Lanthanide-binding helix-turn-helix peptides: Solution structure of a designed metallonuclease

Abstract

A designed lanthanide-binding chimeric peptide based on the strikingly similar geometries of the EF-hand and helix-turn-helix (HTH) motifs was investigated by NMR and CD spectroscopy and found to retain the same overall solution structure of the parental motifs. CD spectroscopy showed that the 33-mer peptide P3W folds on binding lanthanides, with an increase in ␣-helicity from 20% in the absence of metal to 38% and 35% in the presence of excess Eu(III) and La(III) ions, respectively. The conditional binding affinities of P3W for La(III) (5.9 ؎ 0.3 M) and for Eu(III) (6.2 ؎ 0.3 M) (pH 7.8, 5 mM Tris) were determined by tryptophan fluorescence titration. The La(III) complex of peptide P3, which differs from P3W by only one Trp-to-His substitution, has much less signal dispersion in the proton NMR spectra than LaP3W, indicating that the Trp residue is a critical hydrophobic anchor for maintaining a well-folded helixturn-helix structure. A chemical-shift index analysis indicates the metallopeptide has a helix-loop-helix secondary structure. A structure calculated by using nuclear Overhauser effect and other NMR constraints reveals that P3W not only has a tightly folded metalbinding loop but also retains the ␣؊␣ corner supersecondary structure of the parental motifs. Although the solution structure is undefined at both the N and C termini, the NMR structure confirms the successful incorporation of a metal-binding loop into a HTH sequence. One of the great promises of de novo protein design is the possibility of incorporating new reactivity or selectivity into a protein matrix for specific chemical targets or to advance biological understanding. The redesign of a known protein fold is one powerful approach to achieving new function, and the inclusion of metal sites that could confer a range of enzymatic activities is particularly intriguing. Designed metalloproteins have the potential to promote natural enzymatic reactions on unnatural substrates or even to achieve reactivity through mechanisms completely unprecedented in biology. However, to interpret and evaluate their activity and to improve subsequent designs, the resultant constructs must retain a predictable well-folded structure. A structurally well characterized robust scaffold is therefore fundamental to the design of minimalist enzymes.In recent years, several approaches have been taken to engineer novel metalloproteins, including building a metal site and scaffold completely from first principles (1-5), grafting ligands onto known protein scaffolds by point mutations that were identified through modeling or a vector-based algorithm such as DENZYMER (5-9), or redesigning natural metalloproteins for potential new reactivity (10, 11). Alternately, native metal-binding sequences may be used in new contexts (12-14), and we have recently reported the de novo design (or more correctly, redesign) of metallopeptide nucleases based on this premise (12, 15).The structural similarity in their natural protein contexts of the helix-turn-helix (HTH), a DNA-binding motif c...

Lanthanide(III) Complexes with Two Hexapeptides Incorporating Unnatural Chelating Amino Acids: Secondary Structure and Stability

“…[40] In the LBT structures reported in the literature this distance is approximately 7 because the backbone carbonyl of the tryptophan residue is coordinated to the terbium ion. [10,48] In the 10 lowest-energy structures of the complex LaP 2 , the La 3 + ion is located 8.7A C H T U N G T R E N N U N G (AE0.4) from the center of the indole. The distance between the acceptor and the donor is slightly larger in LaP 2 than in the LBTs, mainly because the tryptophan carbonyl is not coordinated to the metal center.…”

“…[48] Lanthanum coordination by P 2 and the establishment of the Ada 2 5 NH···OC Ada 2 2 hydrogen bond as well as the Trp-Pro hydrophobic interaction may thus better be viewed as occurring synergistically. These interactions can reduce the unfavorable conformational energy terms associated with peptide backbone reorganization upon complexation.…”

Lanthanide-binding peptides and the enzymes that Might Have Been

“…This metal-dependant folding can be explained by maximizing critical interhelical hydrophobic interactions upon organizing the metal-binding turn, as well as the formation of an a-helix nucleation site at the C-terminal end of the metal sites as observed for native EF-hand peptides. The solution structure of one metal-bound chimeric peptide (LaP3W, where P3W is a 33-mer peptide of the design depicted in figure 1) was elucidated using NMR spectroscopy and found to be qualitatively similar to the parental EF-hand motif [28]. The metal binding site was confirmed by paramagnetic shifts of loop protons in the EuP3W 1 H-NMR spectrum.…”

“…The structures of the chimeras were investigated using circular dichroism (CD) and NMR spectroscopy [28]. These studies demonstrated that the designed Ln peptides adopt the same secondary and super-secondary structures as the parental motifs.…”

Artificial metalloenzymes as selective catalysts in aqueous media

“…In nature, the former is accomplished by transcription factors and repressor proteins, the latter by the hydrolytic activity metal-ions such as Ca, Mg and Zn. Recent studies of artificial nucleases suggest that lanthanides (Eu, Ce) [74][75][76][77]79,80,[82][83][84][85][86] and transition metals (copper [78], zirconium [81]) are promising metals combined with peptide motifs such as helix-turn-helix (HTH) chimera [74][75][76]78], zinc fingers [79,80] or peptide nucleic acids [81].…”