Bound nucleotide can control the dynamic architecture of monomeric actin

Figures

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  Extended Data Fig. 1:. Expression, purification and δ1–methyl 13C assignments of G-actin. a, Schematics showing steps used for purification of recombinant G-actin from P. pastoris. b-d, SDS-PAGE (15%) showing fractions after Ni-NTA (b) anion exchange (c) and Superdex-200 size-exclusion chromatography (d). In (b), Lane 1, Molecular weight markers; Lane 2, pellet after lysis and centrifugation; Lane 3, supernatant after lysis and centrifugation; lane 4, flow through; lane 5, wash I; lane 6, wash II; lanes 7&8, elution; lane 9, after overnight TEV cleavage reaction. The SDS-PAGE gels are representative of at least 10 independent experiments. e-f, Overlaid 1H/13C HMQC spectra of WT G-actin (red) and mutants (black) I85V & I122V (e) and I175V (f). All assignments were obtained by mutagenesis; missing peaks in the mutant spectra were used to obtain sequence specific assignments. The missing peak in each mutant spectrum is labeled. Overlaid spectra of WT and mutant I175V showing chemical shift perturbation experienced by residues that are away from the position of mutation and propagate to different sub-domains (f). g, Ribbon diagram of G-actin with I175 shown as a red ball and residues altered by its…

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  Extended Data Fig. 2:. Preparation of Ca2+-ADP-actin and effects of metal ion on 1H/13C HMQC spectra. a, Purified Ca2+-ATP-actin was first converted using hexokinase to Mg2+-ADP-actin, which was then dialyzed against buffer containing Ca2+-ADP to produce Ca2+-ADP-actin. See Methods for details. b, 1H/13C HMQC spectra of perdeuterated, 1H/13C- Ile δ1-methyl-labeled Ca2+-ATP-actin (top); overlaid spectra of Ca2+-ATP-actin (red) and Mg2+-ADP-actin (cyan) (middle); overlaid spectra of Mg2+-ADP-actin (cyan) and Ca2+-ADP-actin (black) (bottom). Residues showing chemical shift changes upon metal switching in the ADP state are circled. c-d, Structural comparison between AMPPNP (PDB ID INWK)- and ADP-bound (PDB ID 1J6Z) forms of TMR-labeled actin.

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  Extended Data Fig. 3:. G-actin does not aggregate and is stable during NMR data acquisition at 100 μM. a-b, Full linewidth at half height (νFWHH) in the 13C (a) and 1H (b) dimensions of resolved Ile δ1-methyl resonances in 1H/13C HMQC spectra recorded at 600 MHz on 1H/13C- Ile δ1-methyl-labeled Ca2+-ATP-actin at the indicated protein concentrations. c, S/N (signal to noise) ratio in 1H/13C HMQC spectra of 1H/13C- Ile δ1-methyl-labeled labeled Ca2+-ATP-actin acquired at different times (0, 20 and 40 hours) after sample preparation. No significant changes in S/N were observed over the 40 hour timecourse. Error bars represent SD calculated from the noise in the spectra.

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  Extended Data Fig. 4:. μs-ms dynamics of Ca2+-ATP-actin. a, Multiple quantum relaxation dispersion (ΔR2), the difference in R2eff at lowest and highest pulsing rates, recorded at 800 MHz, of Ile δ1-methyl resonances of 1H/13C- Ile δ1-methyl-labeled Ca2+-ATP-actin. Error bars in ΔR2 represent the SD based on noise levels in NMR spectra and were calculated using standard error propagation methods, b, Magnitude of 13C chemical shift difference (13C|Δω|) between the major (ground) and minor (excited) states for Ile δ1-methyl groups of ATP-actin. c, Thermodynamic (Pb) and kinetic parameters (kex) obtained from two-state global fitting of the relaxation dispersion data. Errors in 13C|Δω|, Pb, and kex were determined from the covariance matrix of the global fits. ΔR2 values near zero were obtained for all Ile δ1-methyl resonances in a 1H single quantum relaxation dispersion experiment as shown for representative residues in Fig. 3 (columns 2 and 4).

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  Extended Data Fig. 5:. μs-ms dynamics in Ca2+-ADP-actin. a, Multiple quantum relaxation dispersion (ΔR2), difference in 1H/13C R2eff between the lowest and highest CPMG field strengths, recorded at 800 MHz of Ile δ1-methyl resonances of 1H/13C- Ile δ1-methyl-labeled Ca2+-ADP-actin. Error bars in ΔR2 represent the SD based on noise levels in NMR spectra and were calculated using standard error propagation methods b, Absolute value of the difference between ground and excited state 13C and 1H chemical shifts |Δω| for Ile δ1-methyl resonances of Ca2+-ADP-actin determined from global fitting of the MQ and 1H SQ CPMG data. c, Thermodynamic (Pb) and kinetic parameters (kex) obtained from two-state global fitting. Errors in |Δω|, Pb, and kex were determined from the covariance matrix of the global fits.

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  Extended Data Fig. 6:. Comparison of dynamics in ATP- and ADP-bound G-actin. a, Comparison of MQ ΔR2 recorded at 800 MHz of Ile δ1-methyl resonances of 1H/13C- Ile δ1-methyl-labeled ATP- (red) and ADP-bound (green) actin. Only residues I64, I71, I75 and I85 showed ΔR2 ≥ 2Hz in ADP-actin, whereas many additional residues showed ΔR2 ≥ 2Hz in ATP-actin. Error bars in ΔR2 represent the SD, based on noise levels in NMR spectra and were calculated using standard error propagation methods b, Although, residues showing ΔR2 ≥ 2Hz are mainly confined to SD1 and SD2, in ADP-actin, the magnitude of ΔR2 was higher due to a greater population of the excited state. The error bars represent the SD (standard deviation), calculated from the two and three replicated data points for 600 and 800 MHz data, respectively. c, Hydrolysis of ATP (red) into ADP (yellow) triggers a series of rearrangements from which multiple hydrogen bonds (shown as dotted line) are broken.

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  Extended Data Fig. 7:. Structural and dynamic analyses of K118N Ca2+-ATP actin. a, Location of K118 on the F-actin structure (PDB ID 6DJM). K118, shown as a red sphere, is not at a subunit interface. b, Comparison of Ile δ1-methyl 13C excited state chemical shifts 13C|Δω| of K118N (light blue) and WT (pink) ATP-actin. Error in 13C|Δω|, Pb, and kex were determined from the covariance matrix of the global fits.

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  Fig. 1:. High quality methyl TROSY NMR spectrum and sequence specific isoleucine δ1-methyl chemical shift assignments of non-polymerizable (DVD) G-actin. a, Cartoon representation of TEV protease cleavable, (His)6 tagged DVD G-actin fused with thymosin β4. b, δ1 13C-methyl labeling of isoleucine in P. pastoris using precursor α–ketobutyrate (4-13C-3,3-D2) (see Methods). c, Ribbon diagram of G-actin (PDB ID 2HF4) showing isoleucine residues as black spheres. Elements of actin are colored as follows: SD1 (cyan), SD2 (magenta), SD3 (yellow), SD4 (corn blue), P1 and P2 nucleotide binding loops (red), sensor loop (blue), hydrophobic plug (green) and WH2 binding motif in the hydrophobic groove (violet). Sites in SD3 mutated to block polymerization (D286A/V287A/D288A) are shown as red spheres. d, 1H/13C methyl TROSY NMR spectrum of perdeuterated, 1H/13C- Ile δ1-methyl-labeled G-actin showing assigned isoleucine residues (see Extended data Fig. 1).

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  Fig. 2:. Chemical shift changes due to the nucleotide switch propagate throughout G-actin. a, Overlaid 1H/13C methyl TROSY NMR spectra of Ca2+-actin in the ATP- (red) and ADP-bound (black) forms. b-c, Chemical shift differences (Δ = [(δ1H)2 + (0.25×δ13C)2]1/2) between the ATP- and ADP-bound forms of actin. Residues showing Δ > 0.04 ppm (black line) are colored red (b), and are labeled in Fig. 2a and shown as red spheres in the wire representation of G-actin (PDB ID 2HF4) (c). SDs are colored as in Fig. 1c. d-e, Comparison of AP (A204E/P243K) mutant G-actin crystal structures in the ATP- (PDB 2HF4, cyan) and ADP-bound (PDB 2HF3, wheat) forms32 (d). The structures in two states are nearly identical except in sensor loop region with an overall backbone rmsd of 0.23 Å (e). Panels (c) – (e) show stereo views.

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  Fig. 3:. μs-ms dynamics measurements of Ca2+-ATP and -ADP-actin. a-d, Representative relaxation dispersion (RD) profiles (effective relaxation rate, R2eff vs CPMG frequency) for isoleucine residues in SD1 (a), SD2 (b), SD3 (c) and SD4 (d). In all panels (a-d) data recorded at 600 MHz and 800 MHz field strengths are black and red, respectively. MQ data (column 1 and 3) report on chemical exchange experienced by both 1H and 13C nuclei, whereas SQ (column 2 and 4) report on chemical exchange experienced by 1H only. The error bars represent the SD (standard deviation), calculated from the two and three replicated data pointsfor 600 and 800 MHz data, respectively).

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  Fig. 4:. G-actin has a different dynamic architecture in its ATP- and ADP-bound forms. a-c, Isoleucine residues with MQ or 1H SQ RD > 2 Hz mapped on the G-actin structure in the ATP- (left panel, red balls, PDB 2HF4) and ADP-bound (right panel, green balls, PDB 2HF3) forms (a). The SDs are colored as in Fig. 1c. Figs. 4a and 4b were generated using Chimera. Three-dimensional (b) and two dimensional (c) depictions of the nucleotide binding site of ATP- and ADP-bound actin, left and right images, respectively. Hydrogen bonds are indicated by dashed lines. In ATP-actin the two halves of the structure are bridged through additional hydrogen bonds between the terminal γ–phosphate and residues in SD3 that are absent in ADP-actin (circled in red). Fig. 4c was generated using LigPlot+.

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  Fig. 5:. Structural models for the dynamics of G-actin. In each panel the left column schematically depicts a potential structural mechanism to explain the observed dynamics (T and D indicate ATP- and ADP-bound actin ground states, respectively; T* and D* indicate the respective excited states), the middle panel plots various ground and excited state chemical shifts against each other, and the right column depicts the mechanistic conclusion from the data (see text). In the middle column each datapoint represents an isoleucine residue. Residues with different ground state chemical shifts in ATP- and ADP-actin are boxed, and are not used to make structural comparisons. a, Model in which actin exists in only two conformational states, which are differentially biased by ATP and ADP (left). In such a model T and D* are conformationally identical and have the same chemical shifts, as are D and T*. Scatter plots (middle) compare the ATP and ADP* 13C chemical shifts (top) and the ADP and ATP* chemical shifts (bottom). Off-diagonal, non-boxed points indicate structural differences between the states (more definitive for the T*/D comparison than T/D* comparison), ruling out a two-state-equilibrium…