Peptide deformylase (a) Ribbon diagram of the EcPDF crystal structure, 1BS5, chain A. The bound metal is blue, metal ligands are Cys⁹⁰, His¹³² and His¹³⁶; the oxygen atom from the water molecule that comprises the fourth metal ligand is shown as a red sphere. Ser¹⁴⁷ marks the C-terminus of the catalytic core used in the present study. (b) Close-up of the actinonin binding site illustrating the intermolecular hydrogen bonds (dashes) and actinonin-metal interactions.

Effect of actinonin on the structure and dynamics of EcPDF. (a) Pairwise backbone atom RMSDs between the mean structures from the three molecules in each of the asymmetric units of the free (1BS5) and actinonin-bound (1G2A) EcPDF crystal structures. (b) Weighted-average ¹H/¹⁵N shift perturbations, Δδ_HN (55) induced by actinonin (bars, left y-axis) graphed with intermolecular closest approach distance, D (lines, right y-axis). (c) Actinonin-induced absolute changes in residual dipolar couplings (rDC) recorded in 20 mg/ml Pf1 phage; rDC values were normalized by the magnitude of the alignment tensor (D_a) prior to subtraction (complex-free). (d) Actinonin-induced change backbone entropy, ΔS (in entropy units, calories), as calculated from the change in order parameter, S² (complex-free). Positive values indicate increased flexibility in the complex. (e) Actinonin-induced change in amide proton stability as measured from hydrogen/deuterium exchange data, with positive values indicating amides stabilized by actinonin binding; red symbols indicate residues whose amides exchange to fast or slow for the effect of actinonin to be measured. Red and blue boxes highlight regions close to and far from actinonin, respectively, for effects of the ligand are observed on ΔRDC, ΔS, ΔCS and ΔΔG.

Spectral perturbations induced by actinonin binding. (a) UV-visible spectra of Co²⁺-EcPDF recorded before (solid line) and after addition of actinonin (dashed line). The change in coordination geometry is evident from the decreased absorbance at 560 nm and the red-shift of the band at 330 nm (79). (b) Overlay of 2D ¹⁵N HSQC spectra of (Zn²⁺) EcPDF recorded in the absence (black) and presence (red) of stoichiometric concentrations of actinonin. (c) Expanded overlays of upfield (black) and downfield (red) components of IPAP spectra (800 MHz) showing the effect of actinonin binding on the rDC of several amide resonances. The top two spectra correspond to IPAP spectra of free EcPDF recorded in isotropic solution (left) or in 20 mg/ml Pf1 phage, while the bottom two spectra are those of the complex. The amide resonances and splittings (in Hz) are as indicated; errors in peak positions were roughly ± 0.2 Hz. (d) Correlation plots showing degree of agreement between experimentally measured rDCs and those calculated from the free (1BS5) and actinonin-bound (1G2A) crystal structures.

Isothermal titration calorimetry of Zn-PDF with actinonin at 37°C. Heats of reaction from each injection were fit to a single binding site model (Origin V.7) to obtain the best-fit values of binding stoichiometry (n = 0.954 ± 0.004), affinity (K_A = 9.02 ± 1.2 × 10⁶ M⁻¹), and enthalpy (ΔH = −1.664 ± 0.012 kcal mol⁻¹).

Effect of actinonin binding on EcPDF structure and dynamics. (a) Actinonin-induced amide shift perturbations. Residues are colored by a linear ramp from grey for unperturbed amides (Δδ = 0) to red for amides perturbed greater than twice the standard deviation from the mean (Δδ > 0.27) (b) Normalized actinonin-induced absolute changes in residual dipolar couplings (| rDC |). Values are mapped via a color ramp from grey (no change) to red (Δ|rDC| > 0.6; ca. three times the standard deviation from the mean). (c) The change in residual conformational entropy, TΔS, mapped onto the crystal structure of the EcPDF-actinonin complex. Residues in grey exhibit no measurable change in ps-ns dynamics, blue residues become more rigid upon actinonin binding, while red residues are more flexible in the complex; the magnitude of the change is represented by linear color ramps. (d) Change in free energy as reported by hydrogen/deuterium exchange rates. Residues in grey exhibit no significant change in rate, blue indicates residues whose amides are more protected in the complex, while red residues are less protected in the complex; the magnitude of the change is represented by linear color ramps. In all cases, values smaller than the error are colored grey in the figures. Residues in green are those for which the data were not available either in the free protein or complex. The metal ion is drawn as a grey sphere, actinonin is shown as sticks with atoms colored by type.

Backbone amide ¹⁵N ‘model-free’ parameters for free (black) and actinonin-bound EcPDF (red). S², square of the generalized order parameter; τ_e, effective correlation time for internal motions; R_ex, phenomenological exchange term contributing to line broadening; Model, best fit extended model-free motional model selected for fitting relaxation data. The mean S² is 0.86 for the free protein and for the complex (dashed line), with standard deviations of 0.06 and 0.05, respectively. Bottom, order parameters (S²) for free EcPDF (left) and the EcPDF-actinonin complex (right) mapped with linear color ramps from grey (mean S² for free PDF, 0.86) to red for amides more dynamic than the mean (S² ≤ 0.77) or blue for amides more rigid than the average (S² ≥ 0.97).