Supporting Information

Siderocalin-mediated recognition, sensitization, and cellular uptake of

actinides

Benjamin E. Allred1, Peter B. Rupert2, Stacey S. Gauny1, Dahlia D. An1, Corie Y. Ralston3,

Manuel Sturzbecher-Hoehne1, Roland K. Strong2 & Rebecca J. Abergel1

1Chemical Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA

2Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109 3Berkeley Center for Structural Biology, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA 4To whom correspondence should be addressed. E-mail: rjabergel@lbl.gov; Fax: +1 510 486 5596; Tel: +1 510 486 5249.

Fluorescence Spectroscopy Detailed Methods. In steady state mode, a continuous xenon lamp (450 W) was used as the light source. For lifetime measurement, a sub-microsecond Xenon flashlamp (Jobin Yvon, 5000XeF) was used as the lightsource, with an input pulse energy (100 nF discharge capacitance) of ca. 50 mJ, yielding optical pulse duration of less than 300 ns at FWHM. Spectral selection was achieved by passage through a double grating excitation monochromator (2.1 nm/mm dispersion, 1200 grooves/mm). Emission was monitored perpendicular to the excitation pulse, again with spectral selection achieved by passage through a double grating excitation monochromator (2.1 nm/mm dispersion, 1200 grooves/mm). A thermoelectrically cooled single photon detection module (HORIBA Jobin Yvon IBH, TBX-04-D) incorporating fast rise time PMT, wide bandwidth preamplifier and picosecond constant fraction discriminator was used as the detector. Signals were acquired using an IBH DataStation Hub photon counting module and data analysis was performed using the commercially available DAS 6 decay analysis software package from HORIBA Jobin Yvon IBH. Goodness of fit was assessed by minimizing the reduced chi squared function, χ2, and a visual inspection of the weighted residuals. Each trace contained at least 5,000 points, and the estimated error on the reported lifetime values is ±10%. Calculation of Energy Transfer Efficiency Ratios. The efficiency of energy transfer E was defined according to Equation 1, where Aacc and Adon are the absorbances of the acceptor (metal ion M) and donor (ligand L or protein:ligand adduct Scn:L), respectively, at the same excitation wavelength. IM and I0

M are the luminescence intensities of the metal ion M in the presence and in the absence of energy transfer.

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E =Aacc

Adon

IMIM0 −1

⎛

⎝ ⎜

⎞

⎠ ⎟ (Eq. 1)

Calculation of Siderocalin Dissociation Constants. The dissociation constants (Kd) reported in Figure 1 were determined according to Equations 2 and 3.

€

Scn+ML↔Scn :ML (Eq. 2)

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Ka =Scn :ML[ ][Scn][ML]

=1Kd

(Eq. 3)

Diffraction Data Analysis. Diffraction data were integrated and scaled with HKL-2000 (1). Initial phases were determined by rigid body positional refinement with Refmac (2) using 3FW5.pdb as a starting structure, or molecular replacement with MolRep (3) using 3FW5.pdb as a search model. Structures were refined through iterative rounds of positional refinement using Refmac (2) alternating with model building using COOT (4), followed by a final round of TLS refinement (5). Residues or

side-chains that did not exhibit clear electron density in 2Fobs-Fcalc Fourier syntheses when contoured at 0.7σ were removed or truncated to the Cβ atom. The quality of the final model was assessed using ProCheck (6) and Molprobity (7). Cellular Uptake Experimental Details. Porcine kidney proximal tubule LLC-PK1 cells (ATCC, CL-101) were grown in Dulbecco's Modified Eagle Medium (DMEM, BioWhittaker) supplemented with 10% fetal bovine serum (FBS, ATCC), 2mM L-glutamine (BioWhittaker) and 1% penicillin-streptomycin (BioWhittaker). Cell cultures were maintained in a humidified atmosphere at 37°C in 5% CO2/95% air. Cells were subcultured by rinsing twice with Ca2+/Mg2+ free Dulbecco's Phosphate-Buffered Saline (DPBS, Life Technologies), incubating with Trypsin-Versene (BioWhittaker) for 5 min at room temperature followed by 5 min at 37°C. Cells were gently re-suspended in fresh medium and aliquoted into flasks before being plated for uptake experiments.

Supporting Table S1. Crystallographic data collection and refinement statistics.

232Th-Ent 232Th-TRENCAM 242Pu-Ent Sm-HOPO 243Am-HOPO 248Cm-HOPO PDB accession code 4ZFX 4ZHC 4ZHD 4ZHH 4ZHG 4ZHF Data collection* Space group P412121 P412121 P412121 P212121 P212121 P212121 Cell dimensions a, b, c (Å) 114.5, 114.5,

119.5 114.7, 114.7, 119.2

114.6, 114.6, 118.8

107.7, 117.8, 121.2

110.7, 115.3, 120.5

107.7, 117.7, 121.2

Wavelength CuKa 1.00000 Å 1.00000 Å 1.00000 Å 1.00000 Å 1.00000 Å Resolution (Å) 50.0-2.55

(2.59-2.55) 50.0-2.04 (2.08-2.04)

50.0-2.05 (2.09-2.05)

50.0-2.4 (2.49-2.45)

50.0-2.04 (2.08-2.04)

50.0-2.05 (2.09-2.05)

Rmerge (%) 8.2 (49.0) 9.7 (48.8) 6.3 (34.8) 13.5 (46.3) 7.1 (20.8) 11.4 (34.8) I / σI 27.8 (5.1) 25.4 (5.6) 38.2 (7.3) 13.7 (3.4) 38.7 (9.8) 16.5 (4.4) Completeness (%) 99.7 (100) 99.7 (97.0) 99.1 (95.3) 98.8 (100) 99.2 (93.2) 99.1 (81.7) Redundancy 7.6 (6.7) 9.6 (8.7) 9.5 (7.3) 4.9 (5.0) 4.8 (4.6) 4.8 (3.6) Refinement

No. reflections 26,474 (1,289) 51,169 (2,430) 49,864 (2,341) 56,438 (2,806) 98,255 (4,564) 96,900 (3,939) Rwork / Rfree (%) 23.9/26.8 20.7/22.9 20.4/22.6 20.3/22.4 17.4/19.9 19.5/21.7 No. atoms Protein 4,166 4,167 4,162 8,562 8,535 8,584 Ligand/ion 34 71 63 330 330 330 Water 26 138 118 326 565 332 B-factors (Å2) Protein 59 50 46 30 28 27 Ligand/ion 48 63 57 38 25 28 Water 41 48 43 29 32 27 R.m.s. deviations Bond lengths (Å) / angles (°) 0.01/1.45 0.01/0.90 0.01/1.15 0.01/0.97 0.01/1.25 0.01/1.19 MolProbity Percentile 100 99 97 99 99 98 Score 1.27 1.37 1.49 1.64 1.28 1.45 Residues in most favored regions (%) 98 97 98 98 98 98 Res. in disallowed regions (%) 0 0 0 0 0 0 Est. coordinate error (max. likelihood

ESUc) (Å) 0.2 0.1 0.1 0.2 0.1 0.1

*One crystal was used per data set. Values in parentheses are for the highest-resolution shell.

Supporting Figure S1. Triplet state measurement of free and Scn-bound [GdIII(HOPO)]-. Quarts tubes containing solutions of [GdIII(HOPO)]- with and without Scn (TBS, pH 7.4, 50% glycerol) were frozen in N2(l). The fluorescence intensity of the resulting solid samples was measured from the front face after excitation at 325 nm.

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Supporting Figure S2. Luminescent crystals of the Siderocalin-HOPO Eu(III) and Cm(III) complexes. a-b, Scn:[EuIII(HOPO)]-; c-d, Scn:[CmIII(HOPO)]-. Crystals are shown under visible (a, c) and UV (b, d) lights.

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b"

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d"

Supporting Figure S3. Changes in [EuIII(HOPO)]- emission spectral features upon Scn-binding. Normalized emission spectra for solutions of free and Scn-bound [EuIII(HOPO)]- (10 µM, 10 µg/mL ubiquitin, 0.1 M HEPES, pH 7.4). Comparisons are shown over the full visible emission spectra (a), and in the 5D0 → 7F0,1 (b), 5D0 → 7F2 (c), and 5D0 → 7F4 (d) spectral regions. Spectral features observed for each species are independent of the excitation wavelength (λexc = 280 nm for Scn excitation and λexc = 325 nm for ligand excitation), while they are significantly altered upon protein binding.

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Supporting Figure S4. Changes in [CmIII(HOPO)]- emission spectral features upon Scn-binding. Normalized emission spectra for solutions of free and Scn-bound [CmIII(HOPO)]- (1 µM, 10 µg/mL ubiquitin, 0.1 M HEPES, pH 7.4). Comparisons are shown over the full visible emission spectra (a), and in the 6D7/2 → 8S7/2 (b) spectral region. Spectral features observed for each species are independent of the excitation wavelength (λexc = 280 nm for Scn excitation and λexc = 325 nm for ligand excitation), while they are significantly altered and red-shifted upon protein binding.

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Supporting Figure S5. Spectral deconvolution of [EuIII(HOPO)]- and [CmIII(HOPO)]- emission peaks for free and Scn-bound complexes. Deconvolution of emission spectra (λexc = 325 nm) for solutions of free (a) and Scn-bound (b) [CmIII(HOPO)]- (1 µM, 10 µg/mL ubiquitin, 0.1 M HEPES, pH 7.4), and free (c, e, g) and Scn-bound (d, f, h) [EuIII(HOPO)]- (10 µM, 10 µg/mL ubiquitin, 0.1 M HEPES, pH 7.4). Comparisons are shown in the 6D7/2 → 8S7/2 spectral regions for the Cm species (a, b); and in the 5D0 → 7F2 (c, d), 5D0 → 7F0,1 (e, f), and 5D0 → 7F4 (g, h) spectral regions for the Eu species. Data are shown in solid blue lines; fits from the deconvolution (pink lines) are shown in dotted blue lines.

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Supporting Table S2. Relative intensities of the different transitions in the spectral deconvolution of [EuIII(HOPO)]- and [CmIII(HOPO)]- emission peaks for free and Scn-bound complexes. Calculated from data presented in Supporting Figure 5.

Deconvoluted Peak 1 Deconvoluted Peak 2 Deconvoluted Peak 3

Compound Transition λmax (nm) Relative Intensity λmax (nm)

Relative Intensity λmax (nm)

Relative Intensity

[Eu(HOPO)] 5D0 → 7F2 611.7 62.4% 617.1 16.8% 613.8 20.8%

Scn-[Eu(HOPO)] 5D0 → 7F2 611.5 67.8% 617.2 15.3% 613.6 16.8%

[Eu(HOPO)] 5D0 → 7F1 594.8 37.5% 591.2 37.5% 587.9 32.8%

Scn-[Eu(HOPO)] 5D0 → 7F1 594.3 39.8% 591.9 30.1% 587.5 30.1%

[Eu(HOPO)] 5D0 → 7F4 702.5 58.1% 694.3 26.2% 689.0 15.8%

Scn-[Eu(HOPO)] 5D0 → 7F4 702.5 58.3% 695.9 16.7% 689.4 25.1%

[Cm(HOPO)] 6D7/2 → 8S7/2 610.6 73.4% 606.3 21.3% 589.5 5.4%

Scn-[Cm(HOPO)] 6D7/2 → 8S7/2 612.8 75.3% 606.3 20.4% 590.1 4.3%

Supporting Figure S6. Job’s plot confirming the formation of 1:1 [M(Ent)] complexes. The absorbance spectra were measured for 11 solutions (TBS, 5% DMSO, pH 7.4) that varied in mole fraction of Ent from 0 -1 at a total concentration of 25 µM ([M]+[Ent]) (top left). The absorbance at 350 nm was corrected by subtracting the absorbance of a blank solution, apo-Ent and uncomplexed metal (top right). The absorbance peak at a mole ratio of 0.5 confirms the 1:1 stoichiometry of the complex formed in situ (bottom). Data shown for the [EuIII(Ent)]3- complex.

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