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A set of computationally designed orthogonal antiparallel homodimers that expands the synthetic coiled-coil toolkit.

ABSTRACT: Molecular engineering of protein assemblies, including the fabrication of nanostructures and synthetic signaling pathways, relies on the availability of modular parts that can be combined to give different structures and functions. Currently, a limited number of well-characterized protein interaction components are available. Coiled-coil interaction modules have been demonstrated to be useful for biomolecular design, and many parallel homodimers and heterodimers are available in the coiled-coil toolkit. In this work, we sought to design a set of orthogonal antiparallel homodimeric coiled coils using a computational approach. There are very few antiparallel homodimers described in the literature, and none have been measured for cross-reactivity. We tested the ability of the distance-dependent statistical potential DFIRE to predict orientation preferences for coiled-coil dimers of known structure. The DFIRE model was then combined with the CLASSY multistate protein design framework to engineer sets of three orthogonal antiparallel homodimeric coiled coils. Experimental measurements confirmed the successful design of three peptides that preferentially formed antiparallel homodimers that, furthermore, did not interact with one additional previously reported antiparallel homodimer. Two designed peptides that formed higher-order structures suggest how future design protocols could be improved. The successful designs represent a significant expansion of the existing protein-interaction toolbox for molecular engineers.

SUBMITTER: Negron C

PROVIDER: S-EPMC4277747 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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A set of computationally designed orthogonal antiparallel homodimers that expands the synthetic coiled-coil toolkit.

Negron Christopher C Keating Amy E AE

Journal of the American Chemical Society 20141113 47

Molecular engineering of protein assemblies, including the fabrication of nanostructures and synthetic signaling pathways, relies on the availability of modular parts that can be combined to give different structures and functions. Currently, a limited number of well-characterized protein interaction components are available. Coiled-coil interaction modules have been demonstrated to be useful for biomolecular design, and many parallel homodimers and heterodimers are available in the coiled-coil ...[more]

PMID: 25337788

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Project description:Deficits in DNA damage-repair pathways are the root cause of several human cancers. In mammalian cells, DNA double-strand break repair is carried out by multiple mechanisms, including homologous recombination (HR). The partner and localizer of BRCA2 (PALB2), which is an essential factor for HR, binds to the breast cancer susceptibility 1 (BRCA1) protein at DNA double-strand breaks. At the break site, PALB2 also associates with the breast cancer susceptibility 2 (BRCA2) protein to form a multiprotein complex that facilitates HR. The BRCA1-PALB2 interaction is mediated by association of predicted helical coiled-coil regions in both proteins. PALB2 can also homodimerize through the formation of a coiled coil by the self-association of helical elements at the N-terminus of the PALB2 protein, and this homodimerization has been proposed to regulate the efficiency of HR. We have produced a segment of PALB2, designated PALB2cc (PALB2 coiled coil segment) that forms ?-helical structures, which assemble into stable homodimers. PALB2cc also forms heterodimers with a helical segment of BRCA1, called BRCA1cc (BRCA1 coiled coil segment). The three-dimensional structure of the homodimer formed by PALB2cc was determined by solution NMR spectroscopy. This PALB2cc homodimer is a classical antiparallel coiled-coil leucine zipper. NMR chemical-shift perturbation studies were used to study dimer formation for both the PALB2cc homodimer and the PALB2cc/BRCA1cc heterodimer. The mutation of residue Leu24 of PALB2cc significantly reduces its homodimer stability, but has a more modest effect on the stability of the heterodimer formed between PALB2cc and BRCA1cc. We show that mutation of Leu24 leads to genomic instability and reduced cell viability after treatment with agents that induce DNA double-strand breaks. These studies may allow the identification of distinct mutations of PALB2cc that selectively disrupt homodimeric versus heterodimeric interactions, and reveal the specific role of PALB2cc homodimerization in HR.

Dataset Information

A set of computationally designed orthogonal antiparallel homodimers that expands the synthetic coiled-coil toolkit.

Publications

A set of computationally designed orthogonal antiparallel homodimers that expands the synthetic coiled-coil toolkit.

Similar Datasets

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