Polypeptides and polynucleotides are natural programmable biopolymers that can self-assemble into complex tertiary structures. We describe a system analogous to designed DNA nanostructures in which protein coiled-coil (CC) dimers serve as building blocks for modular de novo design of polyhedral protein cages that efficiently self-assemble in vitro and in vivo. We produced and characterized >20 single-chain protein cages in three shapes-tetrahedron, four-sided pyramid, and triangular prism-with the largest containing >700 amino-acid residues and measuring 11 nm in diameter. Their stability and folding kinetics were similar to those of natural proteins. Solution small-angle X-ray scattering (SAXS), electron microscopy (EM), and biophysical analysis confirmed agreement of the expressed structures with the designs. We also demonstrated self-assembly of a tetrahedral structure in bacteria, mammalian cells, and mice without evidence of inflammation. A semi-automated computational design platform and a toolbox of CC building modules are provided to enable the design of protein cages in any polyhedral shape.

The coiled-coil dimer is a widespread protein structural motif and, due to its designability, represents an attractive building block for assembling modular nanostructures. The specificity of coiled-coil dimer pairing is mainly based on hydrophobic and electrostatic interactions between residues at positions a, d, e, and g of the heptad repeat. Binding affinity, on the other hand, can also be affected by surface residues that face away from the dimerization interface. Here we show how design of the local helical propensity of interacting peptides can be used to tune the stabilities of coiled-coil dimers over a wide range. By designing intramolecular charge pairs, regions of high local helical propensity can be engineered to form trigger sequences, and dimer stability is adjusted without changing the peptide length or any of the directly interacting residues. This general principle is demonstrated by a change in thermal stability by more than 30 °C as a result of only two mutations outside the binding interface. The same approach was successfully used to modulate the stabilities in an orthogonal set of coiled-coils without affecting their binding preferences. The stability effects of local helical propensity and peptide charge are well described by a simple linear model, which should help improve current coiled-coil stability prediction algorithms. Our findings enable tuning the stabilities of coiled-coil-based building modules match a diverse range of applications in synthetic biology and nanomaterials.

Understanding the molecular basis of ligand–DNA-binding events, and its application to the rational design of novel drugs, requires knowledge of the structural features and forces that drive the corresponding recognition processes. Existing structural evidence on DNA complexation with classical minor groove-directed ligands and the corresponding studies of binding energetics have suggested that this type of binding can be described as a rigid-body association. In contrast, we show here that the binding-coupled conformational changes may be crucial for the interpretation of DNA (hairpin) association with a classical minor groove binder (netropsin). We found that, although the hairpin form is the only accessible state of ligand-free DNA, its association with the ligand may lead to its transition into a duplex conformation. It appears that formation of the fully ligated duplex from the ligand-free hairpin, occurring via two pathways, is enthalpically driven and accompanied by a significant contribution of the hydrophobic effect. Our thermodynamic and structure-based analysis, together with corresponding theoretical studies, shows that none of the predicted binding steps can be considered as a rigid-body association. In this light we anticipate our thermodynamic approach to be the basis of more sophisticated nucleic acid recognition mechanisms, which take into account the dynamic nature of both the nucleic acid and the ligand molecule.

Intrinsically disordered proteins (IDPs) are proteins that lack a unique three-dimensional structure in their native state. Many have, however, been found to fold into a defined structure when interacting with specific binding partners. The energetic implications of such behavior have been widely discussed, yet experimental thermodynamic data is scarce. We present here a thorough thermodynamic and structural study of the binding of an IDP (antitoxin CcdA) to its molecular target (gyrase poison CcdB). We show that the binding-coupled folding of CcdA is driven by a combination of specific intramolecular interactions that favor the final folded structure and a less specific set of intermolecular contacts that provide a desolvation entropy boost. The folded structure of the bound IDP appears to be defined largely by its own amino acid sequence, with the binding partner functioning more as a facilitator than a mold to conform to. On the other hand, specific intermolecular interactions do increase the binding affinity up to the picomolar range. Overall, this study shows how an IDP can achieve very strong and structurally well-defined binding and it provides significant insight into the molecular forces that enable such binding properties.

Autotransporter (AT) proteins provide a diverse array of important virulence functions to Gram-negative bacterial pathogens, and have also been adapted for protein surface display applications. The “autotransporter” moniker refers to early models that depicted these proteins facilitating their own translocation across the bacterial outer membrane. Although translocation is less autonomous than originally proposed, AT protein segments upstream of the C-terminal transmembrane β-barrel have nevertheless consistently been found to contribute to efficient translocation and/or folding of the N-terminal virulence region (the “passenger”). However, defining the precise secretion functions of these AT regions has been complicated by the use of multiple overlapping and ambiguous terms to define AT sequence, structural, and functional features, including “autochaperone”, “linker” and “junction”. Moreover, the precise definitions and boundaries of these features vary among ATs and even among research groups, leading to an overall murky picture of the contributions of specific features to translocation. Here we propose a unified, unambiguous nomenclature for AT structural, functional and conserved sequence features, based on explicit criteria. Applied to 16 well studied AT proteins, this nomenclature reveals new commonalities for translocation but also highlights that the autochaperone function is less closely coupled with a conserved sequence element than previously believed.

Background: Many virulence proteins are secreted using the autotransporter system. Results: Autotransporter proteins do not fold until secreted and are secreted poorly in the absence of folding. Conclusion: Folding helps drive autotransporter secretion, but another energy source is required for its initiation. Significance: Our conclusions reconcile apparent contradictions in the literature and importantly contribute to understanding and manipulating autotransporter secretion.

Folding and unfolding of many biological macromolecules can be characterized thermodynamically, yielding a wealth of information about the stability of various conformations and the interactions that hold them together. The relevant thermodynamic parameters are usually obtained by employing spectroscopic and/or calorimetric techniques and fitting an appropriate thermodynamic model to the experimental data. In this work, we compare the traditional approach of fitting the thermodynamic model to experimental data obtained from each experiment individually and the global approach of simultaneously fitting the model to all available data from different experiments. On the basis of several specific examples of DNA and protein unfolding, we demonstrate that piece-by-piece verification of the proposed thermodynamic model using individual fits is frequently inappropriate and can result in an incorrect mechanism and thermodynamics of the studied unfolding process. We find that while the two approaches are complementary in some aspects of analysis global fitting is essential for the appropriate selection and critical evaluation of the model mechanism. Only a good global fit thus gives us confidence that the obtained thermodynamic parameters of unfolding have real physical meaning.

Bacteria can become transiently tolerant to several classes of antibiotics. This phenomenon known as persistence is regulated by small genetic elements called toxin–antitoxin modules with intricate yet often poorly understood self-regulatory features. Here, we describe the structures of molecular complexes and interactions that drive the transcription regulation of the ccdAB toxin–antitoxin module. Low specificity and affinity of the antitoxin CcdA2 for individual binding sites on the operator are enhanced by the toxin CcdB2, which bridges the CcdA2 dimers. This results in a unique extended repressing complex that spirals around the operator and presents equally spaced DNA binding sites. The multivalency of binding sites induces a digital on-off switch for transcription, regulated by the toxin:antitoxin ratio. The ratio at which this switch occurs is modulated by non-specific interactions with the excess chromosomal DNA. Altogether, we present the molecular mechanisms underlying the ratio-dependent transcriptional regulation of the ccdAB operon.