CCBuilder: an interactive web-based tool for building, designing and assessing coiled-coil protein assemblies.

MOTIVATION: 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. RESULTS: 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. AVAILABILITY AND IMPLEMENTATION: CCBuilder is freely available, without registration, at http://coiledcoils.chm.bris.ac.uk/app/cc_builder/.

Cryo-transmission electron microscopy structure of a gigadalton peptide fiber of de novo design.

Nature presents various protein fibers that bridge the nanometer to micrometer regimes. These structures provide inspiration for the de novo design of biomimetic assemblies, both to address difficulties in studying and understanding natural systems, and to provide routes to new biomaterials with potential applications in nanotechnology and medicine. We have designed a self-assembling fiber system, the SAFs, in which two small α-helical peptides are programmed to form a dimeric coiled coil and assemble in a controlled manner. The resulting fibers are tens of nm wide and tens of µm long, and, therefore, comprise millions of peptides to give gigadalton supramolecular structures. Here, we describe the structure of the SAFs determined to approximately 8 Å resolution using cryotransmission electron microscopy. Individual micrographs show clear ultrastructure that allowed direct interpretation of the packing of individual α-helices within the fibers, and the construction of a 3D electron density map. Furthermore, a model was derived using the cryotransmission electron microscopy data and side chains taken from a 2.3 Å X-ray crystal structure of a peptide building block incapable of forming fibers. This was validated using single-particle analysis techniques, and was stable in prolonged molecular-dynamics simulation, confirming its structural viability. The level of self-assembly and self-organization in the SAFs is unprecedented for a designed peptide-based material, particularly for a system of considerably reduced complexity compared with natural proteins. This structural insight is a unique high-resolution description of how α-helical fibrils pack into larger protein fibers, and provides a basis for the design and engineering of future biomaterials.

A de novo peptide hexamer with a mutable channel.

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.

Bipartite design of a self-fibrillating protein copolymer with nanopatterned peptide display capabilities.

The development of biomatrices for technological and biomedical applications employs self-assembled scaffolds built from short peptidic motifs. However, biopolymers composed of protein domains would offer more varied molecular frames to introduce finer and more complex functionalities in bioreactive scaffolds using bottom-up approaches. Yet, the rules governing the three-dimensional organization of protein architectures in nature are complex and poorly understood. As a result, the synthetic fabrication of ordered protein association into polymers poses major challenges to bioengineering. We have now fabricated a self-assembling protein nanofiber with predictable morphologies and amenable to bottom-up customization, where features supporting function and assembly are spatially segregated. The design was inspired by the cross-linking of titin filaments by telethonin in the muscle sarcomere. The resulting fiber is a two-protein system that has nanopatterned peptide display capabilities as shown by the recruitment of functionalized gold nanoparticles at regular intervals of ∼ 5 nm, yielding a semiregular linear array over micrometers. This polymer promises the uncomplicated display of biologically active motifs to selectively bind and organize matter in the fine nanoscale. Further, its conceptual design has high potential for controlled plurifunctionalization.

Structural and kinetic studies on native intermediates and an intermediate analogue in benzoylformate decarboxylase reveal a least motion mechanism with an unprecedented short-lived predecarboxylation intermediate.

The thiamin diphosphate- (ThDP-) dependent enzyme benzoylformate decarboxylase (BFDC) catalyzes the nonoxidative decarboxylation of benzoylformic acid to benzaldehyde and carbon dioxide. To date, no structural information for a cofactor-bound reaction intermediate in BFDC is available. For kinetic analysis, a chromophoric substrate analogue was employed that produces various absorbing intermediates during turnover but is a poor substrate with a 10(4)-fold compromised kcat. Here, we have analyzed the steady-state distribution of native intermediates by a combined chemical quench/1H NMR spectroscopic approach and estimated the net rate constants of elementary catalytic steps. At substrate saturation, carbonyl addition of the substrate to the cofactor (k' approximately 500 s-1 at 30 degrees C) and elimination of benzaldehyde (k' approximately 2.400 s-1) were found to be partially rate-determining for catalysis, whereas decarboxylation of the transient 2-mandelyl-ThDP intermediate is 1 order of magnitude faster with k' approximately 16.000 s-1, the largest rate constant of decarboxylation in any thiamin enzyme characterized so far. The X-ray structure of a predecarboxylation intermediate analogue was determined to 1.6 A after cocrystallization of BFDC from Pseudomonas putida with benzoylphosphonic acid methyl ester. In contrast to the free acid, for which irreversible phosphorylation of active center Ser26 was reported, the methyl ester forms a covalent adduct with ThDP with a similar configuration at C2alpha as observed for other thiamin enzymes. The C2-C2alpha bond of the intermediate analogue is out of plane by 7degrees, indicating strain. The phosphonate part of the adduct forms hydrogen bonds with Ser26 and His281, and the 1-OH group is held in place by interactions with His70 and the 4'-amino group of ThDP. The phenyl ring accommodates in a hydrophobic pocket formed by Phe464, Phe397, Leu109, and Leu403. A comparison with the previously determined structure of BFDC in noncovalent complex with the inhibitor (R)-mandelate suggests a least motion mechanism. Binding of benzoylphosphonic acid methyl ester to BFDC was further characterized by CD spectroscopy and stopped-flow kinetics, indicating a two-step binding mechanism with a 200-fold slower carbonyl addition to ThDP than determined for benzoylformic acid, in line with the observed slight structural reorganization of Phe464 due to steric clashes with the phosphonate moiety.

Activation of anthranilate phosphoribosyltransferase from Sulfolobus solfataricus by removal of magnesium inhibition and acceleration of product release .

Anthranilate phosphoribosyltransferase from the hyperthermophilic archaeon Sulfolobus solfataricus (ssAnPRT) is encoded by the sstrpD gene and catalyzes the reaction of anthranilate (AA) with a complex of Mg(2+) and 5'-phosphoribosyl-alpha1-pyrophosphate (Mg.PRPP) to N-(5'-phosphoribosyl)-anthranilate (PRA) and pyrophosphate (PP(i)) within tryptophan biosynthesis. The ssAnPRT enzyme is highly thermostable (half-life at 85 degrees C = 35 min) but only marginally active at ambient temperatures (turnover number at 37 degrees C = 0.33 s(-1)). To understand the reason for the poor catalytic proficiency of ssAnPRT, we have isolated from an sstrpD library the activated ssAnPRT-D83G + F149S double mutant by metabolic complementation of an auxotrophic Escherichia coli strain. Whereas the activity of purified wild-type ssAnPRT is strongly reduced in the presence of high concentrations of Mg(2+) ions, this inhibition is no longer observed in the double mutant and the ssAnPRT-D83G single mutant. The comparison of the crystal structures of activated and wild-type ssAnPRT shows that the D83G mutation alters the binding mode of the substrate Mg.PRPP. Analysis of PRPP and Mg(2+)-dependent enzymatic activity indicates that this leads to a decreased affinity for a second Mg(2+) ion and thus reduces the concentration of enzymes with the inhibitory Mg(2).PRPP complex bound to the active site. Moreover, the turnover number of the double mutant ssAnPRT-D83G + F149S is elevated 40-fold compared to the wild-type enzyme, which can be attributed to an accelerated release of the product PRA. This effect appears to be mainly caused by an increased conformational flexibility induced by the F149S mutation, a hypothesis which is supported by the reduced thermal stability of the ssAnPRT-F149S single mutant.

Construction of a Chassis for a Tripartite Protein-Based Molecular Motor.

Improving our understanding of biological motors, both to fully comprehend their activities in vital processes, and to exploit their impressive abilities for use in bionanotechnology, is highly desirable. One means of understanding these systems is through the production of synthetic molecular motors. We demonstrate the use of orthogonal coiled-coil dimers (including both parallel and antiparallel coiled coils) as a hub for linking other components of a previously described synthetic molecular motor, the Tumbleweed. We use circular dichroism, analytical ultracentrifugation, dynamic light scattering, and disulfide rearrangement studies to demonstrate the ability of this six-peptide set to form the structure designed for the Tumbleweed motor. The successful formation of a suitable hub structure is both a test of the transferability of design rules for protein folding as well as an important step in the production of a synthetic protein-based molecular motor.

New currency for old rope: from coiled-coil assemblies to α-helical barrels.

α-Helical coiled coils are ubiquitous protein-protein-interaction domains. They share a relatively straightforward sequence repeat, which directs the folding and assembly of amphipathic α-helices. The helices can combine in a number of oligomerisation states and topologies to direct a wide variety of protein assemblies. Although in nature parallel dimers, trimers and tetramers dominate, the potential to form larger oligomers and more-complex assemblies has long been recognised. In particular, complexes above pentamer are interesting because they are barrel-like, having central channels or pores with well-defined dimensions and chemistry. Recent empirical and rational design experiments are beginning to chart this potential new territory in coiled-coil space, leading to intriguing new structures, and possibilities for functionalisation and applications.

The intracellular Ig fold: a robust protein scaffold for the engineering of molecular recognition.

Protein scaffolds that support molecular recognition have multiple applications in biotechnology. Thus, protein frames with robust structural cores but adaptable surface loops are in continued demand. Recently, notable progress has been made in the characterization of Ig domains of intracellular origin--in particular, modular components of the titin myofilament. These Ig belong to the I(intermediate)-type, are remarkably stable, highly soluble and undemanding to produce in the cytoplasm of Escherichia coli. Using the Z1 domain from titin as representative, we show that the I-Ig fold tolerates the drastic diversification of its CD loop, constituting an effective peptide display system. We examine the stability of CD-loop-grafted Z1-peptide chimeras using differential scanning fluorimetry, Fourier transform infrared spectroscopy and nuclear magnetic resonance and demonstrate that the introduction of bioreactive affinity binders in this position does not compromise the structural integrity of the domain. Further, the binding efficiency of the exogenous peptide sequences in Z1 is analyzed using pull-down assays and isothermal titration calorimetry. We show that an internally grafted, affinity FLAG tag is functional within the context of the fold, interacting with the anti-FLAG M2 antibody in solution and in affinity gel. Together, these data reveal the potential of the intracellular Ig scaffold for targeted functionalization.

A basis set of de novo coiled-coil peptide oligomers for rational protein design and synthetic biology.

Protein engineering, chemical biology, and synthetic biology would benefit from toolkits of peptide and protein components that could be exchanged reliably between systems while maintaining their structural and functional integrity. Ideally, such components should be highly defined and predictable in all respects of sequence, structure, stability, interactions, and function. To establish one such toolkit, here we present a basis set of de novo designed α-helical coiled-coil peptides that adopt defined and well-characterized parallel dimeric, trimeric, and tetrameric states. The designs are based on sequence-to-structure relationships both from the literature and analysis of a database of known coiled-coil X-ray crystal structures. These give foreground sequences to specify the targeted oligomer state. A key feature of the design process is that sequence positions outside of these sites are considered non-essential for structural specificity; as such, they are referred to as the background, are kept non-descript, and are available for mutation as required later. Synthetic peptides were characterized in solution by circular-dichroism spectroscopy and analytical ultracentrifugation, and their structures were determined by X-ray crystallography. Intriguingly, a hitherto widely used empirical rule-of-thumb for coiled-coil dimer specification does not hold in the designed system. However, the desired oligomeric state is achieved by database-informed redesign of that particular foreground and confirmed experimentally. We envisage that the basis set will be of use in directing and controlling protein assembly, with potential applications in chemical and synthetic biology. To help with such endeavors, we introduce Pcomp, an on-line registry of peptide components for protein-design and synthetic-biology applications.