<Supplemental information>

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1 The Structural Basis of Endosomal Anchoring of KIF16B Kinesin Nichole R. Blatner, Michael I. Wilson, Cai Lei, Wanjin Hong, Diana Murray, Roger L. Williams, and Wonhwa Cho <Supplemental information> Protein expression and purification. The C-terminally His 6 -tagged human KIF16B PX domain was overexpressed in an E. coli methionine auxotrophic strain, B834(DE3)pLysS (Novagen), transformed with a pet21a vector (Novagen) containing the construct. Cultures were grown at 18 C in M9 minimal growth media supplemented with 2 mm MgSO 4, 0.4 % glucose, 25 µg/ml FeSO 4 7H 2 O, 1 µg/l vitamins (riboflavin, niacinamide, pyridoxine, thiamine), 100 µg/l ampicillin, a mixture of 19 L-amino acids (without L-methionine) added to a final concentration of 40 mg/l for each amino acid, and 40 mg/l seleno-l-methionine. When the optical density (600 nm) of the culture reached 1.0, they were induced by adding 0.3 mm isopropyl-dthiogalactopyranoside (Sigma). Cultures were grown further for 12 hours at 18 C. Cells were collected by centrifugation at 4500 rpm for 15 minutes, pellets were frozen in liquid N 2 and stored at -80 C. The pellets were thawed on ice in lysis buffer (20 mm Tris ph 8.0 at 25 C; 500 mm NaCl; 15 mm imidazole) and sonicated 3 minutes, then passed through a French press at 4 C. The lysate was clarified by centrifugation at x g for 30 minutes at 4 C and the supernatant then passed through a 0.2 µm filter. The His 6 -tagged human KIF16B PX was purified by passing the supernatant through 2-tandem 5 ml HisTrap FF columns (GE Healthcare). The bound protein was then eluted in imidazole buffer (20 mm Tris ph 8.0 at 25 C; 500 mm NaCl; 15 mm imidazole) using a linear gradient of mm imidazole over 20 column volumes. Fractions containing KIF16B-PX, determined by SDS-polyacrylamide gel electrophoresis, were pooled and diluted 8-fold with chilled ion-exchange buffer (20 mm Tris ph 8.0 at 25 C; 2 mm DTT). The solution was passed through a HiTrap SP 5ml HP cation exchange column (GE Healthcare), the column was rinsed with 10 volumes of ion-exchange buffer and eluted using a linear gradient of 0-1 M NaCl over 20 column volumes. Fractions containing KIF16B-PX, determined by SDS-polyacrylamide gel electrophoresis, were pooled and concentrated to 2 ml for injection onto a Superdex 75 16/60 gel-filtration column (GE Healthcare) equilibrated with gel-filtration buffer (20 mm Tris, ph 8.0 at 25 C; 100 mm NaCl; 2 1

2 mm DTT). Fractions containing KIF16B-PX, determined by SDS-polyacrylamide gel electrophoresis were pooled and concentrated to 7.4 mg/ml, flash frozen in 100 µl aliquots in liquid N 2 and stored at -80 C. Monolayer measurements. Surface pressure (π) was measured using a Wilhelmy plate as described previously (Bittova et al., 1999). After 5-10 µl of phospholipid solution in ethanol/hexane (1:9 (v/v)) was spread onto 5 ml of subphase (20 mm Tris-HCl, ph 7.4, 0.16 M KCl) to form a monolayer with a given π 0 the protein solution was injected into the subphase and Δπ was measured as a function of time. The maximal Δπ value at a given π 0 depended on the protein concentration and thus protein concentrations in the subphase were maintained high enough to ensure that the observed Δπ represented a maximal value. The protein concentration in the subphase that gave the saturating Δπ varied between 0.5 and 1 µg/ml depending on the π 0 of the lipid monolayer. The π c was determined by extrapolating the Δπ versus π 0 plot to the x-axis. It has been shown that Δπ caused by a protein is mainly due to the penetration of the protein into the lipid monolayer (Bittova et al., 1999). SPR measurements. All SPR measurements were performed at 23 C and in 20 mm Tris-HCl, ph 7.4, containing 0.16 M KCl using a lipid-coated L1 chip in the BIACORE X system as described (Stahelin and Cho, 2001). To the washed sensor chip surface, 90 µl of lipid vesicles (POPC/POPE/PI (77:20:3)) were injected at 5 µl/min to give a response of 5500 resonance units (RU). Similarly, a control surface was coated with POPC/POPE (80:20) vesicles, to give the same response as the active binding surface. Under our experimental conditions, no binding was detected to this control surface beyond the refractive index change for all proteins. Each lipid layer was washed with 10 µl of 50 mm NaOH three times at 100 µl/min. Typically, no decrease in lipid signal was seen after the first injection. Equilibrium SPR measurements were done at the flow rate of 5 µl/min to allow sufficient time for the R values of the association phase to reach near-equilibrium values (R eq ). After sensorgrams were obtained for 5 or more different concentrations of each protein within a 10-fold range of K d, each of the sensorgrams was corrected for refractive index change by subtracting the control surface response from it. R eq values were then plotted versus protein concentrations (C), and the K d value was determined by a nonlinear least-squares analysis of the binding isotherm using an equation, R eq = R max /(1 + K d /C). 2

3 Each data set was repeated three or more times to calculate average and standard deviation values. For kinetic SPR measurements, the flow rate was maintained at 30 µl/min for both association and dissociation phases. Supplemental Figure 1. (a). Section of electron density map calculated using experimental phases obtained after solvent-flattening, contoured at 1σ, showing PP II -α2 loop residues (b). The stereodiagram of the same electron density map. Supplemental Figure 2. Structural and sequence alignment of the KIF16B PX domain (NM_024704) with PX domains from p40 phox (accession number: NM_013416), PI3K-C2α (NM_002645), SNX8 (NM_013321), SNX9 (NM_016224), SNX11 (NM_013323) and SNX14 (NM_153816). KIF16B was aligned to p40 phox (r.m.s. deviation of 1.5 Å for 88 Cα) and PI3K- C2α was aligned to KIF16B (r.m.s. deviation of 1.3 Å for 83 Cα) using LSQMAN (Kleywegt and Jones, 1997). Other sequences were aligned using JALVIEW (Clamp et al., 2004). The secondary structure of KIF16B according to the definition by Kabsch and Sander (Kabsch and Sander, 1983) is shown above the alignment. The red stars above the alignment indicate positions of PtdIns(3)P-interacting residues in p40 phox. Residues L1247 and F1248 that form a hydrophobic stalk on the PP II -α2 loop of KIF16B along with G1250 are boxed with corresponding residues in the aligned sequences. Residue positions that are very conserved across the alignment are colored. <References> Bittova, L., Sumandea, M. and Cho, W. (1999) A structure-function study of the C2 domain of cytosolic phospholipase A2. Identification of essential calcium ligands and hydrophobic membrane binding residues. J Biol Chem, 274, Clamp, M., Cuff, J., Searle, S.M. and Barton, G.J. (2004) The Jalview Java alignment editor. Bioinformatics, 20, Kabsch, W. and Sander, C. (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers, 22, Kleywegt, G.J. and Jones, T.A. (1997) Detecting folding motifs and similarities in protein structures. Methods Enzymology, 277, Stahelin, R.V. and Cho, W. (2001) Differential roles of ionic, aliphatic, and aromatic residues in membrane-protein interactions: a surface plasmon resonance study on phospholipases A2. Biochemistry, 40,

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