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1 Supporting Information Xiao et al /pnas SI Methods Protein Expression and Purification. Bacmid was generated and recombinant baculovirus amplified using standard procedures (Bac-to-Bac; Invitrogen). Hi5 cells were infected at a density of cells/ml. After 48 h, the conditioned medium was collected, cleared of cell debris, and concentrated using a tangential flow filtration device (Vivaflow 200; Sartorius). The fusion protein was purified by nickel nitrilotriacetic acid (Ni-NTA) affinity chromatography, and tobacco etch virus (TEV) protease was used to remove the N-terminal fusion tag. Untagged cefam20 protein was further purified using ion-exchange chromatography (RESOURCEQcolumn;GEHealthcare) and size-exclusion chromatography (Superdex 200 column; GE Healthcare). To generate selenomethionine (Se-Met)-substituted protein, Hi5 cells were adapted to methionine-free medium (ESF921 methionine-deficient; Expression Systems), infected with baculovirus, and supplemented twice with 100 mg/l Se-Met (Acros) at 12 and 36 h postinfection. The Se-Met substituted protein was purified as described above. Human Fam20C and Fam20B were immunopurified from conditioned medium of HEK293T cells as described previously (1). Enzyme Assays. For kinetic analyses using β28 40, reactions were performed in 50 mm Hepes (ph 7), 0.5 mg/ml BSA, and 2.5 mm β (Stock solutions of β28 40 were prepared in 10 mm Hepes, with ph adjusted to 7 with NaOH.) For experiments using different cations, 10 mm MnCl 2 or 10 mm MgCl 2 was used, at an enzyme concentration of μg/ml for Mn 2+ reactions or 1 μg/ml for Mg 2+ reactions. Reactions were initiated with varying amounts of [γ 32 P]ATPandincubatedfor10minat30 C. Incorporated radioactivity was quantified by the phosphocellulose filter paper method and scintillation counting as described previously (1). Mutants of Fam20C were transiently expressed in HEK293T cells and immunopurified from the conditioned medium as described previously (1). The relative levels of the mutant proteins were quantified based on protein immunoblotting and comparison with a standard curve using known amounts of Flag-tagged WT Fam20C. Activity was monitored by the incorporation of 32 Pfrom[γ- 32 P]ATP into β28 40 as described previously (1). Kinase assays using GlcAβ1 3Galβ1 3Galβ1 4Xylβ1-O-benzyl (1 mm) were performed in 50 mm Hepes (ph 7.4), 10 mm MnCl 2, 100 μm [γ- 32 P]ATP (specific activity, 100 cpm/pmol), and 10 μg/ ml kinase at 30 C for 10 min. Reactions were terminated with 15 mm EDTA and 2 mm ATP, loaded onto Sep-Pack C18 cartridges (Waters) preequilibrated with 0.2 M (NH 4 ) 2 SO 4. Columns were washed five times with 2 ml of 0.2 M (NH 4 ) 2 SO 4, and the substrate was eluted with 1 ml of methanol. Incorporated radioactivity was quantified by liquid scintillation counting. Crystallization and Structure Determination. Recombinant cefam20 in 20 mm Hepes (ph 7.5) and 150 mm NaCl was concentrated to 7 mg/ml. The cefam20 crystals were grown at 20 C by the sittingdrop vapor diffusion method, using a 1:1 ratio of protein:reservoir solution containing % PEG 4000, 200 mm imidazolemalate (4:1), and 3 6 mm NiCl 2. Crystals grew to full size in several days, and were then dehydrated by serial transfer to solutions containing increasing concentration of PEG (a 2% increase at each step, with a 30-min incubation). Finally, the crystals were transferred to a solution containing 28% PEG 4000, 200 mm imidazole-malate (4:1), 4 mm NiCl 2, and 20% ethylene glycol before being flash-frozen in liquid nitrogen. The Se-Met crystals were grown and treated similarly. To obtain the ADP-bound structure, 5 mm MnCl 2 and 5 mm ADP were kept in the dehydration solutions during crystal transfer. Diffraction data were collected at the Advanced Light Source beamline and processed with HKL2000 (HKL Research). The nucleotide-free (apo) structure was determined by the singlewavelength dispersion method using data collected from a Se-Met crystal. Heavy atom search, phase calculation and refinement, density modification, and initial model building were carried out with Phenix (2). The structural model was traced in Coot (3) and refined using Phenix, with 5% randomly selected reflections used for cross-validation (4). The final working R and free R factors were 21.2% and 24.8%, respectively (Table S1). The Mn/ADPbound structure was determined by molecular replacement with Phaser (5), using the Apo structure as the search model, and refined as described above. The final working R and free R factors were 21.5% and 25.3%, respectively (Table S1). 1. Tagliabracci VS, et al. (2012) Secreted kinase phosphorylates extracellular proteins that regulate biomineralization. Science 336(6085): Adams PD, et al. (2010) PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66(Pt 2): Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66(Pt 4): Brünger AT, et al. (1998) Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54(Pt 5): McCoy AJ, et al. (2007) Phaser crystallographic software. J Appl Cryst 40(Pt 4): Sievers F, et al. (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7: Waterhouse AM, Procter JB, Martin DM, Clamp M, Barton GJ (2009) Jalview Version 2 a multiple sequence alignment editor and analysis workbench. Bioinformatics 25(9): of6

2 Fig. S1. Structural comparison of cefam20 with some atypical kinases and protein kinase A (PKA). The structures of cefam20, cell translocating kinase A (CtkA) [Protein Data Bank (PDB) ID code 3AKK], high persistence factor A (HipA) (PDB ID code 3TPE), actin-fragmin kinase (AFK) (PDB ID code 1CJA), phosphoinositide 3-kinase (PI3K) (PDB ID code 3LS8), and PKA (PDB ID code 3E8C) are shown in the same spatial orientation. The N- and C-lobes are colored in magenta and teal, respectively. The αc helices are highlighted in orange. Other structural elements are in white. cefam20 can be superimposed onto CtkA, HipA, AFK, PI3K, and PKA with rmsd over 200 Cα atoms of 3.5, 4.2, 3.9, 4.4, and 4.9 Å, respectively. Fig. S2. Electron density map of a glycosylated Asn. This composite omit map (contoured at 1.0 σ) depicts the glycosylated Asn113 in the Apo structure of cefam20. 2of6

3 Fig. S3. Comparison of the N- and C-lobes of cefam20 and PKA. (A) Structural superposition of the N-lobes of cefam20 (white, labels in black) and PKA (colored). (B) Structural superposition of the C-lobes of cefam20 (white) and PKA (colored). Fig. S4. Sequence alignment of the Fam20 family. Multiple sequence alignment of the kinase domains of human Fam20A, Fam20B, Fam20C, and cefam20. Conserved residues are highlighted. The alignment was generated using ClustalO (6) and Jalview (7). 3of6

4 Fig. S5. Kinetic analysis of human Fam20C and cefam20. Shown is the concentration dependence of ATP on the rate of phosphate incorporation into β28 40 (2.5 mm) using human Fam20C (0.075 μg/ml) and Mn 2+ ATP (A), human Fam20C (1 μg/ml) and Mg 2+ ATP (B), cefam20 (0.075 μg/ml) and Mn 2+ ATP (C), and cefam20 (1 μg/ml) and Mn 2+ ATP (D). The reactions were carried out for 10 min at 30 C. Data from three independent experiments are shown. Fig. S6. Mn/ADP-bound structure of cefam20. (A) Structural superposition of Apo (white) and ADP-bound (green) structures of cefam20 reveals little conformational changes on nucleotide binding. The ADP molecule and the two Mn 2+ ions are shown as sticks and spheres, respectively. (B) Composite omit map (contoured at 1.0 σ) confirms the presence of ADP and Mn 2+ ions in the Mn/ADP-bound cefam20 structure. Fig. S7. Staurosporine likely is not accommodated in the nucleotide-bound pocket of cefam20. cefam20 is depicted as a surface representation (green). Attempts to model staurosporine (spheres) into the nucleotide-binding pocket of cefam20 revealed several potential clashes. The complex structure between PKA (cyan) and staurosporine is shown on the right for reference (PDB ID code 1STC). 4of6

5 Fig. S8. cefam20 does not appear to contain an activation loop. cefam20 (Left) and PKA (Right) are shown as ribbon diagrams. The region following the DFG motif in cefam20 (brown) that corresponds to the activation loop in PKA (red) does not contain a phosphorylated residue (pt197 in PKA), suggesting that cefam20 does not require an activating phosphorylation event. Fig. S9. Structure modeling provides insight into Fam20A-related human disorders. The homology structure model of human Fam20A is shown as a ribbon diagram. Disease mutations are highlighted in red. 5of6

6 Table S1. Data collection and refinement statistics Apo (SeMet) ADP-bound Data collection Space group P P Cell dimensions, Å 78.79, , , , Wavelength, Å Resolution, Å R sym or R merge 7.4 (47.5) 7.3 (68.0) I/σI 18.4 (2.0) 20.6 (2.0) Completeness, % 98.8 (96.1) 96.9 (89.5) Multiplicity 4.7 (3.9) 6.6 (5.7) Wilson B-factor Refinement No. of reflections 64,343 18,179 R work /R free 21.2/ /25.3 No. of atoms Protein 14,118 7,041 Ligand/ion 2 59 Water Protein residue B-factors 1, Macromolecules Water 41.6 rmsd Bond lengths, Å Bond angles, Ramachandran Favored, % Outliers, % Each dataset was collected from a single crystal. Values in parentheses are for the highest-resolution shell. Table S2. Missense disease mutations in human Fam20C and Fam20A, and their corresponding residues in cefam20 Fam20C cefam20 Fam20A Val146 Leu173(Arg) Ile258(Asn) Ile165 Thr268(Met) Thr175 Gly280(Arg) Lys187 Pro328(Ser) Pro235 Gly379(Glu,Arg) Gly285 Gly331(Asp) Leu388(Arg) Val294 Asp451(Asn) Asp359 Arg549(Trp) Arg458 Gly280 in Fam20C is not conserved in cefam20. Rather, the cefam20 contains a Lys at this position. Nevertheless, the local environment surrounding the Gly in the human protein is hydrophobic, whereas Lys187 is surrounded by negatively charged residues (Asp132 and Glu135) in cefam20. 6of6