Supplementary Figure 1 (a
) Monomer of the KChIP1/T1N fusion23
. KChIP1 amino acids are in slate blue with the exception of the H10 helix which is green. T1N (residues 1-21) (light orange) and N-terminal residue T1N are indicated. Calcium ions are shown as magenta spheres. (b)
Ribbon diagram of a superposition of the Frequenin EF2 (dark blue), which is calcium bound, and KChIP1 EF2 in the KChIP1/Kv4.3 T1 domain complex (cyan). T1N (orange) is shown. Frequenin side chains that participate in calcium co-ordination are shown in stick representation. (c
) Frequenin structure27
(skyblue). Three polyethylene glycol molecules bound in the hydrophobic groove are shown as sticks with carbon (skyblue) and oxygen (red) atoms indicated. Calcium ions are shown as magenta spheres.
Supplementary Figure 2 T1N helix and linker structure. (a) 2Fo-Fc omit map (green) contoured at 1.0 σ showing the density for the T1N aromatic residues (orange) and KChIP1 (cyan). Labels indicate select residues. Similar quality density was seen for Trp19. (b) NCS averaged 2Fo-Fc omit map density (green) derived for the T1N linker contoured at 1.0 σ from a 4.2 Å dataset. This dataset was used as the map shows better density for this region. (c) Selenomethionine anomalous signal shown from NCS averaged anomalous difference maps at 4.2 Å. Maps are contoured at 3.5 σ (pink), 4.0 σ (hot pink), and 4.5 σ (black). Kv4.3 and KChIP1 elements are labeled and colored as before. Methionine side chains are shown as sticks.
Supplementary Figure 3 Solution analysis of the KChIP1/Kv4.3 T1 complex. (a) SAXS data (top) and distribution function plot (bottom) for the complex. s = 4πsin(θ)/λ, where λ is the X-ray wavelength. (b) Top two panels, ab initio model of the KChIP1/Kv4.3 T1 complex (spheres). Bottom panel, comparison of the data and the calculated scattering from the ab initio model (χ2 = 3.46). (c), Dynamic light scattering data for the KChIP1/Kv4.3 T1 domain complex (16 mg ml-1 in a buffer of 50 mM KCl, 1 mM CaCl2, 50 μM ZnCl2, 30 mM DTT, 10 mM Na+-HEPES, pH 7.4) shows a single, monodisperse species in solution. The measured molecular weight, 148 kDa is in excellent agreement with the expected molecular weight of a (KChIP1)4(Kv4.3 T1 domain)4 complex (151 kDa). (d, e, and f) Comparison of the SAXS data with the calculated profiles for d, the T1 tetramer alone (χ2= 12.41), e, KChIP1 alone (χ2= 10.32), and f, the hexadecamer seen in the asymmetric unit of the KChIP1/Kv4.3 T1 domain crystal structure (χ2= 9.15).
Supplementary Figure 4 (a) Superdex 200 gel filtration elution profiles for purified KChIP1 37-216 and EF mutants. Molecular weights were determined using six proteins of known mass as standards. EF2, EF4 and EF2/EF4 mutants behave similar to wild-type (blue line). EF3 behaves similar to the EF2/EF3 mutant (green line). EF3/EF4 behaves similarly to EF2/EF3/EF4 (red line). Expected molecular weights are: KChIP1 27-216, 21 kDa; DnaK 70 kDa. (b) Top, 15% SDS-PAGE of KChIP1, EF3/EF4, EF2/EF3/EF4, and wild-type peak fractions from gel filtration. Bottom, Western-blot of the top panel using an anti-DnaK antibody. (c) Dynamic Light Scattering analysis of wild-type KChIP1 37-216 in the presence and absence of calcium.
Protein expression and purification
Kv4.3 1-143/KChIP1 37-216 complex
Rat Kv4.3 1-1431 was cloned into a modified pET28 vector containing a His6 tag, maltose binding protein, and a TEV cleavage site on the N-terminus of the protein sequence (pET28-HMT)2. hKChIP1 (residues 37-216) (IMAGE clone) was cloned into pEGST vector lacking the GST tag3. Vectors were co-transformed in BL21(DE3)pLysS E. coli, grown in 2YT media at 37° C, and induced (0.6-1.0 OD600 nm) with 0.4 mM IPTG for 4 hrs. Cells were resuspended in lysis buffer (150 mM KCl, 10% w/v sucrose, 25 mM n-octyl-β-D-glucopyranoside, 20 ng/μl lysozyme, 25 ng/μl Dnase I, 5 mM MgCl2, 1 mM CaCl2, 50 μM ZnCl2, 1 mM PMSF, 100 mM Tris-HCl, pH 8.8), disrupted by sonication, and centrifuged (30’, 31000 g, 4 °C, Beckman JA 25.50). Protamine sulfate at a final concentration of 2 mg/ml was added to the supernatant, incubated on ice for 10’, centrifuged (15’, 12000g, 4 °C, Beckman JA 12), and the supernatant loaded into a Poros20MC Ni2+ column (Applied Biosystem) equilibrated with buffer A (250 mM KCl, 1mM CaCl2, 50 μM ZnCl2, 10 mM HEPES-NaOH, pH 7.4). Protein was eluted in buffer A supplemented with 300 mM imidazole using a linear gradient over one column volume. 10 mM β-mercaptoethanol was added to the protein solution and the affinity tag was removed with TEV protease4 (overnight, room temperature). Cleavage reaction was dialyzed against buffer B (50 mM KCl, 1 mM CaCl2, 50 μM ZnCl2, 10 mM β-mercaptoethanol, 10 mM TRIS-HCl, pH 8.8), loaded onto a HiLoad Q 16/10 column (GE Healthcare), and eluted in a linear gradient from 50 mM -0.5 M KCl over twelve column volumes. The complex was further purified on an amylose column (New England Biolabs) in buffer A supplemented with 10 mM β-mercaptoethanol to remove traces of MBP and followed by gel filtration on a Superdex 200 (GE Healthcare) in buffer A + 10 mM β-mercaptoethanol. Protein was concentrated to ~15 mg/ml and stored at 4° C in 50 mM KCl, 1 mM CaCl2, 50 μM ZnCl2, 30 mM DTT, 10 mM HEPES-NaOH, pH 7.4. Point mutations were made by QuikChange (Stratagene) and expression and purification were performed as described for the wild-type complex.
Selenium methionine complex
Selenium methionine substituted complex was expressed in BL21(DE3)pLysS using autoinduction media5 (40 hrs., 37°C). Purification was performed as described for the native complex except that all buffers contained 5 mM methionine to prevent selenium oxidation.
hKChIP1 37-216 and EF mutants were cloned into pET28-HMT. Expression and purification were performed as described for the complex with the difference that buffers did not contain Zn2+ and β-mercaptoethanol. Proteins were stored at 4° C in 250 mM KCl, 1 mM CaCl2, and 10 mM HEPES-NaOH, pH = 7.4.
rKv4.3 1-143 and hKChIP1 (1-216) or EF mutants were co-expressed in E.coli and lysates were prepared as described above. Cell lysates were loaded on Amylose (New England Biolabs) resins equilibrated with buffer A (250 mM KCl, 1 mM KCl, 50 μM ZnCl2, and 10 mM HEPES-NaOH, pH 7.4). The complex was eluted in buffer A supplemented with 10 mM maltose and samples were loaded on 15% SDS-PAGE and stained with comassie brilliant blue R250.
Crystallization and Structure Determination
Prior to crystallization samples were centrifuged (30’, 70000g, 4°C, Beckman TLA 120.2). Dynamic light scattering (Dynapro, Wyatt Technologies) indicated that samples used for crystallization had <15% polydispersion. Crystals of the Kv4.3 1-143/KChIP1 37-216 K160A/K167A and selenomethionine derivative complexes were obtained at room temperature by hanging drop vapor diffusion using 1 μl of protein solution and 1 μl of 10-18% PEG 3000, 200 mM NaCl, 100 mM Bis-Tris, pH 6.5. Crystals used for data collection appeared in 7-10 days. Crystals were flash frozen in mother liquor supplemented with 20% glycerol. Data were collected at <100K at the Advanced Light Source (ALS) beamline 8.3.1 at the Lawrence Berkeley National Laboratories (LBNL). Diffraction images were indexed using MOSFLM6 and DENZO7 and scaled using SCALA8. Initial phases were obtained by using molecular replacement MOLREP9 with the Kv4.3 T1 domain tetramer (1S1G). Data collection wavelengths were 1.116 Å for native data and 0.9796 Å for SeMet data. Manual rebuilding correctly identified the position of the KChIP H2 helix. At this stage, we used the KChIP1 monomer (1S6C) with residues 159-189 deleted for molecular replacement. Refinement was carried out by iterative cycles of manual building in O910 and restrained refinement in Refmac58. Residues 3-20 and 39-140 of the T1 domain and residues 38-158 and 171-211 were refined using tight NCS restraints; the remaining loops were refined with medium restraints. Omit maps were generated using CNS11. Figures were displayed and rendered with Pymol12. The Ramachandran statistics for the structure are as follows: Most favored regions, 82.4 %; Additional allowed regions, 16.1%; Generously allowed regions, 1.5%, Disallowed regions, 0.0 %.
Circular Dichroism and Dynamic Light Scattering analysis
For the CD and DLS experiments, protein concentrations were determined by absorbance13.
Small angle X-ray scattering (SAXS)
Small-Angle X-Ray Scattering (SAXS) data were collected at ALS beamline 12.3.1 LBNL Berkeley, California. Complexes were 8 mg ml-1 and 16 mg ml-1 in a buffer containing 50 mM KCl, 1 mM CaCl2, 50 μM ZnCl2, 30 mM DTT, 10 mM Na+-HEPES, pH 7.4. Buffer scattering was subtracted and low- and high-angle curves were merged over the s range of 0.01-0.3 Å-1 using PRIMUS14. Radius of gyration (Rg) was evaluated using the Guinier approximation and from the entire scattering curve with GNOM15. GNOM also provides the distribution function p(r). To determine Dmax, the p(r) function was computed while constraining the function to go to zero at rmax, where rmax was varied from 100 to 200 Å in 5 Å increments. The rmax that yielded the highest “total estimate” value in combination with a plausible p(r) function was taken as Dmax. Ab initio models were generated using GASBOR16. Ten independent runs were performed imposing P1, P2 and P4 symmetry. Models were aligned and averaged using DAMAVER17. Crystal structures of Kv4.3 29-143 (1S1G) and KChIP1 37-216 (1S1E) were manually fit into the model using XtalView18. Theoretical scattering curves and fits to the experimental scattering curve from atomic coordinates were calculated using CRYSOL19.
Wild-type KChIP1 and EF3/EF4 and EF2/EF3/EF4 mutants were run on a 15% SDS-PAGE and afterwards transferred to a nitrocellulose membrane. Samples were probed with anti-DnaK antibodies (Stressgen biotechnologies) and detected with ECL (GE Healthcare).
1. Tsaur, M.L., Chou, C.C., Shih, Y.H. & Wang, H.L. Cloning, expression and CNS distribution of Kv4.3, an A-type K+ channel alpha subunit. FEBS Lett 400, 215-20 (1997).
2. Van Petegem, F., Clark, K.A., Chatelain, F.C. & Minor, D.L., Jr. Structure of a complex between a voltage-gated calcium channel beta-subunit and an alpha-subunit domain. Nature 429, 671-5 (2004).
3. Kholod, N. & Mustelin, T. Novel vectors for co-expression of two proteins in E. coli. Biotechniques 31, 322-3, 326-8. (2001).
4. Kapust, R.B. et al. Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng 14, 993-1000. (2001).
5. Studier, F.W. Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41, 207-34 (2005).
6. Leslie, A.G.W. Recent changes to the MOSFLM package for processing film and image plate data. Joint CCP4 + ESF-EAMCB Newsletter of Protein Crystallography 26(1992).
7. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Meth. Enzy. 276, 307-326 (1997).
8. Collaborative Computational Project, N. The CCP4 suite: Programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50, 760-763. (1994).
9. Vagin, A. & Teplyakov, A. MOLREP: an automated program for molecular replacement. J. Appl. Cryst. 30, 1022-1025 (1997).
10. Kleywegt, G.J. & Jones, T.A. Efficient rebuilding of protein structures. Acta Crystallogr D Biol Crystallogr 52, 829-32 (1996).
11. Brunger, A.T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54, 905-21. (1998).
12. DeLano, W.L. The PyMOL Molecular Graphics System. (DeLano Scientific, San Carlos, CA, 2002).
13. Edelhoch, H. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6, 1948-54. (1967).
14. Konarev, P.V., Volkov, V.V., Sokolova, A.V., Koch, M.H.J. & Svergun, D.I. PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J. Appl. Cryst. 36, 1277-1282 (2003).
15. Svergun, D.I. Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J. Appl. Cryst. 25, 495-503 (1992).
16. Svergun, D.I., Petoukhov, M.V. & Koch, M.H. Determination of domain structure of proteins from X-ray solution scattering. Biophys J 80, 2946-53 (2001).
17. Volkov, V.V. & Svergun, D.I. Uniqueness of ab initio shape determination in small-angle scattering. J. Appl. Cryst. 36, 860-864 (2003).
18. McRee, D.E. XtalView/Xfit--A versatile program for manipulating atomic coordinates and electron density. J Struct Biol 125, 156-65. (1999).
19. Svergun, D.I., Barberato, C. & Koch, M.H.J. CRYSOL - a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J. Appl. Cryst. 28, 768-773 (1995).