Dynamics connect substrate recognition to catalysis in protein kinase A

Transcription

1 Supplementary Information for Dynamics connect substrate recognition to catalysis in protein kinase A Larry R. Masterson 1,2, Cecilia Cheng 3, Tao Yu 2, Lei Shi 2, Marco Tonelli 3, Yi Wang 2, Susan S. Taylor 4*, and Gianluigi Veglia 1,2 Departments of 1 Biochemistry, Molecular Biology, and Biophysics and 2 Chemistry, University of Minnesota, Minneapolis, Minnesota , and the 3 Department of Chemistry and Biochemistry, University of San Diego, San Diego CA , and 4 National Magnetic Resonance Facility at Madison, Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin

2 Supplementary Results Table 1. Data collection and refinement statistics for Protein Data Bank accession number 3O7L. Phospholamban1-19 : C-subunit complex Data collection Space group P6 1 Cell dimensions a, b, c (Å) 92.2, 92.2, α, β, γ ( ) 90.0, 90.0, Resolution (Å) 2.8 R sym or R merge (0.456) * I / σi 38.8 (5.1) Completeness (%) 99.5 (100) Redundancy 10.5 (10.1) No. reflections No. molecules per 2 asymmetrical unit Refinement Resolution (Å) R work / R free / Number of protein atoms 5210 Number of water molecules 21 Wilson B-factors (Å 2 ) 51.6 R.m.s. deviations Bond lengths (Å) Bond angles ( ) Ramachandran angles (%) most favored 85.8 disallowed none *Highest-resolution shell ( Å) is shown in parentheses. 2

3 Table 2. Mean and trimmed mean of backbone amide 15 N R 1, R 2, and H-X NOE for PKA-C. Parameter Apo AMP-PNP Bound AMP-PNP/PLN 1-20 Bound R 1 (s -1 ) ± ± ± Trimmed R 1 (s -1 ) ± ± ± R 2 (s -1 ) 27.8 ± ± ± 2.9 Trimmed R 2 (s -1 ) 51.9 ± ± ± 3.2 Trimmed R 2 /R ± ± ± 4.2 H-X NOE 0.71 ± ± ± 0.32 Trimmed H-X NOE 0.76 ± ± ± 0.16 H-X NOE (Glycine-loop) 0.76 ± ± ± 0.05 H-X NOE (DFG/Activation/Peptide-loop) 0.65 ± ± ± 0.10 % of Residues with R ex >8 Hz 4.9% 15.7% 12.6% 3

4 Fig. 1: Steady-state phosphorylation kinetics of (a) PLN 1-20 and (b) full-length PLN (AFA mutant which is monomeric) in DMPC/DHPC isotropic bicelles. Michaelis-Menten pararmeters provided in the plots indicate no major differences in catalytic efficiency between the two substrates. Therefore, the use of the peptide corresponding to the cytoplasmic portion of PLN was used as a model for the full-length protein. 4

5 Fig. 2: Details of stabilizing interactions between PKA-C and PLN Hydrophobic packing (a) between the apo enzyme of PKA-C and the N-terminal region of the PLN 1-19 peptide was observed in the crystal structure. However, the conserved recognition sequence of the substrate (residues 12-17) makes key electrostatic interactions in the ternary complex (b). The positioning of the γ-po4 of AMP-PNP toward the hydroxyl of Ser16, and the interaction of Gly200 with Thr17 of PLN 1-19 are also shown in a. The interactions of the essential P-2 (Arg14) and P-3 (Arg13) of PLN 1-19 are depicted in b. 5

6 Fig. 3: Mapping of B-factors for the (a) PKA-C apo state, (b) PKA-C ternary complex, and (c) PLN 1-19 bound to PKA-C. 6

7 Fig. 4: Nuclear spin dynamics of 2 H, 15 N-labeled PLN 1-20 in the free state (black) and in the ternary complex with PKA-C (red). (a) H-X NOE, (b) R 2, (c) R 1, and (d) R 2 /R 1 values are plotted for both of these states. 7

8 Fig. 5: Nuclear spin dynamics of 2H, 15N-labeled PKA-C along its pathway to the ternary complex: (a) Apo-enzyme, (b) nucleotide bound, and (c) bound to both nucleotide and PLN

9 Fig. 6: Amide resonance inverse peak heights mapped for PKA-C in the (a) Apo, (b) AMP-PNP bound, and (c) AMP-PNP/PLN 1-20 bound states. Large values of inverse peak heights are indicative of conformational exchange on the µs-ms timescale (R ex ) and qualitative agreement between the residues displaying R ex and large inverse peak heights are expected to occur. 9

10 Fig. 7: Conformational interconversion of the highly conserved C-helix and Mg 2+ -loop in the AMP-PNP (shown in blue) bound state of PKA-C. Residues highlighted in red spheres are those which experienced significant R ex contributions. Both regions depicted here exhibited dynamics on a similar timescale of ~25 s

11 Fig. 8: Details of residues which were synchronous with opening and closing (black) and those which were not (red). Dynamics of the AMP-PNP bound (a) and AMP-PNP/PLN 1-20 bound (b) states are shown. 11

12 Supplementary Methods Peptide synthesis. Peptides were synthesized using standard Fmoc chemistry starting with Fmoc- Glu(OtBu)-PEG-PS resin (0.4 g, 0.5 mmol/g). Side chain protecting groups were 2,2,5,78- pentamethylchroman-6-sulfonyl (Pmc) for arginine, N ω -triphenylmethyl (Trt) for asparagine and glutamine, tert-butyl etster (OtBu) for glutamic acid, and tert-butyl ethers (tbu) for serine, threonine, and tyrosine. Deprotection of the resin-bound peptide was done using Reagent K (82.5% TFA, 5% phenol, 5% thioanisole, 2.5% 1,2-ethandiol, and 5% water) for 3 hours at 298 K. The resin mixture was washed three times (2 ml each) using the same cocktail and filtrate was collected. The peptide was precipitated overnight at 273 K in 80 ml of diethyl ether, then collected by centrifugation and washing the pellet 3 times with 30 ml of diethyl ether. The crude peptide was then purified by preparative HPLC using a Waters C18 reversed-phase cartridge (2.5 x 10 cm, 15 µm, 300 Ǻ) with 0.1% TFA and CH 3 CN as eluents and detection at 220 nm. A linear gradient of 100:0 to 70:30 over 30 minutes at 5 ml/min was used. Purities of pooled fractions were >90% as determined by analytical HPLC using a Vydac C18 column (0.46 x 25 cm) and confirmed by ESI-TOF (PLN 1-19, calculated m/z, found m/z). Aliquots of peptide were prepared in water from the same purified stock, lyophilized, and concentrations of these solutions were verified by amino acid analysis. Kinetic Assays. The steady-state enzymatic rate of PLN 1-20 phosphorylation was measured spectrophotometrically at 299 K following the procedure by Cook, et al 1. The values of V max, k cat, and K M were determined from non-linear fitting of initial velocities versus substrate concentration according to: 12

13 v = V K [ S] [ S] max (1) M + Where v is the initial velocity, [S] is the concentration of substrate, V max is the maximum velocity for the reaction, and K M is the Michaelis-Menten constant. Determination of k cat values was made by dividing V max by the total enzyme concentration. Reactions for full-length PLN were conducted using isotropic bicelles (q=0.5, 5% C L ) composed of DMPC/DHPC (Avanti Polar Lipids, Inc.). X-ray Crystallography. Optimization of crystallization conditions The mixture containing PKA-C/PLN 1-19 /AMP-PNP was incubated on ice then filtered with 0.2 μm centrifugal devices (Life Sciences). Crystallization trials were setup on CrystalClear Duo sitting drop plates (Douglas Instruments) using the Oryx8 Protein Crystallization Robot (Douglas Instruments, UK). Commercial HT crystal screens were used to determine initial crystallization conditions (PACT, JCSG, Proplex, Structure Screen I&II, Clear Strategy I, and Clear Strategy II, Molecular Dimensions). Screens were done with 0.5 μl drops, testing both 50% and 70% protein concentrations and 4 C and 22.5 C temperatures. Initial crystal hits were optimized using the Oryx8 robot. The final condition was 4.3 mg/ml PKA-C, 0.09 M sodium malonate (ph 7.0), 15.5% PEG 3350, which produced μm sized tetragonal crystals at 22.5 C. Data refinement All ambiguous main chains and side chains were removed and manually rebuilt using Coot 2, followed by iterative cycles of structure refinement using REFMAC in the CCP4 suite 3. Upon inspection of the F o -F c maps, electron density in the N-terminal lobe for one of the two molecules did not match with the model so residues were removed. Both peptide and 13

14 nucleotide appeared to be absent in this molecule, so an apo form was assumed. Alignment of the large lobes of the apo structure (PDB code 1J3H) 4 and our second molecule to obtain a rough estimate of the small lobe position. Coot was utilized in subsequent model building and REFMAC for refinement. The phospholamban peptide was not manually built until all atoms in PKA-C were satisfactorily refined. TLS refinement 5 was implemented for each lobe. Simulated annealing via the program Phenix 6,7 was used in the final stages of refinement because geometric restraints were too loose with REFMAC. Water molecules were manually incorporated. 14

15 References 1. Cook, P. F., Neville,M.E., Vrana,K.E., Hartl,F.T. and Roskoshi,R. Adenosine cyclic 3'5'- monophosphate dependent protein kinase: Kinetic mechanism for the bovine skeletal muscle catalytic subunit. Biochemistry. 21, (1982). 2. Emsley, P. and Cowtan,K. Coot: Model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, (2004). 3. Murshudov, G. N., Vagin,A.A. and Dodson,E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, (1997). 4. Akamine, P., Madhusudan, Wu,J., Xuong,N.H., Ten Eyck,L.F. and Taylor,S.S. Dynamic features of camp-dependent protein kinase revealed by apoenzyme crystal structure. J. Mol. Biol. 327, (2003). 5. Winn, M. D., Isupov,M.N. and Murshudov,G.N. Use of TLS parameters to model anisotropic displacements in macromolecular refinement. Acta Crystallogr. D Biol. Crystallogr. 57, (2001). 6. Adams, P. D., et al. PHENIX: Building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, (2002). 7. Adams, P. D., et al. Recent developments in the PHENIX software for automated crystallographic structure determination. J. Synchrotron Radiat. 11, (2004). 15