Fully Selective Pb(II) Templated Heterotrimeric Redox and Hydrolytic Metalloenzymes

Abstract

The design and characterization of the first fully selective, self-assembling, asymmetric de novo designed three stranded coiled coils and their use to induce non-3-fold symmetric transition metal coordination for hydrolytic and redox catalysis is presented. This work was done in a peptide scaffold of the sequence Ac-G-(LaKbAcLdEeEfKg)5-G-NH2 where subscripts indicate position in the heptad. Leu residues are substituted with metal binding residues to both induce asymmetry at one site and perform transition metal catalysis at a second, distant site. Full selectivity for both A2B- and AB2-type heterotrimers was achieved using a d-site Cys, Pb(II), and an adjacent layer (toward the C terminus) of reduced steric bulk with mixed Leu and Ala residues. This work presents a significant improvement over a reported scaffold using a-site Cys residues that was only selective for A2B-type (2 Ala) heterotrimers. To maintain stability, these scaffolds are limited to 3 Leu substitutions per strand. These d-site scaffolds allow for both X1His3 and X2His3 transition metal sites, expanding the scope of asymmetry that can be explored. The selectivity of heterotrimers with d-site Cys was determined via 207Pb NMR. Homotrimeric peptides had a single resonance at 5762 ppm (3 Ala) and 5789 ppm (3 Leu) and heterotrimeric mixtures had a single resonance at 5875 ppm (2 Ala, 1 Leu) and 5803 ppm (1 Ala, 2 Leu). Crystallographic comparison of the homotrimers found that the hydrogen bonding network present in the Ala3 homotrimer was significantly different for a- and d-site Cys residues, where solvent was able to hydrogen bond two a-site Cys residues at 3.0 and 3.4 Å while only one solvent molecule could hydrogen bond a d-site Cys at 3.4 Å. The energetic differences between hydrogen bonding and leucine packing in the adjacent layer yields the observed selectivity between a- and d-site Cys heterotrimers. A distant His3 site can be incorporated into the scaffold and can coordinate a transition metal without perturbing the Pb(II)Cys3 site, or the heterotrimer stoichiometry, as determined by 207Pb NMR. As such, this work utilized a-site His residues with adjacent Glu to investigate Zn(II) hydrolysis as found in carbonic anhydrase. A potential hydrogen bonding residue with a long side chain assessed whether the Zn(II)His3OH coordination environment previously characterized was maintained, while also affording a second sphere hydrogen bond as found in the native system. Co(II) spectroscopy determined that an asymmetric 5-coordinate environment was generated with 1 or 2 Glu residues while a symmetric 6-coordinate environment was observed for 1 or 3 Glu. Ultimately, Zn(II) coordinated Glu1His3 (kcat/KM=11.4 M-1s-1), Glu2His3 (22 M-1s-1), and Glu3His3 (18.7 M-1s-1) did not improve upon the most efficient His3 model (23.3 M-1s-1), likely because the Glu residue was instead able to hydrogen bond directly to a His residue, precluding Zn(II)His3(H2O) coordination. Redox catalysis, modelling Cu-only superoxide dismutase, was found at pH 7.5 with a variety of His3, His4, and mixed Carboxy/His binding environments. This work describes the best peptidic models of this system. The d-site His3 (kMcF=4.5x107 M-1s-1) was ~7.5x faster than a-site His3 (6.7x106 M-1s-1) despite having a less ideal reduction potential (695 vs 541 mV vs NHE, respectively). The fastest construct contained 2 His and 1 Asp in the d-site and 1 His in the a-site (kMcF= 6.2x107 M-1s-1). These experiments could only be completed by using these new asymmetric heterotrimers.