Hydrolytic catalysis and structural stabilization in a designed metalloprotein

Abstract

Metal ions are an important part of many natural proteins, providing structural, catalytic and electron transfer functions. Reproducing these functions in a designed protein is the ultimate challenge to our understanding of them. Here, we present an artificial metallohydrolase, which has been shown by X-ray crystallography to contain two different metal ions-a Zn(II) ion, which is important for catalytic activity, and a Hg(II) ion, which provides structural stability. This metallohydrolase displays catalytic activity that compares well with several characteristic reactions of natural enzymes. It catalyses p-nitrophenyl acetate (pNPA) hydrolysis with an efficiency only ~100-fold less than that of human carbonic anhydrase (CA)II and at least 550-fold better than comparable synthetic complexes. Similarly, CO(2) hydration occurs with an efficiency within ~500-fold of CAII. Although histidine residues in the absence of Zn(II) exhibit pNPA hydrolysis, miniscule apopeptide activity is observed for CO(2) hydration. The kinetic and structural analysis of this first de novo designed hydrolytic metalloenzyme reveals necessary design features for future metalloenzymes containing one or more metals.

Figure 1. Ribbon diagrams of the [Hg(II)]_S[Zn(II)(H₂O/OH )]_N(CSL9PenL23H)₃ⁿ⁺ parallel 3SCC (one of two different 3-helix bundles present in the asymmetric unit) at pH 8.5

Shown are the main chain atoms represented as helical ribbons (cyan) and the Pen and His side chains in stick form (sulphur = yellow, nitrogen = blue, oxygen = red). a, One of two trimers found in the asymmetric unit of the crystal structure. b, a top down view of the structural trigonal thiolate site, Hg(II)S₃, confirming the proposed structure of Hg(II) in Cys-containing TRI peptides. This metal site should mimic well the structural site in the metalloregulatory protein MerR. c, a side view of the tetrahedral catalytic site, Zn(II)N₃O, which closely mimics carbonic anhydrase and matrix metalloproteinase active sites. All figures are shown with 2F_o-F_c electron density contoured at 1.5 σ overlaid.

Figure 2. Overlay of the Zn(II)N₃O site in [Hg(II)]_S[Zn(II)(H₂O/OH⁻)]_N(CSL9PenL23H)₃ⁿ⁺ with the active site of human CAII

[Hg(II)]_S[Zn(II)(H₂O/OH⁻)]_N(CSL9PenL23H)₃ⁿ⁺ is shown in cyan (pdb 3PBJ) and CAII in tan (pdb 2CBA). The solvent molecule associated with [Hg(II)]_S[Zn(II)(H₂O/OH⁻)]_N(CSL9PenL23H)₃ⁿ⁺ is shown in red and that associated with CAII lies underneath. The model displays an excellent structural overlay for the first coordination sphere atoms with CAII; however, the orientation of the imidazoles differs between the two proteins. Another subtle difference is that the present structure has three ε amino nitrogens bound to the Zn(II) ion whereas CAII has a mixed two ε and one δ coordination sphere. Hence, the present structure better mimics the MMP adamalysin II which also has three ε amino nitrogens bound to Zn(II). Overlay was performed manually in Pymol. See Supplementary Fig. S2 for a side-on view.

Figure 3. pH-dependency of pNPA hydrolysis by [Hg(II)]_S[Zn(II)(H₂O/OH⁻)]_N(TRIL9CL23H)₃ⁿ⁺

Plots of a, k_cat/K_M vs. pH and b, k_cat vs. pH for the hydrolysis of pNPA by [Hg(II)]_S[Zn(II)(H₂O/OH⁻)]_N(TRIL9CL23H)₃ⁿ⁺ (10 μM). pK_a values of 8.82 ± 0.11 and 8.77 ± 0.08 for plots a and b, respectively, can be determined from the fittings and presumably represent the deprotonation of Zn-OH₂ to form an active Zn-OH⁻ nucleophile, as with CAII. See Supplementary Methods for a description of the fitting and error analysis.