CCBuilder: an interactive web-based tool for building, designing and assessing coiled-coil protein assemblies.
ABSTRACT: The ability to accurately model protein structures at the atomistic level underpins efforts to understand protein folding, to engineer natural proteins predictably and to design proteins de novo. Homology-based methods are well established and produce impressive results. However, these are limited to structures presented by and resolved for natural proteins. Addressing this problem more widely and deriving truly ab initio models requires mathematical descriptions for protein folds; the means to decorate these with natural, engineered or de novo sequences; and methods to score the resulting models.We present CCBuilder, a web-based application that tackles the problem for a defined but large class of protein structure, the α-helical coiled coils. CCBuilder generates coiled-coil backbones, builds side chains onto these frameworks and provides a range of metrics to measure the quality of the models. Its straightforward graphical user interface provides broad functionality that allows users to build and assess models, in which helix geometry, coiled-coil architecture and topology and protein sequence can be varied rapidly. We demonstrate the utility of CCBuilder by assembling models for 653 coiled-coil structures from the PDB, which cover >96% of the known coiled-coil types, and by generating models for rarer and de novo coiled-coil structures.CCBuilder is freely available, without registration, at http://coiledcoils.chm.bris.ac.uk/app/cc_builder/.
Project description:Because the space of folded protein structures is highly degenerate, with recurring secondary and tertiary motifs, methods for representing protein structure in terms of collective physically relevant coordinates are of great interest. By collapsing structural diversity to a handful of parameters, such methods can be used to delineate the space of designable structures (i.e., conformations that can be stabilized with a large number of sequences)-a crucial task for de novo protein design. We first demonstrate this on natural ?-helical coiled coils using the Crick parameterization. We show that over 95% of known coiled-coil structures are within 1-Å C(?) root mean square deviation of a Crick-ideal backbone. Derived parameters show that natural geometric space of coiled coils is highly restricted and can be represented by "allowed" conformations amidst a potential continuum of conformers. Allowed structures have (1) restricted axial offsets between helices, which differ starkly between parallel and anti-parallel structures; (2) preferred superhelical radii, which depend linearly on the oligomerization state; (3) pronounced radius-dependent a- and d-position amino acid propensities; and (4) discrete angles of rotation of helices about their axes, which are surprisingly independent of oligomerization state or orientation. In all, we estimate the space of designable coiled-coil structures to be reduced at least 160-fold relative to the space of geometrically feasible structures. To extend the benefits of structural parameterization to other systems, we developed a general mathematical framework for parameterizing arbitrary helical structures, which reduces to the Crick parameterization as a special case. The method is successfully validated on a set of non-coiled-coil helical bundles, frequent in channels and transporter proteins, which show significant helix bending but not supercoiling. Programs for coiled-coil parameter fitting and structure generation are provided via a web interface at http://www.gevorggrigoryan.com/cccp/, and code for generalized helical parameterization is available upon request.
Project description:Motivation:To understand protein structure, folding and function fully and to design proteins de novo reliably, we must learn from natural protein structures that have been characterized experimentally. The number of protein structures available is large and growing exponentially, which makes this task challenging. Indeed, computational resources are becoming increasingly important for classifying and analyzing this resource. Here, we use tools from graph theory to define an Atlas classification scheme for automatically categorizing certain protein substructures. Results:Focusing on the ?-helical coiled coils, which are ubiquitous protein-structure and protein-protein interaction motifs, we present a suite of computational resources designed for analyzing these assemblies. iSOCKET enables interactive analysis of side-chain packing within proteins to identify coiled coils automatically and with considerable user control. Applying a graph theory-based Atlas classification scheme to structures identified by iSOCKET gives the Atlas of Coiled Coils, a fully automated, updated overview of extant coiled coils. The utility of this approach is illustrated with the first formal classification of an emerging subclass of coiled coils called ?-helical barrels. Furthermore, in the Atlas, the known coiled-coil universe is presented alongside a partial enumeration of the 'dark matter' of coiled-coil structures; i.e. those coiled-coil architectures that are theoretically possible but have not been observed to date, and thus present defined targets for protein design. Availability and implementation:iSOCKET is available as part of the open-source GitHub repository associated with this work (https://github.com/woolfson-group/isocket). This repository also contains all the data generated when classifying the protein graphs. The Atlas of Coiled Coils is available at: http://coiledcoils.chm.bris.ac.uk/atlas/app.
Project description:Peptide-based structures can be designed to yield artificial proteins with specific folding patterns and functions. Template-based assembly of peptide units is one design option, but the use of two orthogonal self-assembly principles, oligonucleotide triple helix and a coiled coil protein domain formation have never been realized for de novo protein design. Here, we show the applicability of peptide-oligonucleotide conjugates for self-assembly of higher-ordered protein-like structures. The resulting nano-assemblies were characterized by ultraviolet-melting, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering and transmission electron microscopy. These studies revealed the formation of the desired triple helix and coiled coil domains at low concentrations, while a dimer of trimers was dominating at high concentration. CD spectroscopy showed an extraordinarily high degree of ?-helicity for the peptide moieties in the assemblies. The results validate the use of orthogonal self-assembly principles as a paradigm for de novo protein design.
Project description:Short, alpha-helical coiled coils provide a simple, modular method to direct the assembly of proteins into higher order structures. We previously demonstrated that by genetically fusing de novo-designed coiled coils of the appropriate oligomerization state to a natural trimeric protein, we could direct the assembly of this protein into various geometrical cages. Here, we have extended this approach by appending a coiled coil designed to trimerize in response to binding divalent transition metal ions and thereby achieve metal ion-dependent assembly of a tetrahedral protein cage. Ni2+ , Co2+ , Cu2+ , and Zn2+ ions were evaluated, with Ni2+ proving the most effective at mediating protein assembly. Characterization of the assembled protein indicated that the metal ion-protein complex formed discrete globular structures of the diameter expected for a complex containing 12 copies of the protein monomer. Protein assembly could be reversed by removing metal ions with ethylenediaminetetraacetic acid or under mildly acidic conditions.
Project description:The increased availability of user-friendly and accessible computational tools for biomolecular modeling would expand the reach and application of biomolecular engineering and design. For protein modeling, one key challenge is to reduce the complexities of 3D protein folds to sets of parametric equations that nonetheless capture the salient features of these structures accurately. At present, this is possible for a subset of proteins, namely, repeat proteins. The ?-helical coiled coil provides one such example, which represents ? 3-5% of all known protein-encoding regions of DNA. Coiled coils are bundles of ? helices that can be described by a small set of structural parameters. Here we describe how this parametric description can be implemented in an easy-to-use web application, called CCBuilder 2.0, for modeling and optimizing both ?-helical coiled coils and polyproline-based collagen triple helices. This has many applications from providing models to aid molecular replacement for X-ray crystallography, in silico model building and engineering of natural and designed protein assemblies, and through to the creation of completely de novo "dark matter" protein structures. CCBuilder 2.0 is available as a web-based application, the code for which is open-source and can be downloaded freely. http://coiledcoils.chm.bris.ac.uk/ccbuilder2.We have created CCBuilder 2.0, an easy to use web-based application that can model structures for a whole class of proteins, the ?-helical coiled coil, which is estimated to account for 3-5% of all proteins in nature. CCBuilder 2.0 will be of use to a large number of protein scientists engaged in fundamental studies, such as protein structure determination, through to more-applied research including designing and engineering novel proteins that have potential applications in biotechnology.
Project description:The design of new proteins that expand the repertoire of natural protein structures represents a formidable challenge. Success in this area would increase understanding of protein structure and present new scaffolds that could be exploited in biotechnology and synthetic biology. Here we describe the design, characterization and X-ray crystal structure of a new coiled-coil protein. The de novo sequence forms a stand-alone, parallel, six-helix bundle with a channel running through it. Although lined exclusively by hydrophobic leucine and isoleucine side chains, the 6-Å channel is permeable to water. One layer of leucine residues within the channel is mutable, accepting polar aspartic acid and histidine side chains, which leads to subdivision and organization of solvent within the lumen. Moreover, these mutants can be combined to form a stable and unique (Asp-His)(3) heterohexamer. These new structures provide a basis for engineering de novo proteins with new functions.
Project description:Protein crystals have catalytic and materials applications and are central to efforts in structural biology and therapeutic development. Designing predetermined crystal structures can be subtle given the complexity of proteins and the noncovalent interactions that govern crystallization. De novo protein design provides an approach to engineer highly complex nanoscale molecular structures, and often the positions of atoms can be programmed with sub-? precision. Herein, a computational approach is presented for the design of proteins that self-assemble in three dimensions to yield macroscopic crystals. A three-helix coiled-coil protein is designed de novo to form a polar, layered, three-dimensional crystal having the P6 space group, which has a "honeycomb-like" structure and hexameric channels that span the crystal. The approach involves: (i) creating an ensemble of crystalline structures consistent with the targeted symmetry; (ii) characterizing this ensemble to identify "designable" structures from minima in the sequence-structure energy landscape and designing sequences for these structures; (iii) experimentally characterizing candidate proteins. A 2.1 ? resolution X-ray crystal structure of one such designed protein exhibits sub-? agreement [backbone root mean square deviation (rmsd)] with the computational model of the crystal. This approach to crystal design has potential applications to the de novo design of nanostructured materials and to the modification of natural proteins to facilitate X-ray crystallographic analysis.
Project description:The regular arrangements of ?-strands around a central axis in ?-barrels and of ?-helices in coiled coils contrast with the irregular tertiary structures of most globular proteins, and have fascinated structural biologists since they were first discovered. Simple parametric models have been used to design a wide range of ?-helical coiled-coil structures, but to date there has been no success with ?-barrels. Here we show that accurate de novo design of ?-barrels requires considerable symmetry-breaking to achieve continuous hydrogen-bond connectivity and eliminate backbone strain. We then build ensembles of ?-barrel backbone models with cavity shapes that match the fluorogenic compound DFHBI, and use a hierarchical grid-based search method to simultaneously optimize the rigid-body placement of DFHBI in these cavities and the identities of the surrounding amino acids to achieve high shape and chemical complementarity. The designs have high structural accuracy and bind and fluorescently activate DFHBI in vitro and in Escherichia coli, yeast and mammalian cells. This de novo design of small-molecule binding activity, using backbones custom-built to bind the ligand, should enable the design of increasingly sophisticated ligand-binding proteins, sensors and catalysts that are not limited by the backbone geometries available in known protein structures.
Project description:The STIL protein is essential for centriole replication and for the non-templated, de novo centriole biogenesis that is required for mammalian embryogenesis. Here we performed quantitative biophysical and structural analysis of the central short coiled coil domain (CCD) of STIL that is critical for its function. Using biophysical, biochemical and cell biology approaches, we identified the specific residues in the CCD that mediate the oligomerization, centrosomal localization and protein interactions of STIL. We characterized the structural properties of the coiled coil peptide using circular dichroism spectroscopy and size exclusion chromatography. We identified two regions in this domain, containing eight hydrophobic residues, which mediate the coiled coil oligomerization. Mutations in these residues destabilized the coiled coil thermodynamically but in most cases did not affect its secondary structure. Reconstituting mouse embryonic fibroblasts lacking endogenous Stil, we show that STIL oligomerization mediated by these residues is not only important for the centrosomal functions of STIL during the canonical duplication process but also for de-novo formation of centrosomes.
Project description:The assembly of individual protein subunits into large-scale symmetrical structures is widespread in nature and confers new biological properties. Engineered protein assemblies have potential applications in nanotechnology and medicine; however, a major challenge in engineering assemblies de novo has been to design interactions between the protein subunits so that they specifically assemble into the desired structure. Here we demonstrate a simple, generalizable approach to assemble proteins into cage-like structures that uses short de novo designed coiled-coil domains to mediate assembly. We assembled eight copies of a C3-symmetric trimeric esterase into a well-defined octahedral protein cage by appending a C4-symmetric coiled-coil domain to the protein through a short, flexible linker sequence, with the approximate length of the linker sequence determined by computational modeling. The structure of the cage was verified using a combination of analytical ultracentrifugation, native electrospray mass spectrometry, and negative stain and cryoelectron microscopy. For the protein cage to assemble correctly, it was necessary to optimize the length of the linker sequence. This observation suggests that flexibility between the two protein domains is important to allow the protein subunits sufficient freedom to assemble into the geometry specified by the combination of C4 and C3 symmetry elements. Because this approach is inherently modular and places minimal requirements on the structural features of the protein building blocks, it could be extended to assemble a wide variety of proteins into structures with different symmetries.