Supporting Information Boehr et al. 10.1073/pnas.0914163107 SI Text Materials and Methods. R 2 relaxation dispersion experiments. 15 NR 2 relaxation dispersion data measured at 1 H Larmor frequencies of 500 and 800 MHz were fit with the following series of equations describing conformational exchange between two sites, A and B: R 2 ð1 τ CP Þ ¼ R 0 2 in which, þ 1 2 k ex 1 cosh 1 ½D τ þ coshðη þ Þ D cosðη ÞŠ CP [S1] D ¼ 1 2 1 þ ψ þ 1 2; 2Δω2 ðψ 2 þ ζ 2 Þ 1 2 [S2] η ¼ τ h i CP 1 2; ψ þðψ 2 þ ζ 2 Þ 1 2 [S3] 2 where ψ ¼ k 2 ex Δω 2, ζ ¼ 2Δωk ex ðp A p B Þ, τ cp is the time between successive 180 pulses in the CPMG pulse train, R 0 2 is the R 2 relaxation rate in the absence of conformational exchange, p A and p B are the populations of the ground- and excited-state conformations respectively (p A þ p B ¼ 1), and Δω is the chemical shift difference between substates A and B. Rate constants for the ground-to-excited state (k AB ) and excited-to-ground state (k BA ) transitions can be determined by p B :k ex and p A :k ex, respectively. Residues reporting on the same conformational exchange process have similar k ex and p A p B values, but different Δω values. In the initial fitting procedure, residues were placed into one of three clusters the cofactor-binding cleft, the active-site loops and the C-terminal associated region (Table S1). The k ex and p A p B values for the cofactor-binding cleft and active-site loop clusters were nearly identical and so only two clusters, the combined cofactor-binding cleft/active-site region and the C-terminal associated region, were used in the final fits. The two clusters showed significantly different k ex and p A p B values (Table S2). The Δω values are listed in Table S3. However, in a few select cases, χ 2 cluster χ2 individual > 2, so we report only the Δω value for the individual fit. The sign of Δω was determined by comparing HSQC and HMQC spectra (1). Temperature dependence of μs-ms timescale dynamics. The enthalpy (ΔH) and entropy ( TΔS) differences between the excited- and ground-state conformations were estimated from the temperature dependence of the conformational exchange equilibrium constant using van t Hoff analysis, ln K ¼ ΔH RT þ ΔS R; [S4] where K ¼ k BA k AB. A linear plot of ln K vs. 1 T will yield a slope of ΔH R and a y-intercept of ΔS/R. Reported errors are based on jackknife simulations. The activation barriers were estimated using transition-state theory and the Eyring equation, lnðk TÞ ¼ ΔH RT þ lnðk B hþþδs R; [S5] where k is the rate constant measured from R 2 relaxation dispersion (k AB ), k B is Boltzmann s constant, h is Planck s constant, R is the universal gas constant, T is temperature, ΔS is the entropy of activation, and ΔH is the enthalpy of activation. A linear plot of ln (k T) vs. 1 T will yield a slope of ΔH R and a y-intercept of lnðk B hþþδs R. An alternative to transition-state theory is the phenomenological Ferry law (2, 3) that describes a lower energy barrier with a rough enthalpic surface ln k ¼ ln C ðδh 0 RTþ < H2 > ðrtþ 2 Þ; [S6] where k is the rate constant measured from R 2 relaxation dispersion (k AB ), C is a constant, R is the universal gas constant, T is temperature, ΔH 0 is the enthalpy of activation associated with the smooth Arrhenius-like barrier, and <H 2 > 1 2 is the enthalpy due to the ruggedness of the barrier. If ΔH 0 is set to 0, a linear plot of ln k vs. 1 ðrtþ 2 will yield a slope of <H 2 > from which <H 2 > 1 2 can be calculated. 1 H- 15 N steady-state heteronuclear NOE measurements. Two spectra, with or without proton pre-saturation, were acquired in an interleaved manner to minimize systematic error. A total of three pairs of spectra were recorded for E:THF at 306 K and analyzed according to (4). 1. Skrynnikov NR, Dahlquist FW, Kay LE (2002) Reconstructing NMR spectra of "invisible" excited protein states using HSQC and HMQC experiments. J Am Chem Soc 124:12352 12360. 2. Ferry JD, Grandine LDJ, Fitzgerald ER (1953) The relaxation distribution function of polyisobutylene in the transition from rubber-like to glass-like behavior. J Appl Phys 24:911 921. 3. Denisov VP, Peters J, Hörlein HD, Halle B (1996) Using buried water molecules to explore the energy landscape of proteins. Nature Struct Biol 3:505 509. 4. Osborne MJ, Schnell J, Benkovic SJ, Dyson HJ, Wright PE (2001) Backbone dynamics in dihydrofolate reductase complexes: Role of loop flexibility in the catalytic mechanism. Biochemistry 40:9846 9859. 5. Boehr DD, McElheny D, Dyson HJ, Wright PE (2006) The dynamic energy landscape of dihydrofolate reductase catalysis. Science 313:1638 1642. 6. McElheny D, Schnell JR, Lansing JC, Dyson HJ, Wright PE (2005) Defining the role of active-site loop fluctuations in dihydrofolate reductase catalysis. Proc Natl Acad Sci USA 102:5032 5037. 7. Boehr DD, Dyson HJ, Wright PE (2008) Conformational relaxation following hydride transfer plays a limiting role in dihydrofolate reductase catalysis. Biochemistry 47:9227 9233. 1of7
Fig. S1. Per-residue chemical shift difference (A) between E:FOL and E:THF, calculated according to jδδj ¼ððΔδ HN Þ 2 þðδδ N 5Þ 2 Þ 1 2, where Δδ x ¼ δ x (E:FOL) δ x (E:THF), (B) Chemical shift difference, calculated according to jδδj ¼ððΔδ HN Þ 2 þðδδ N 5Þ 2 Þ 1 2, where Δδ x ¼ δ x (E:THF:NADP + ) δ x (E:THF:NADPH), between E: THF:NADP + and E:THF:NADPH. Nearly all chemical shift differences are less than 0.1 ppm. Fig. S2. (A) Correlation between dynamic chemical shifts (Δω) for residues in the cofactor-binding cleft of E:FOL and E:THF complexes (Slope equals 1.1, R 2 ¼ 0.96). Circles indicate residues for which the sign of Δω could be determined for both complexes; squares indicate that the sign could only be determined for one complex (E:FOL or E:THF). (B) Correlation plot for dynamic chemical shifts for residues in the active-site loops of E:FOL to the ground-state chemical shift differences between occluded and closed conformations of DHFR. Closed red circles equal residues where sign could be determined, open red circles, sign could not be determined, red line equals linear least squares fit to all data, (slope equals 0.76, R 2 ¼ 0.81). Black dashed line shows a 1:1 correlation. Fig. S3. 1 H- 15 N steady-state heteronuclear NOE for E:THF at 306 K. 2of7
Fig. S4. R 2 relaxation dispersion curves for E:folate and E:dihydrofolate complexes of DHFR at 300 K. Data measured at 1 H frequencies of 500 MHz (Black) and 800 MHz (Red). 3of7
Fig. S5. (Red). R 2 relaxation dispersion curves for E:tetrahydrofolate complex of DHFR at 300 K. Data measured at 1 H frequencies of 500 MHz (Black) and 800 MHz Table S1. Comparison of R 2 relaxation dispersion fits for E:FOL at 303 K Residue Fit Number 1 Cofactor Binding Cleft Fit Number 2 Active-Site Loops Fit Number 3 All Residues Except C-Terminus k ex =510±24s -1 k ex =482±19s -1 k ex =510±14s -1 p Ap B = 0.026 ± 0.001 p Ap B = 0.028 ± 0.001 p Ap B = 0.026 ± 0.001 Δω (ppm) χ 2 cluster/χ 2 ind. Δω (ppm) χ 2 cluster/χ 2 ind. Δω (ppm) χ 2 cluster/χ 2 ind. Leu8 0.76 1.15 Val10 1.00 1.02 1.32 1.01 Gly15 1.32 1.08 1.43 1.07 Glu17 2.17 1.48 2.41 1.35 Asn18 1.27 1.16 1.35 1.27 Ala19 1.02 1.07 1.09 1.10 Trp22 1.31 1.13 1.40 1.14 Asn23 1.26 1.66 1.35 1.68 Leu24 1.56 1.13 1.67 1.13 Met42 1.29 1.11 1.28 1.11 Thr46 1.04 1.04 1.03 1.04 Ile50 1.15 1.05 Gly51 0.96 1.07 Ser63 3.33 1.09 3.33 1.09 Gly67 0.79 1.31 Ser77 3.57 1.08 3.57 1.09 Gly95 1.30 1.09 Gly96 2.86 1.65 2.85 1.65 Val99 1.76 1.53 1.76 1.53 Glu101 1.03 1.07 1.03 1.07 Phe103 0.47 1.05 0.47 1.05 Glu118 1.53 1.04 1.64 1.06 Glu120 1.10 1.23 1.18 1.22 Gly121 1.74 2.02 1.93 1.81 Asp122 1.10 1.07 1.19 1.07 Thr123 1.55 1.01 His149 1.52 2.93 1.62 3.35 4of7
Table S2. 15 NR 2 relaxation dispersion kinetic and thermodynamic fitting parameters for E.coli DHFR bound with different folate-analogs Cluster 1 Active-Site Cluster 2 C-Terminal Associated Region k ex (s -1 ) p Ap B k ex (s -1 ) p Ap B E:FOL - 294 K 430 ± 69 0.016 ± 0.002 295 ± 65 0.019 ± 0.014-297 K 456 ± 53 0.020 ± 0.002 310 ± 70 0.021 ± 0.003-300 K 490 ± 33 0.023 ± 0.003 429 ± 21 0.025 ± 0.001-303 K 510 ± 24 0.026 ± 0.001 440 ± 91 0.027 ± 0.003-306 K 537 ± 32 0.029 ± 0.001 480 ± 92 0.030 ± 0.003 E:DHF - 297 K 554 ± 40 0.017 ± 0.001 298 ± 81 0.022 ± 0.004-300 K 620 ± 56 0.023 ± 0.001 343 ± 72 0.026 ± 0.004-306 K 682 ± 64 0.034 ± 0.002 482 ± 123 0.041 ± 0.004 E:THF - 297 K n.d. 1 n.d. 1 297 ± 32 0.021 ± 0.001-300 K 277 ± 12 0.071 ± 0.003 395 ± 17 0.023 ± 0.001-303 K 322 ± 11 0.069 ± 0.002 452 ± 46 0.027 ± 0.002-306 K 430 ± 11 0.063 ± 0.001 479 ± 21 0.030 ± 0.001-309 K 462 ± 10 0.049 ± 0.001 527 ± 27 0.034 ± 0.001 5of7
Table S3. Dynamic chemical shift differences [Δω (ppm)] between lowest and higher energy conformational substates in substrate/product-bound E.coli DHFR as determined by 15 NR 2 relaxation dispersion NMR spectroscopy (error in values is approximately 5 20%) Cluster 1 Active-Site (Loops and Cofactor Binding Cleft) E:FOL E:DHF E:THF Res.* 294 K 297 K 300 K 303 K 306 K 297 K 300 K 306 K 297 K 300 K 303 K 306 K 309 K Leu8 2.09 1.19 1.07 0.76 0.77 0.71 0.54 0.68 Val10 1.04 0.89 1.08 0.87 0.51 0.52 Asp11 2 0.97 Val13 0.46 Ile14 1.34 0.94 Gly15 1.67 1.51 1.43 0.98 Glu17 2.18 2.63 2.41 2.30 Asn18 1.29 1.06 1.35 1.40 Ala19 0.89 1.09 0.50 0.52 0.52 Met20 2.27 2.33 Trp22 1.73 0.88 1.09 1.40 1.51 Asn23 1.65 1.04 1.29 1.35 0.93 1.35 Leu24 1.97 1.50 1.67 1.62 Met42 1.37 1.20 1.28 1.05 1.59 1.08 1.07 1.16 1.24 Arg44 4.25 0.43 0.42 Thr46 1.06 1.46 1.03 0.58 0.48 0.69 Ile50 1.01 1.16 1.16 1.38 1.13 1.09 0.82 1.02 Gly51 0.96 v 0.80 0.72 0.79 0.86 Leu62 3.80 v Ser63 3.90 3.48 3.61 3.33 3.34 4.56 4.23 4.40 4.41 4.60 3.51 4.16 Gln65 2.47 1.22 0.89 Gly67 0.88 0.89 0.79 1.03 1.66 0.76 0.83 0.82 Thr68 1.71 Asp69 1.56 1.70 1.84 0.91 2.55 Arg71 0.91 0.47 0.49 Trp74 2.38 2.00 2.63 Ser77 3.45 3.36 3.57 3.07 4.23 3.20 3.12 3.70 3.88 3.76 4.20 Val78 0.49 0.48 Gly95 1.55 1.11 1.30 1.65 0.45 Gly96 3.96 2.35 2.85 3.55 3.15 2.46 Gly97 0.78 1.04 Val99 1.23 1.76 1.75 1.65 1.75 1.75 2.05 Glu101 0.98 0.91 1.10 1.03 1.80 1.04 1.14 1.04 0.96 1.10 Phe103 0.98 1.05 0.68 1.06 0.85 0.79 0.91 Glu118 1.65 2.23 1.74 Val119 1.50 1.52 0.40 Glu120 1.30 0.89 v 1.18 1.14 1.84 1.68 1.32 Gly121 1.29 1.94 2.31 2.36 2.20 1.43 0.46 Asp122 1.23 0.89 1.24 1.19 1.47 1.61 1.07 1.30 Thr123 1.58 1.08 1.55 1.12 1.22 1.11 1.12 0.95 0.86 0.90 0.90 His149 1.22 0.98 1.41 1.52 1.46 1.08 1.18 1.11 Cluster 2 C-terminal associated residues Glu129 2.52 2.35 2.16 1.57 1.37 1.22 1.14 0.89 0.59 Asp131 2.46 2.81 2.67 2.97 2.84 2.42 3.18 2.18 2.82 2.85 2.79 2.97 2.64 Trp133 2.82 2.85 2.49 3.20 2.21 3.24 2.74 2.89 3.33 3.30 3.00 3.36 Glu134 1.35 1.32 1.23 1.16 1.02 0.89 0.82 0.68 Arg158 2.96 2.93 2.09 2.50 2.34 2.36 2.17 2.07 1.87 1.69 * Active site residues (red), cofactor binding cleft (green), C-terminal associated residues (Blue). Not determined, conformational exchange is apparent but poor quality R 2 relaxation dispersion curve, poor fit or resonance is severely broadened. Not determined because conformational exchange is not detectable. 6of7
Table S4. Thermodynamic parameters governing conformational exchange processes in E. coli DHFR Complex Energy Differences between Substates (kcal/mol) Energy Barriers between Substates (kcal/mol) van t Hoff analysis (temp = 300 K) Eyring plot (temp = 300 K) Ferry law ΔG ΔH TΔS ΔG ΔH TΔS <H 2 > 1/2 E:NADPH 2 Active site (2.2) n.d. n.d. (14.6) n.d. n.d. n.d. C-term 2.0 n.d. n.d. 15.8 n.d. n.d. n.d. E:FOL Active site 2.2 9.1 6.8 16.1 11.6-4.5 1.9 C-term 2.2 7.3 5.1 16.2 14.4-1.8 2.1 E:DHF 1 Active site 2.3 14.4 12.1 16.0 n.d. n.d. n.d. C-term 2.1 n.d. n.d. 16.3 n.d. n.d. n.d. E:THF Active site 1.4-8.4-9.9 15.7 8.8-6.9 1.7 C-term 2.2 7.9 5.7 16.2 15.2-1.0 2.2 E:FOL:NADP +3 Active site 2.0 15.6 13.6 16.1 21.2 5.1 2.4 C-term (2.0) n.d. n.d. (15.5) n.d. n.d. n.d. E:THF:NADP +4 Active site 1 2.6 13.9 11.3 15.9 20.3 4.4 2.6 Active site 2 2.1 n.d. n.d. 15.2 n.d. n.d. n.d. C-term 2.3 15.5 13.2 16.1 18.5 2.4 2.4 E:THF:NADPH 2 Active site 2.2 n.d. n.d. 15.9 n.d. n.d. n.d. C-term 2.4 n.d. n.d. 16.1 n.d. n.d. n.d. It is important to note that the active site dynamics may include contributions from amino acid residues in the cofactorbinding cleft, the substrate/product-binding pocket and/or the active site loops depending on the complex. For the identity of amino acids undergoing conformational exchange, consult Figs. 2 and 4 and Table S3, and similar figures and/or tables in refs. 5 7. The C-term region includes amino acids 129 134 and 155 159. 1 Data for E:DHF was collected at only three temperatures and was of insufficient quality for complete thermodynamic analysis. 2 Data for E:NADPH and E:THF:NADPH determined using a single temperature using the values in (5) or from previously unpublished data. ΔG and ΔG values for E:NADPH active site determined at 281 K. 3 Data for E:FOL:NADP+ based on parameters previously reported in ref. 6. ΔG andδg C-term values determined at 303 K. 4 Data for E:THF:NADP+ based on parameters previously reported in ref (7). Active site 1 reports on conformational exchange in the active site loops associated with the closed-occluded conformational change. Active site 2 reports on conformational exchange in the cofactor-binding cleft. Active site 2 parameters were determined using data only at 300 K. 7of7