Supporting Information

Transcription

1 Supporting Information A Streptavidin Binding Site Mutation Yields an Unexpected Result: An Ionized Asp128 Residue is Not Essential for Strong Biotin Binding. Loren Baugh, Isolde Le Trong, Patrick S. Stayton, Ronald E. Stenkamp &, Terry P. Lybrand* Departments of Bioengineering, Biological Structure, and Biochemistry, University of Washington, Seattle, Washington 98195, and Center for Structural Biology and Department of Chemistry, Vanderbilt University, Nashville, Tennessee S1

2 CRYSTALLOGRAPHIC METHODS, DATA COLLECTION AND REFINEMENT STATISTICS Crystallization. The D128N mutant of core streptavidin (1) was co-crystallized with biotin using hanging drop vapor diffusion techniques. Crystals were obtained by mixing protein (18 mg/ml in water with a two-fold molar excess of biotin. The reservoir solution was 0.1 M HEPES, ph 7.5 with 15% PEG Drops of protein and ligand solution were mixed with equal volumes of reservoir solution before equilibration. Crystals were transferred to a crystallization solution containing 30% ethylene glycol as a cryoprotectant before freezing at 100 K in a nitrogen stream for diffraction data collection. Diffraction data collection. Diffraction data for D128N were collected at Stanford Synchrotron Radiation Lightsource beamline 12-2 (λ = 1.0 Å) at 100K using a Pilatus detector and were processed using XDS (2). The space group for the D128N crystals is P2 1, with a streptavidin tetramer in the asymmetric unit. Data set statistics are shown in Table S1A. Structure solution and refinement. The initial structural model for D128N was obtained from an isomorphous structure, the biotin complex of the double mutant S45A/D128A of core-streptavidin (PDB entry 1MEP) (3). The structural models were refined using REFMAC-5 (4) in the CCP4 suite (5). R free (6) was calculated using 5% of the data in the test sets. All atoms were refined with anisotropic temperature factors. Riding hydrogen atoms were added to the models, and Babinet scaling was used to account for bulk solvent effects. Sigma A weighted Fo - Fc and 2 Fo - Fc electron density maps (7) were viewed with XtalView (8) and COOT (9) for graphical evaluation of the model and electron density maps. The final model for the biotin complex of D128N streptavidin contains four polypeptide chains (A: residues , B: , C: , D: ), four biotin molecules, 379 fully-occupied water molecules, and 28 partially occupied waters. MolProbity (10) was used for model validation. Table S1B contains refinement statistics for the structures. Coordinates and structure factors have been deposited for the D128N mutant in the Protein Data Bank with identifier 4YVB. Molecular Dynamics Simulations Starting coordinates for the D128N-biotin complex simulations were taken from the current x-ray structure. To model the complex at neutral ph conditions, all eight histidine residues in the tetramer were singly protonated. Hydrogen atoms were added to all protein heavy atoms using the Leap module in AMBER The full complex was solvated in a truncated octahedral box with 12,212 water molecules, and one sodium counterion was included to maintain charge neutrality for the system. We used the simulation methods and protocol reported previously for solution phase simulations of WT streptavidin. 12 Briefly, all calculations were performed using the AMBER ff99 force field 13,14 with modifications by Simmerling and co-workers, 15 the SPC/E water model, 16 a sodium cation model from Åqvist 17 and biotin parameters from previous work by Israilev and co-workers. 18 Force calculations were performed with periodic boundary conditions, a 9.0 Å cutoff on real space interactions, smooth particle-mesh Ewald for long-range electrostatics, 19 and a homogeneity assumption to approximate the contributions of long-range Lennard-Jones forces to the virial tensor. Covalent bonds involving hydrogen atoms were constrained at equilibrium bond lengths using the SHAKE algorithm 20 and the rigid SPC/E water internal geometry was constrained with the SETTLE algorithm. 21 The simulation temperature was maintained at 298K using a Langevin thermostat 22 with a 3 ps -1 collision frequency. All energy minimizations and dynamics were performed with the PMEMD module of AMBER Prior to beginning MD simulations, all hydrogen atoms, water molecules and sodium atoms were first relaxed by 2000 steps of steepest-descent energy minimization while crystallographically resolved protein atoms were held in place by 1000 kcal/mol-å 2 position restraints. The protein heavy atoms were then energy minimized while solvent particles were tightly restrained to their new positions, and finally all components of the system were energy-minimized with no restraints. Next, restrained dynamics of the system were conducted for a total of 450 ps, beginning with a 0.5 fs time step in the constant volume, constant temperature ensemble and 16.0 kcal/mol-å 2 restraints on all crystallographically observed protein atoms. The restraints were gradually reduced to 1.0 kcal/mol-å 2 over the first 150 ps before switching to the constant pressure ensemble, increasing the time step to 1.5 fs, and reducing the restraints to kcal/mol-å 2 over the next 300 ps. Production dynamics were run in the constant pressure ensemble with a 1.5 fs time step for 500 ns with no position restraints. Biotin Dissociation Rate Measurements For the cold-case radiometric assay, solutions of 10 nm 3 H-biotin and 30 μm D128N streptavidin in 50 mm sodium phosphate buffer and 100 mm NaCl at ph 7.0 were equilibrated at 25 C for 2 h, then a large excess of unlabeled biotin (50 μm final concentration) was added and mixed rapidly. 200 μl aliquots were removed periodically and immediately ultrafiltered using chilled, 30k Microcon filters. The filtrate was counted to quantify the amount of 3 H-biotin released as a function of time. The dissociation rate constant, k off, was determined by fitting the dissociation data (Figure S2) to a one-term exponential decay. S2

3 Table S1. A. Diffraction data statistics unit cell a, b, c (Å), β ( ) space group P2 1 Overall Inner Outer Shell Shell Low resolution limit (Å) High resolution limit (Å) Rmerge Number unique reflections <I/σ(I)> Completeness (%) Redundancy B. Refinement statistics Resolution Å Number of reflections, working set Number of reflections, test set 5081 R cryst, all data R cryst, working set R free, test set Bond RMS (restrained) Å Number of protein atoms 3670 Number of ligand and solvent atoms 471 Ramachandran outliers as 0.2% determined by Molprobity Ramachandran favored 97.9% PDB accession code 4YVB S3

4 FIGURE S1. Correlation of equilibrium constant K a versus biotin dissociation rate k off for a collection of conservative point mutations. REFERENCES 1. Sano, T., Pandori, M. W., Chen, X., Smith, C. L., and Cantor, C. R. (1995) Recombinant core streptavidins, J. Biol. Chem. 270, Kabsch, W. (2010) XDS, Acta Cryst. D66, Hyre, D., Le Trong, I., Merritt, E. A., Eccleston, J. F., Green, N. M., Stenkamp, R. E., and Stayton, P. S. (2006) Cooperative hydrogen-bond interactions in the streptavidin-biotin system., Protein Science 15, Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Refinement of macromolecular structures by the maximum-likelihood method., Acta Cryst. D53, Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., Keegan, R. M., Krissinel, E. B., Leslie, A. G. W., McCoy, A., McNicholas, S. J., Murshudov, G. N., Pannu, N. S., Potterton, E. A., Powell, H. R., Read, R. J., Vagin, A., and Wilson, K. S. (2011) Overview of the CCP4 suite and current developments, Acta Cryst. D67, Brünger, A. T. (1993) Assessment of phase accuracy by cross validation: the free R value. Methods and applications, Acta Cryst. D49, Read, R. J. (1986) Improved Fourier Coefficients for Maps Using Phases from Partial Structures with Errors, Acta Cryst. A42, McRee, D. E. (1999) XtalView Xfit - A versatile program for manipulating atomic coordinates and electron density., J. Structural Biology. 125, Emsley, P., and Cowtan, K. (2004) Coot: model-building tools for molecular graphics, Acta Cryst. D60, Davis, I. W., Leaver-Fay, A., Chen, V. B., Block, J. N., Kapral, G. J., Wang, X., Murray, L. W., Arendall, W. B. I., Sinoeyink, J., Richardson, J. S., and Richardson, D. C. (2007) MolProbity: all-atom contacts and structure validation for proteins and nucleic acids, Nucleic Acids Research 35, W375-W383. S4

5 11. Case, D. A., Darden, T. A., Cheatham III, T. E., Simmerling, C. L., Wang, J., Duke, R. E., Luo, R., Walker, R.C., Zhang, W., Merz, K. M., Roberts, B., Hayik, S., Roitberg, A., Seabra, G., Swails, J., Götz, A. W., Kolossváry, I., Wong, K. F., Paesani, F., Vanicke, J., Wolf, R. M., Liu, J., Wu, X., Brozell, S. R., Steinbrecher, T., Gohlke, H., Cai, Q., Ye, X., Wang, J., Hsieh, M.-J., Cui, G., Roe, D. R., Mathews, D. H., Seetin, M. G., Salomon-Ferrer, R., Sagui, C., Babin, V., Luchko, T., Gusarov, S.,Kovalenko, S., and Kollman, P. A. (2012) AMBER12, University of California, San Franciso. 12. Cerutti, D. S., Le Trong, I., Stenkamp, R. E., and Lybrand, T. P. (2009) Dynamics of the streptavidin-biotin complex in solution and in its crystal lattice: Distinct behavior revealed by molecular simulations. J. Phys. Chem. B 113, Cornell, W. D., Cieplak, P., Bayly, C. I., Gould, I. R., Merz, K. M., Jr., Ferguson, D. M., Spellmeyer, D. C., Fox, T., Caldwell, J. W., and Kollman, P. A. (1995) A Second Generation Force Field for the Simulation of Proteins, Nucleic Acids, and Organic Molecules. J. Am. Chem. Soc. 117, Wang, J., Cieplak, P., and Kollman, P. A. (2000) How well does a restrained electrostatic potential (RESP) model perform in calculating conformational energies of organic and biological molecules? J. Comput. Chem 21, Hornak, V., Abel, R., Okur, A., Strockbine, B., Roitberg, A., and Simmerling, C. (2006) Comparison of multiple Amber force fields and development of improved protein backbone parameters. Proteins 65, Berendsen, H. J. C., Grigera, J. R., and Straatsma, T. P. (1987) The missing term in effective pair potentials. J. Phys. Chem. 91, Åqvist, J. (1990) Ion-water interaction potentials derived from free energy perturbation simulations. J. Chem. Phys. 94, Israilev, S., Stepaniants, S., Balsera, M., Oono, Y., and Schulten, K. (1997) Molecular dynamics study of unbinding of the avidin-biotin complex. Biophys. J. 72, Essmann, U., Perera, L., Berkowitz, M. L., Darden, T., Lee, H., Pedersen, L. H. (1995) e A smooth particle mesh Ewald method. J. Chem. Phys. 103, Ryckaert, J. P., Ciccotti, G., Berendsen, H. J. C., Hirasawa, K. (1977) Numerical Integration of the Cartesian Equations of Motion of a System with Constraints: Molecular Dynamics of n-alkanes. J. Comput. Phys. 23, Miyamoto, S., Kollman, P. A. (1992) Settle: An analytical version of the SHAKE and RATTLE algorithm for rigid water models. J. Comput. Chem. 13, Izaguirre, J. A., Catarello, D. P., Wozniak, J. M., Skeel, R. D. (2001) Langevin stabilization of molecular dynamics. J. Chem. Phys. 114, S5