Making or Breaking Metal‐Dependent Catalytic Activity: The Role of Stammers in Designed Three‐Stranded Coiled Coils

Abstract

While many life-critical reactions would be infeasibly slow without metal cofactors, a detailed understanding of how protein structure can influence catalytic activity remains elusive. Using de novo designed three-stranded coiled coils (TRI and Grand peptides formed using a heptad repeat approach), we examine how the insertion of a three residue discontinuity, known as a stammer insert, directly adjacent to a (His) 3 metal binding site alters catalytic activity. The stammer, which locally alters the twist of the helix, significantly increases copper-catalyzed nitrite reductase activity (CuNiR). In contrast, the well-established zinc-catalyzed carbonic anhydrase activity (p-nitrophenyl acetate, pNPA) is effectively ablated. This study illustrates how the perturbation of the protein sequence using non-coordinating and non-acid base residues in the helical core can perturb metalloenzyme activity through the simple expedient of modifying the helical pitch adjacent to the catalytic center.

Open Reading Frame 1 Protein of the Human Long Interspersed Nuclear Element 1 Retrotransposon Binds Multiple Equivalents of Lead

“…This peptide is structurally analogous to the two a -site Cys layers in ORF1p­CCD (Figure A). We note that this model lacks the three-residue stammer insertion discontinuity, which we recently suggested influences proximal metal binding sites . Lead titrations to GRCS ­L16CL23C show the characteristic 343 nm peak, again indicative of PbS 3 formation within the 3SCC interior.…”

“…38 Additionally, we have shown that adding a stammer discontinuity to a 3SCC alters the reactivity of both Zn and Cu designed enzymesin one case diminishing and in the other enhancing activitysuggesting that metal-based function can be modulated by sequence discontinuities in 3SCCs. 22 Taken together, this evidence demonstrates the possibility of Pb(II) interference to the human LINE-1 ORF1pCCD. The formation of the trigonal pyramidal Pb(II)S 3 complexes in the interior tristhiolate sites of the trimer could alter coiled-coil stability and dynamics, which are factors that have been shown to be critical in retrotransposition of L1.…”

“…We note that this model lacks the three-residue stammer insertion discontinuity, which we recently suggested influences proximal metal binding sites. 22 Lead titrations to GRCSL16CL23C show the characteristic 343 nm peak, again indicative of PbS 3 formation within the 3SCC interior. The change in absorbance at 343 nm as a function of added Pb(II) is linear to 2 equiv per 3SCC, and the maximum extinction coefficient (ε 343 nm ) of 7830 M −1 cm −1 (Figure 1C) is characteristic of two PbS 3 chromophores.…”

“…We have previously reviewed possible mechanisms of metal insertion into folded coiled-coils and the impact of metal binding on intrastrand dynamics . Additionally, we have shown that adding a stammer discontinuity to a 3SCC alters the reactivity of both Zn and Cu designed enzymesin one case diminishing and in the other enhancing activitysuggesting that metal-based function can be modulated by sequence discontinuities in 3SCCs . Taken together, this evidence demonstrates the possibility of Pb­(II) interference to the human LINE-1 ORF1p­CCD.…”

Helicity-driven chiral self-sorting supramolecular polymerization with Ag+: right- and left-helical aggregates

“…Chiral metal-coordinated supramolecular assemblies have recently received much attention and are among the most promising structures in supramolecular chemistry, [1][2][3][4][5][6][7][8][9][10] because metal-coordinated systems play essential roles in the formation of superstructures such as coiled-coil helix bundle proteins, 11,12 DNA superhelices, 13 and protein-DNA hybrid superstructures. 14,15 Thus, better understanding of emergent chiral phenomena and the origin of chirality at the supramolecular level are essential for biomedical applications such as drug delivery, [16][17][18] gene delivery, 19 tissue engineering, 20,21 imaging, 22 and sensing.…”