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A switch from parallel to antiparallel strand orientation in a coiled-coil X-ray structure via two core hydrophobic mutations.

ABSTRACT: The coiled-coil is one of the most ubiquitous and well studied protein structural motifs. Significant effort has been devoted to dissecting subtle variations of the typical heptad repeat sequence pattern that can designate larger topological features such as relative ?-helical orientation and oligomer size. Here we report the X-ray structure of a model coiled-coil peptide, HA2-Del-L2seM, which forms an unanticipated core antiparallel dimer with potential sites for discrete higher-order multimerization (trimer or tetramer). In the X-ray structure, a third, partially-ordered ?-helix is weakly associated with the antiparallel dimer and analytical ultracentrifugation experiments indicate the peptide forms a well-defined tetramer in solution. The HA2-Del-L2seM sequence is closely related to a parent model peptide, HA2-Del, which we previously reported adopts a parallel trimer; HA2-Del-L2seM differs by only hydrophobic leucine to selenomethione mutations and thus this subtle difference is sufficient to switch both relative ?-helical topology and number of ?-helices participating in the coiled-coil. Comparison of the X-ray structures of HA2-Del-L2seM (reported here) with the HA2-Del parent (reported previously) reveals novel interactions involving the selenomethionine residues that promote antiparallel coiled-coil configuration and preclude parallel trimer formation. These novel atomic insights are instructive for understanding subtle features that can affect coiled-coil topology and provide additional information for design of antiparallel coiled-coils.

SUBMITTER: Malashkevich VN

PROVIDER: S-EPMC4768791 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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A switch from parallel to antiparallel strand orientation in a coiled-coil X-ray structure via two core hydrophobic mutations.

Malashkevich Vladimir N VN Higgins Chelsea D CD Almo Steven C SC Lai Jonathan R JR

Biopolymers 20150501 3

The coiled-coil is one of the most ubiquitous and well studied protein structural motifs. Significant effort has been devoted to dissecting subtle variations of the typical heptad repeat sequence pattern that can designate larger topological features such as relative α-helical orientation and oligomer size. Here we report the X-ray structure of a model coiled-coil peptide, HA2-Del-L2seM, which forms an unanticipated core antiparallel dimer with potential sites for discrete higher-order multimeri ...[more]

PMID: 25753192

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Project description:The alpha-helical coiled coil is a structurally simple protein oligomerization or interaction motif consisting of two or more alpha helices twisted into a supercoiled bundle. Coiled coils can differ in their stoichiometry, helix orientation, and axial alignment. Because of the near degeneracy of many of these variants, coiled coils pose a challenge to fold recognition methods for structure prediction. Whereas distinctions between some protein folds can be discriminated on the basis of hydrophobic/polar patterning or secondary structure propensities, the sequence differences that encode important details of coiled-coil structure can be subtle. This is emblematic of a larger problem in the field of protein structure and interaction prediction: that of establishing specificity between closely similar structures. We tested the behavior of different computational models on the problem of recognizing the correct orientation--parallel vs. antiparallel--of pairs of alpha helices that can form a dimeric coiled coil. For each of 131 examples of known structure, we constructed a large number of both parallel and antiparallel structural models and used these to assess the ability of five energy functions to recognize the correct fold. We also developed and tested three sequence-based approaches that make use of varying degrees of implicit structural information. The best structural methods performed similarly to the best sequence methods, correctly categorizing approximately 81% of dimers. Steric compatibility with the fold was important for some coiled coils we investigated. For many examples, the correct orientation was determined by smaller energy differences between parallel and antiparallel structures distributed over many residues and energy components. Prediction methods that used structure but incorporated varying approximations and assumptions showed quite different behaviors when used to investigate energetic contributions to orientation preference. Sequence based methods were sensitive to the choice of residue-pair interactions scored.

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 switch from parallel to antiparallel strand orientation in a coiled-coil X-ray structure via two core hydrophobic mutations.

Publications

A switch from parallel to antiparallel strand orientation in a coiled-coil X-ray structure via two core hydrophobic mutations.

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