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

ABSTRACT: 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).

SUBMITTER: Lim S

PROVIDER: S-EPMC2242603 | biostudies-literature | 2006 Sep

REPOSITORIES: biostudies-literature

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

Lim Sunghyuk S Franklin Sonya J SJ

Protein science : a publication of the Protein Society 20060901 9

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-mut ...[more]

PMID: 16943445

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Project description:BACKGROUND: The pyridoxal-5'-phosphate (PLP)-dependent or vitamin B6-dependent enzymes that catalyze manifold reactions in the metabolism of amino acids belong to no fewer than four evolutionarily independent protein families. The multiple evolutionary origin and the essential mechanistic role of PLP in these enzymes argue for the cofactor having arrived on the evolutionary scene before the emergence of the respective apoenzymes and having played a dominant role in the molecular evolution of the B6 enzyme families. Here we report on an attempt to re-enact the emergence of a PLP-dependent protoenzyme. The starting protein was pancreatic ribonuclease A (RNase), in which active-site Lys41 or Lys7 readily form a covalent adduct with PLP. RESULTS: We screened the PLP adduct of wild-type RNase and two variant RNases (K7R and K41R) for catalytic effects toward L- and D-amino acids. RNase(K41R)-PLP, in which the cofactor is bound through an imine linkage to Lys7, qualifies for a model proto-B6 enzyme by the following criteria: (1) covalent linkage of PLP (internal aldimine); (2) catalytic activity toward amino acids that depends on formation of an imine linkage with the substrate (external aldimine); (3) adjoining binding sites for the cofactor and amino acid moiety that facilitate the transimination reaction of the internal to the external aldimine and stabilize the resulting noncovalent complex of the coenzyme-substrate adduct with the protein; (4) reaction specificity, the only detectable reactions being racemization of diverse amino acids and beta-decarboxylation of L-aspartate; (5) acceleration factors for racemization and beta-decarboxylation of >103 over and above that of PLP alone; (6) ribonuclease activity that is 103-fold lower than that of wild-type RNase, attenuation of a pre-existing biological activity being indispensable for the further evolution as a PLP-dependent protoenzyme. CONCLUSION: A single amino acid substitution (Lys41Arg) and covalent binding of PLP to active-site Lys7 suffice to turn pancreatic ribonuclease A into a protein catalyst that complies with all plausible criteria for a proto-B6 enzyme. The study thus retraces in a model system what may be considered the committed step in the molecular evolution of a potential ancestor of a B6 enzyme family.

Dataset Information

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

Publications

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

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