strains used in this work. Strain Growth Mediuma Source Escherichia coli CFT073 TSB ATCC 700928 Acinetobacter baumannii TSB ATCC 17961 Staphylococcus aureus USA 300 JE2 TSB NARSA repository Staphylococcus epidermidis NRS101 BHI NARSA repository Listeria monocytogenes BHI ATCC 19115 Lactobacillus plantarum WCFS1 MRS ATCC a The medium used for culturing and diluted with AMA buffer for the AMA assays. Supporting Figures kda kda 25 25 22 22 17 17 11 11 hcp-ser mcp Figure S1. Purity of recombinant proteins by SDS-PAGE (15% Tris-glycine). The leftmost lane in each gel is a P7712S molecular weight ladder (New England Biolabs). S7
[Θ] λ (deg cm 2 dmol -1 ) 4 10 4 3 10 4 2 10 4 1 10 4 0-1 10 4-2 10 4-3 10 4 200 210 220 230 240 250 260 Wavelength (nm) Figure S2. CD spectra of in the absence (black) and presence (red) of 2 mm Ca(II) (1 mm Tris-HCl, ph 7.5, 25 o C). Figure 2 of the main text presents the CD spectra of mcp. A mcp mcp +Ca(II) B +Ca(II) 1.0 1.0 Fraction Unfolded 0.8 Fraction Unfolded 0.8 20 30 40 50 60 70 80 90 100 20 30 40 50 60 70 80 90 100 T ( C) T ( C) Figure S3. Representative thermal denaturation plots for 10 µm of (A) mcp or (B) in the absence (black) and presence (red) of 3 mm Ca(II) (1 mm Tris-HCl, 1 mm DTT, ph 7.5). Each trace was normalized to the CD signal at 222 nm at 95 C to obtain the fraction of unfolded protein. S8
A mcp B E. coli CFT073 E. coli CFT073 0 200 400 A. 600 baumannii 800 17961 1000 S. aureus USA300 JE2 0 200 400S. epidermidis 600 800 NRS101 1000 0 200 400 L. monocytogenes 600 800 19115 1000 A. baumannii 17961 S. aureus USA300 JE2 0 200 400S. epidermidis 600 800 NRS101 1000 0 200 400 L. monocytogenes 600 800 19115 1000 0 200 400 L. 600 plantarum 800 WCFS1 1000 0 200 400 L. 600 plantarum 800 WCFS1 1000 [mcp] µg/ml [] µg/ml Ca(II) +Ca(II) Figure S4. mcp inhibits growth of various bacterial species. (A) Growth inhibitory activity of mcp. (B) Growth inhibitory activity of. Both proteins were assayed in the absence (black) and presence (red) of a 2-mM Ca(II) supplement in the AMA medium (Tris:TSB, T = 37 o C, 20 h) (mean ± SDM, n ³ 3).Figure 5 of the main text contains data from the same assay at the 8-h timepoint. S9