Structure of the full-length insecticidal protein Cry1Ac reveals intriguing details of toxin packaging into in vivo formed crystals.

For almost half a century, the structure of the full-length Bacillus thuringiensis (Bt) insecticidal protein Cry1Ac has eluded researchers, since Bt-derived crystals were first characterized in 1965. Having finally solved this structure we report intriguing details of the lattice-based interactions between the toxic core of the protein and the protoxin domains. The structure provides concrete evidence for the function of the protoxin as an enhancer of native crystal packing and stability.

DNA targeting and cleavage by an engineered metalloprotein dimer.

Nature has illustrated through numerous examples that protein dimerization has structural and functional advantages. We previously reported the design and characterization of an engineered "metallohomeodomain" protein (C2) based on a chimera of the EF-hand Ca-binding motif and the helix-turn-helix motif of homeodomains (Lim and Franklin in Protein Sci. 15:2159-2165, 2004). This small metalloprotein binds the hard metal ions Ca(II) and Ln(III) and interacts with DNA with modest sequence preference and affinity, yet exhibits only residual DNA cleavage activity. Here we have achieved substantial improvement in function by constructing a covalent dimer of this C2 module (F2) to create a larger multidomain protein. As assayed via fluorescence spectroscopy, this F2 protein binds Ca(II) more avidly (25-fold) than C2 on a per-domain basis; in gel shift selection experiments, metallated F2 exhibits a specificity toward 5'-TAATTA-3' sequences. Finally, Ca(2)F2 cleaves plasmid DNA and generates a linear product in a Ca(II)-dependent way, unlike the CaC2 monomer. To the best of our knowledge this activation of Ca(II) in the context of an EF-hand binding motif is unique and represents a significant step forward in the design of artificial metallonucleases by utilizing biologically significant metal ions.

Dicamba monooxygenase: structural insights into a dynamic Rieske oxygenase that catalyzes an exocyclic monooxygenation.

Dicamba (2-methoxy-3,6-dichlorobenzoic acid) O-demethylase (DMO) is the terminal Rieske oxygenase of a three-component system that includes a ferredoxin and a reductase. It catalyzes the NADH-dependent oxidative demethylation of the broad leaf herbicide dicamba. DMO represents the first crystal structure of a Rieske non-heme iron oxygenase that performs an exocyclic monooxygenation, incorporating O(2) into a side-chain moiety and not a ring system. The structure reveals a 3-fold symmetric trimer (alpha(3)) in the crystallographic asymmetric unit with similar arrangement of neighboring inter-subunit Rieske domain and non-heme iron site enabling electron transport consistent with other structurally characterized Rieske oxygenases. While the Rieske domain is similar, differences are observed in the catalytic domain, which is smaller in sequence length than those described previously, yet possessing an active-site cavity of larger volume when compared to oxygenases with larger substrates. Consistent with the amphipathic substrate, the active site is designed to interact with both the carboxylate and aromatic ring with both key polar and hydrophobic interactions observed. DMO structures were solved with and without substrate (dicamba), product (3,6-dichlorosalicylic acid), and either cobalt or iron in the non-heme iron site. The substitution of cobalt for iron revealed an uncommon mode of non-heme iron binding trapped by the non-catalytic Co(2+), which, we postulate, may be transiently present in the native enzyme during the catalytic cycle. Thus, we present four DMO structures with resolutions ranging from 1.95 to 2.2 A, which, in sum, provide a snapshot of a dynamic enzyme where metal binding and substrate binding are coupled to observed structural changes in the non-heme iron and catalytic sites.

Investigation of the affinity and selectivity of avian prion hexarepeat peptides for physiological divalent metal ions.

To further the understanding of the biological importance of metal-binding by avian prion proteins, we have investigated the affinity and selectivity of peptides Hx1 [Ac-HNPGYP-nh] and Hx2 [Ac-NPGYPHNPGYPH-nh] with a range of physiological metals via electrospray ionization mass spectrometry and tyrosine fluorescence emission spectroscopy. Both the hexamer Hx1 and the "dimer" peptide Hx2 bind only one equivalent of Cu(II), although only the latter peptide binds copper with significant affinity (Hx1 K(d)=150+/-35 microM; Hx2 K(d)=1.07+/-0.78 microM, pH 7.0 in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer). Both peptides are selective for Cu(II) over divalent Ca, Co, Mg, Mn, Ni, and Zn. Cyclic voltammetry was used to estimate Cu(II/I) solution potentials at pH 6.8, which were very similar for the two peptides (CuHx1 E degrees'=+350 mV, CuHx2 E degrees'=+320 mV vs. normal hydrogen electrode). These results suggest similar binding modes for the two peptides, and relative stabilization of Cu(I) relative to similar His-Gly-rich peptides in the literature.

Of folding and function: understanding active-site context through metalloenzyme design.

In the emerging field of biomolecular design, the introduction of metal-binding sites into loop or turn regions of known protein scaffolds has been utilized to create unique metalloprotein and metallopeptide systems for study. This Forum Article highlights examples of the modular-turn-substitution approach to design and the range of structural and mechanistic questions to which this tool can be applied. Examples from the authors' laboratory are given to show that lanthanide-binding metallopeptides, and now a full metallohomeodomain, can be generated by modular substitution of a Ca-binding EF-hand loop into the unrelated scaffold, the engrailed helix-turn-helix motif. We have previously shown that these peptides bind trivalent Ln(III) ions and promote DNA and phosphate hydrolysis, the targeted function. Here, a series of chimeric peptides are presented that differ only in the ninth loop position [given in parentheses; Peptides P3N (Asn), P3E (Glu), P3A (Ala), and P3W(D) (Asp]. This residue, a putative second-shell ligand stabilizing a coordinated water, was found to influence not only metal affinity but also peptide folding. The affinity for Tb(III) was determined by Trp-Tb fluorescence resonance energy transfer and followed the order Ka = P3W(D) > P3A approximately P3E > P3N. However, circular dichroism (CD) titrations with EuCl3 showed that only P3W(D) and P3N folded to any extent upon metal binding, indicating that the Asp/Asn side chains stabilize the central loop structure and thus propagate folding of the peripheral helices, whereas neither Ala nor Glu appears to be interacting with the metal to organize the loop. Finally, we investigated the longer range context of a given loop substitution by cloning and expressing a lanthanide-binding homeodomain (C2), whose loop insertion sequence is analogous to that of peptide P3W(D). We find by CD that apo-C2 has a significant helical structure (approximately 25% alphahelicity), which increases further upon the addition of Tb(III) (approximately 32% alpha helicity). The protein's Tb(III) affinity is similar to that of the chimeric peptides. However, unlike previously reported metallopeptides, we find that EuC2 does not appreciably promote phosphate or DNA cleavage, which suggests a difference in metal accessibility in the context of the full domain. We have demonstrated that substituting beta turns with metal-binding turns does not necessarily require homologous parental scaffolds or small flexible peptides but rather relies on the structural similarity of the motifs flanking the turn.

Engineered lanthanide-binding metallohomeodomains: designing folded chimeras by modular turn substitution.

A series of chimeric metallohomeodomains are described, engineered by rational design of a flexible Ca/Ln binding site into a DNA-binding scaffold. A modular turn-substitution approach was used to create proteins that both bind DNA and lanthanide ions, while retaining the secondary structure of the full homeodomain (determined by circular dichroism [CD]). Four similar metallohomeodomains were designed (C1-C4), their structural stability predicted by molecular dynamics (MD) simulation of loop-mutations into the known homeodomain structure, and each designed protein cloned, expressed, and purified using standard molecular biology techniques. Two of the four loop insertions resulted in folded, metal- and DNA-binding proteins (EuC2 Kd = 2.1 +/- 0.4 microM; EuC4 Kd = 3.2 +/- 1.0 microM). These results show the successful incorporation of a metal site into a full protein domain, without compromising long-range structure. This is an important achievement in biomolecular design, as it provides a critical starting point for exploring metallonuclease function and substrate accessibility in a well-organized chimeric protein domain (rather than only in small HTH peptide systems).

Sequence preference in DNA binding: de novo designed helix-turn-helix metallopeptides recognize a family of DNA target sites.

The DNA-binding behavior and target sequences of two designed metallopeptides have been investigated with an iterative electrophoresis mobility shift assay followed by PCR amplification, and by circular dichroism spectroscopy. Peptides P3W and P5b were designed based on the structural similarity of the helix-turn-helix motif of homeodomains and the EF-hand motifs of calmodulin, as previously described for P3W. Like P3W, P5b binds both Eu(III) (K(d) = 12.6 +/- 1.9 microM) and Ca(II) (K(d) = 70 +/- 8 microM) with reasonable affinity. Binding selection from a library of randomized 8-mer DNA oligonucleotide sequences identified one target family for CaP5b [5'-pur-T-pur-G-(G/C)-3'], and two target sites for CaP3W [5'-(A/T)-G-G-G-(T/C)-3' and 5'-A-T-(G/T)-T-G-3']. Circular dichroism studies indicate that unlike EuP3W, EuP5b is poorly folded in the absence of DNA. In the presence of DNA containing target-binding sites for both peptides, both EuP3W and EuP5b increase in helical content, in the latter case significantly. These results suggest that EuP5b binding to target DNA involves an induced-fit mechanism. These small chimeric metallopeptides have been found to bind selectively to DNA targets, analogous to natural protein-DNA interactions. This corroborates our earlier conclusions (J. Am. Chem. Soc. 125:6656, 2003) that sequence-preferential DNA cleavage by Ce(IV)P3W was due to sequence recognition.

De novo design of a copper(II)-binding helix-turn-helix chimera: the prion octarepeat motif in a new context.

A chimeric Cu-binding peptide has been designed on the basis of a turn substitution of the prion (PrP) octarepeat Cu-binding site into the engrailed homeodomain helix-turn-helix motif (HTH). This system is a model for the investigation of a single PrP Cu-binding site in a defined protein context. The 28-mer Cu-HTH peptide P7 spectroscopically mimics the PrP octarepeat (P7 = TERRRQQLSHGGGWGEAQIKIWFQNKRA). The Cu(II)-binding affinity of P7 was determined by ESI-MS and tryptophan fluorescence titrations to be K(d) = 2.5 +/- 0.7 microM at pH = 7.0. The quenching of fluorescence of the Trp within the binding loop (underlined above) is pH dependent and highly specific for Cu(II). No Trp quenching was observed in the presence of divalent Zn, Mn, Co, Ni, or Ca ions, and ESI-MS titrations confirmed that these divalent ions do not appreciably bind to P7. The EPR spectrum of Cu(II)-P7 shows that the Cu environment is axial and consistent with 6-coordinate N(3)O(H(2)O)(2) or N(4)(H(2)O)(2) coordination (A( parallel) = 172 x10(-)(4) cm(-)(1); g( parallel) = 2.27), very similar to that of the PrP octarepeat itself. Also like PrP, circular dichroism studies show that apo P7 is predominantly disordered in solution, and the structure is slightly enhanced by Cu binding. These data show the Cu-PrP HTH peptide reproduces the Cu-binding behavior of a single PrP octarepeat in a new context.

Gadolinium-binding helix-turn-helix peptides: DNA-dependent MRI contrast agents.

A de novo designed gadolinium metallopeptide was found to be a very efficient relaxation agent, with 100% increase in relaxivity upon binding to DNA.

Europium luminescence of EF-hand helix-turn-helix chimeras: impact of pH and DNA-binding on europium coordination.

A series of Eu(III) metallopeptides, designed on the basis of the structural similarity of the helix-turn-helix and EF-hand motifs, have been studied by Eu(III) (7)F(0) --> (5)D(0) excitation spectroscopy. The impact of EF-hand ligand set differences on the hydration number and Eu(III) coordination environment are compared among the peptides. The conditional binding affinities were determined by Eu titration (P3, log K(a) = 6.0 +/- 0.4; P3W, log K(a) = 5.9 +/- 0.2; P5b, log K(a) = 5.3 +/- 0.1). Two similar coordination environments occur in each case, consistent with structural flexibility about the metal site. The coordination environments are consistent with 8- or 9-coordinate Eu(III), including six peptide-based ligands and two to three water molecules (P3, q = 1.9 +/- 0.2; P3W, q = 2.3 +/- 0.2; P4a, q = 1.9 +/- 0.3; P5b, q = 2.6 +/- 0.2). The Eu(III) (7)F(0) --> (5)D(0) excitation spectra are pH-dependent, as reported for several EF-hand proteins (oncomodulin, parvalbumin). A higher energy transition occurs at pH > 6, and has been assigned to deprotonation of coordinated water. The pK(a) leading to this new transition is dependent on Eu(III) Lewis acidity, which varies with the inner and outer sphere ligand set. The noncoordinating ninth position of the Eu-binding loop, which is poised to make second-sphere contacts to the coordinated water, stabilizes the deprotonated form of the coordinated solvent more effectively when it is Thr (P5b) than Asp (P3W). Upon DNA-binding by the metallopeptides, the pK(a) of the pH-dependent peak increases, but no new DNA-dependent transitions are observed. This indicates no DNA-based Eu(III) ligands are introduced, such as phosphate oxygen atoms of the DNA backbone. The hydration number decreases in the presence of DNA (P3W + DNA, q = 1.9 +/- 0.2; P5b + DNA, q = 1.7 +/- 0.2), indicating that DNA-binding by the metallopeptides organizes rather than compromises the Eu-binding site within the peptide.

Sequence-selective DNA cleavage by a chimeric metallopeptide.

A chimeric metallopeptide derived from the sequences of two structurally superimposable motifs was designed as an artificial nuclease. Both DNA recognition and nuclease activity have been incorporated into a small peptide sequence. P3W, a 33-mer peptide comprising helices alpha2 and alpha3 from the engrailed homeodomain and the consensus EF-hand Ca-binding loop binds one equivalent of lanthanides or calcium and folds upon metal binding. The conditional formation constants (in the presence of 50 mM Tris) of P3W for Eu(III) (K(a) = (2.1 +/- 0.1) x 10(5) M(-1)) and Ce(IV) (K(a) = (2.6 +/- 0.1) x 10(5) M(-1)) are typical of isolated EF-hand peptides. Circular dichroism studies show that 1:1 CeP3W is 26% alpha-helical and EuP3W is up to 40% alpha-helical in the presence of excess metal. The predicted helicity of the folded peptide based on helix length and end effects is about 50%, showing the metallopeptides are significantly folded. EuP3W has considerably more secondary structure than our previously reported chimeras (Welch, J. T.; Sirish, M.; Lindstrom, K. M.; Franklin, S. J. Inorg. Chem. 2001, 40, 1982-1984). Eu(III)P3W and Ce(IV)P3W nick supercoiled DNA at pH 6.9, although EuP3W is more active at pH 8. CeP3W cleaves linearized, duplex DNA as well as supercoiled plasmid. The cleavage of a 5'-(32)P-labeled 121-mer DNA fragment was followed by polyacrylamide gel electrophoresis. The cleavage products are 3'-OPO(3) termini exclusively, suggesting a regioselective or multistep mechanism. In contrast, uncomplexed Ce(IV) and Eu(III) ions produce both 3'-OPO(3) and 3'-OH, and no evidence of 4'-oxidative cleavage termini with either metal. The complementary 3'-(32)P-labeled oligonucleotide experiment also showed both 5'-OPO(3) and 5'-OH termini were produced by the free ions, whereas CeP3W produces only 5'-OPO(3) termini. In addition to apparent regioselectivity, the metallopeptides cut DNA with modest sequence discrimination, which suggests that the HTH motif binds DNA as a folded domain and thus cleaves selected sequences. The de novo artificial nuclease LnP3W represents the first small, underivatized peptide that is both active as a nuclease and sequence selective.

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

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 alpha-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 microM) and for Eu(III) (6.2 +/- 0.3 microM) (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 helix-turn-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 metal-binding loop but also retains the alpha-alpha 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.

Hydrolytically active Eu(III) and Ce(IV) EF-hand peptides.

A chimeric peptide (P4) has been designed to incorporate an EF-hand metal-binding loop into the context of the helix-turn-helix DNA binding motif of the engrailed homeodomain. This construct binds lanthanides, and in the presence of these metals, promotes the cleavage of supercoiled DNA and model phosphate esters (bisnitrophenyl phosphate). P4 binds lanthanides with moderate affinities (Eu(III), log K(a)=4.85; and Ce(IV), log K(a)=5.23). The structure of P4 is enhanced by metal binding, but the increase in secondary structure observed by CD is small, and suggests the metallopeptide is also quite flexible. Despite this flexibility, the efficient cleavage of DNA at low concentrations is dependent on the metallopeptide, and not on peptide or metal alone. This enhanced reactivity suggests the designed DNA-binding EF-hand peptides deliver the metal to the DNA for catalysis, even without rigid secondary structure.

Kinetics of the Formation of Macrocyclic Polyaminocarboxylate Ligand Complexes: A Laser-Excited Luminescence Study of the Eu(3+)-dtpa-dien System.

Excitation spectroscopy of the (7)F(0) --> (5)D(0) transition of Eu(3+) is used to detect and characterize a kinetic intermediate in the formation of a complex between Eu(3+) and the macrocyclic ligand dtpa-dien (1,4,7-tris(carboxymethyl)-9,17-dioxo-1,4,7,10,13,16-hexaazacyclooctadecane). Both the long-lived intermediate and the final product, [Eu(Hdtpa-dien)(H(2)O)](+), are formed immediately upon mixing the components, as evidenced by separate peaks in the excitation spectrum. The transformation of the intermediate to the final product, monitored by excitation spectroscopy, occurs by both proton-assisted and non-proton-assisted pathways. It is proposed that the intermediate represents a "blind alley" in the pathway to a final nine-coordinate tricapped trigonal prismatic product. The intermediate with an "up, down, up" configuration of carboxylate arms must undergo some decoordination to form the final complex. Molecular mechanics calculations and excited state lifetimes suggest that the intermediate is eight-coordinate with one coordinated water molecule. The stability constant of the final complex is found to be log K = 14.11 +/- 0.05.