Coiled-Coil Interaction Toolbox for Engineering Mammalian Cells.

Protein interactions play a crucial role in a variety of biological processes. Therefore, regulation of these interactions has received considerable attention in terms of synthetic biology tool development. Of those, a toolbox of small peptides known as coiled coils (CCs) represents a unique effective tool for mediating protein-protein interactions because their binding specificity and affinity can be designed and controlled. CC peptides have been used as a building module for designing synthetic regulatory circuits in mammalian cells, construction of fast response to a signal, amplification of the response, and localization and regulation of function of diverse proteins. In this chapter, we describe a designed set of CCs used for mammalian cell engineering and provide a protocol for the construction of CC-mediated logic circuits in mammalian cells. Ultimately, these tools could be used for diverse biotechnological and therapeutic applications.

Construction of Reconfigurable and Polymorphic DNA Origami Assemblies with Coiled-Coil Patches and Patterns.

DNA origami nanodevices achieve programmable structure and tunable mechanical and dynamic properties by leveraging the sequence-specific interactions of nucleic acids. Previous advances have also established DNA origami as a useful building block to make well-defined micron-scale structures through hierarchical self-assembly, but these efforts have largely leveraged the structural features of DNA origami. The tunable dynamic and mechanical properties also provide an opportunity to make assemblies with adaptive structures and properties. Here the integration of DNA origami hinge nanodevices and coiled-coil peptides are reported into hybrid reconfigurable assemblies. With the same dynamic device and peptide interaction, it is made multiple higher-order assemblies (i.e., polymorphic assembly) by organizing clusters of peptides into patches or arranging single peptides into patterns on the surfaces of DNA origami to control the relative orientation of devices. The coiled-coil interactions are used to construct circular and linear assemblies whose structure and mechanical properties can be modulated with DNA-based reconfiguration. Reconfiguration of linear assemblies leads to micron scale motions and ≈2.5-10-fold increase in bending stiffness. The results provide a foundation for stimulus-responsive hybrid assemblies that can adapt their structure and properties in response to nucleic acid, peptide, protein, or other triggers.

Co-expression of distinct L1 retrotransposon coiled coils can lead to their entanglement.

L1 (LINE1) non-LTR retrotransposons are ubiquitous genomic parasites and the dominant transposable element in humans having generated about 40% of their genomic DNA during their ~ 100 million years (Myr) of activity in primates. L1 replicates in germ line cells and early embryos, causing genetic diversity and defects, but can be active in some somatic stem cells, tumors and during aging. L1 encodes two proteins essential for retrotransposition: ORF2p, a reverse transcriptase that contains an endonuclease domain, and ORF1p, a coiled coil mediated homo trimer, which functions as a nucleic acid chaperone. Both proteins contain highly conserved domains and preferentially bind their encoding transcript to form an L1 ribonucleoprotein (RNP), which mediates retrotransposition. However, the coiled coil has periodically undergone episodes of substantial amino acid replacement to the extent that a given L1 family can concurrently express multiple ORF1s that differ in the sequence of their coiled coils. Here we show that such distinct ORF1p sequences can become entangled forming heterotrimers when co-expressed from separate vectors and speculate on how coiled coil entanglement could affect coiled coil evolution.

X-ray structure of the metastable SEPT14-SEPT7 coiled coil reveals a hendecad region crucial for heterodimerization.

Septins are membrane-associated, GTP-binding proteins that are present in most eukaryotes. They polymerize to play important roles as scaffolds and/or diffusion barriers as part of the cytoskeleton. α-Helical coiled-coil domains are believed to contribute to septin assembly, and those observed in both human SEPT6 and SEPT8 form antiparallel homodimers. These are not compatible with their parallel heterodimeric organization expected from the current model for protofilament assembly, but they could explain the interfilament cross-bridges observed by microscopy. Here, the first structure of a heterodimeric septin coiled coil is presented, that between SEPT14 and SEPT7; the former is a SEPT6/SEPT8 homolog. This new structure is parallel, with two long helices that are axially shifted by a full helical turn with reference to their sequence alignment. The structure also has unusual knobs-into-holes packing of side chains. Both standard seven-residue (heptad) and the less common 11-residue (hendecad) repeats are present, creating two distinct regions with opposite supercoiling, which gives rise to an overall straight coiled coil. Part of the hendecad region is required for heterodimerization and therefore may be crucial for selective septin recognition. These unconventional sequences and structural features produce a metastable heterocomplex that nonetheless has enough specificity to promote correct protofilament assembly. For instance, the lack of supercoiling may facilitate unzipping and transitioning to the antiparallel homodimeric state.

New protein families with hendecad coiled coils in the proteome of life.

Coiled coils are a widespread and well understood protein fold. Their short and simple repeats underpin considerable structural and functional diversity. The vast majority of coiled coils consist of 7-residue (heptad) sequence repeats, but in essence most combinations of 3- and 4-residue segments, each starting with a residue of the hydrophobic core, are compatible with coiled-coil structure. The most frequent among these other repeat patterns are 11-residue (hendecad, 3 + 4 + 4) repeats. Hendecads are frequently found in low copy number, interspersed between heptads, but some proteins consist largely or entirely of hendecad repeats. Here we describe the first large-scale survey of these proteins in the proteome of life. For this, we scanned the protein sequence database for sequences with 11-residue periodicity that lacked ß-strand prediction. We then clustered these by pairwise similarity to construct a map of potential hendecad coiled-coil families. Here we discuss these according to their structural properties, their potential cellular roles, and the evolutionary mechanisms shaping their diversity. We note in particular the continuous amplification of hendecads, both within existing proteins and de novo from previously non-coding sequence, as a powerful mechanism in the genesis of new coiled-coil forms.

Affinity-controlled capture and release of engineered monoclonal antibodies by macroporous dextran hydrogels using coiled-coil interactions.

Long-term delivery is a successful strategy used to reduce the adverse effects of monoclonal antibody (mAb)-based treatments. Macroporous hydrogels and affinity-based strategies have shown promising results in sustained and localized delivery of the mAbs. Among the potential tools for affinity-based delivery systems, the de novo designed Ecoil and Kcoil peptides are engineered to form a high-affinity, heterodimeric coiled-coil complex under physiological conditions. In this study, we created a set of trastuzumab molecules tagged with various Ecoil peptides and evaluated their manufacturability and characteristics. Our data show that addition of an Ecoil tag at the C-termini of the antibody chains (light chains, heavy chains, or both) does not hinder the production of chimeric trastuzumab in CHO cells or affect antibody binding to its antigen. We also evaluated the influence of the number, length, and position of the Ecoil tags on the capture and release of Ecoil-tagged trastuzumab from macroporous dextran hydrogels functionalized with Kcoil peptide (the Ecoil peptide-binding partner). Notably, our data show that antibodies are released from the macroporous hydrogels in a biphasic manner; the first phase corresponding to the rapid release of residual, unbound trastuzumab from the macropores, followed by the affinity-controlled, slow-rate release of antibodies from the Kcoil-functionalized macropore surface.

Chain Sliding versus ß-Sheet Formation upon Shearing Single α-Helical Coiled Coils.

Coiled coils (CCs) are key building blocks of biogenic materials and determine their mechanical response to large deformations. Of particular interest is the observation that CC-based materials display a force-induced transition from α-helices to mechanically stronger ß-sheets (αßT). Steered molecular dynamics simulations predict that this αßT requires a minimum, pulling speed-dependent CC length. Here, de novo designed CCs with a length between four to seven heptads are utilized to probe if the transition found in natural CCs can be mimicked with synthetic sequences. Using single-molecule force spectroscopy and molecular dynamics simulations, these CCs are mechanically loaded in shear geometry and their rupture forces and structural responses to the applied load are determined. Simulations at the highest pulling speed (0.01 nm ns-1 ) show the appearance of ß-sheet structures for the five- and six-heptad CCs and a concomitant increase in mechanical strength. The αßT is less probable at a lower pulling speed of 0.001 nm ns-1 and is not observed in force spectroscopy experiments. For CCs loaded in shear geometry, the formation of ß-sheets competes with interchain sliding. ß-sheet formation is only possible in higher-order CC assemblies or in tensile-loading geometries where chain sliding and dissociation are prohibited.

Cohesin organization, dynamics, and subdomain functions revealed by genetic suppressor screening.

Cohesin is a heteropentameric protein complex that contributes to various aspects of chromosome structure and function, such as sister chromatid cohesion, genome compaction, and DNA damage response. Previous studies have provided abundant information on architecture and regional structures of the cohesin complex, but the configuration and structural dynamics of the whole cohesin complex are still largely unknown, partly due to flexibility of its coiled coils. We studied cohesin organization and dynamics using in vivo functional mutation compensation. Specifically, we developed and applied genetic suppressor screening methods to identify second mutations in cohesin complex genes that rescue lethality caused by various site-specific abnormalities in the cohesin complex. Functional analysis of these missense suppressor mutations revealed novel features of cohesin. Here, we summarize recent genetic suppressor screening results and insights into: 1) cohesin's structural organization when holding chromosomal DNAs; 2) interaction between cohesin head-kleisin and hinge; 3) ATP-driven cohesin conformational changes for genome packaging.

A Modular Scaffold for Controlling Transcriptional Activation via Homomeric Protein-Protein Interactions.

Protein-protein interactions (PPIs) have been extensively utilized in synthetic biology to construct artificial gene networks. However, synthetic regulation of gene expression by PPIs in E. coli has largely relied upon repressors, and existing PPI-controlled transcriptional activators have generally been employed with heterodimeric interactions. Here we report a highly modular, PPI-dependent transcriptional activator, cCadC, that was designed to be compatible with homomeric interactions. We describe the process of engineering cCadC from a transmembrane protein into a soluble cytosolic regulator. We then show that gene transcription by cCadC can be controlled by homomeric PPIs and furthermore discriminates between dimeric and higher-order interactions. Finally, we demonstrate that cCadC activity can be placed under small molecule regulation using chemically induced dimerization or ligand dependent stabilization. This work should greatly expand the scope of PPIs that can be employed in artificial gene circuits in E. coli and complements the existing repertoire of tools for transcriptional regulation in synthetic biology.

Bioactive Fibronectin-III10-DNA Origami Nanofibers Promote Cell Adhesion and Spreading.

The integration of proteins with DNA nanotechnology would enable materials with diverse applications in biology, medicine, and engineering. Here, we describe a method for the incorporation of bioactive fibronectin domain proteins with DNA nanostructures using two orthogonal coiled-coil peptides. One peptide from each coiled-coil pair is attached to a DNA origami cuboid in a multivalent fashion by attaching the peptides to DNA handles. These structures can then be assembled into one-dimensional arrays through the addition of a fibronectin domain linker genetically fused with the complementary peptides to those on the origami. We validate array formation using two different self-assembly protocols and characterize the fibers by atomic force and electron microscopy. Finally, we demonstrate that surfaces coated with the protein-DNA nanofibers can serve as biomaterial substrates for fibroblast adhesion and spreading with the nanofibers showing enhanced bioactivity compared to that of the monomeric protein.

An M protein coiled coil unfurls and exposes its hydrophobic core to capture LL-37.

Surface-associated, coiled-coil M proteins of Streptococcus pyogenes (Strep A) disable human immunity through interaction with select proteins. However, coiled coils lack features typical of protein-protein interaction sites, and it is therefore challenging to understand how M proteins achieve specific binding, for example, with the human antimicrobial peptide LL-37, leading to its neutralization. The crystal structure of a complex of LL-37 with M87 protein, an antigenic M protein variant from a strain that is an emerging threat, revealed a novel interaction mode. The M87 coiled coil unfurled and asymmetrically exposed its hydrophobic core to capture LL-37. A single LL-37 molecule was bound by M87 in the crystal, but in solution additional LL-37 molecules were recruited, consistent with a 'protein trap' neutralization mechanism. The interaction mode visualized crystallographically was verified to contribute significantly to LL-37 resistance in an M87 Strep A strain and was identified to be conserved in a number of other M protein types that are prevalent in human populations. Our results provide specific detail for therapeutic inhibition of LL-37 neutralization by M proteins.

We share our environment with many different bacteria. Some are beneficial for our health, like gut bacteria, but others can cause severe disease if they infect and spread within the body's tissues. For example, the bacterium Streptococcus pyogenes can cause conditions ranging from skin infections to a rapidly spreading deep-tissue infection, giving it the nickname "flesh-eating bacterium". To prevent infection, our bodies have developed defence mechanisms that target disease-causing bacteria. These include antimicrobial molecules, such as LL-37, which is a small protein produced on the skin. LL-37 kills bacteria by puncturing their cell membrane (the bacterial equivalent of our skin); in other words, it acts like a tiny chemical dart that 'pops' the bacterial cell. However, some bacteria, including S. pyogenes, can disarm these defences. S. pyogenes captures LL-37 on its surface with so called M proteins, which prevent LL-37 from reaching and destroying the underlying membrane. However, it was unknown how exactly the two proteins interact, especially since LL-37 is a simple molecule that lacks the structural features that allow most proteins to bind to each other. Kolesinski et al. set out to determine how the M protein can 'grab' LL-37. A technique called X-ray crystallography allowed them to visualise the molecules atom by atom and to examine the configuration of the M protein after it had captured LL-37. The M protein selected for these experiments (M87) came from a strain associated with particularly severe disease, considered to be an emerging health threat. The results showed that M87 uncurled itself, thereby exposing specific parts that normally remain hidden. This way, it could capture LL-37, like a hand opening to grab an object. Kolesinski et al. have revealed a key molecular mechanism that enables a disease-causing bacterium to invade our immune defences. Identifying which regions of M87 are involved in capturing LL-37 may help design more effective therapies to combat S. pyogenes infections.

Development of Thermoresponsive Protein Complexes for Targeting CD20 Receptors.

Therapeutics targeting cell receptors can elicit biological responses in situ. However, the ability to dictate when and where these responses occur is a current challenge. Therapeutic proteins can be combined with stimuli-responsive peptides to increase targeting and stimuli responsive behavior. To this end the authors genetically engineered an elastinlike polypeptide (ELP) fusion protein for selective ELPylation. The addition of a charged, foldable region provides these protein subunits with varied thermoresponsive properties from their parent ELPs. These subunits have responsive secondary structures, dependent on pH, indicating the capability to form coiled-coils with a complementary peptide tag. A Rituximab conjugate is generated herein, containing the complementary peptide. Upon mixing of the ELP and Rituximab subunits, the resulting protein complexes can target CD20 receptors on Raji B cells, resulting in at least twofold increase in mean fluorescent intensities. These ELP subunits fold in vitro with the complimentary generated Rituximab conjugate. This work provides the basis for the design of a therapeutic stimuli-responsive biomacromolecule for targeting receptors.

Single amino acid substitutions in hydrophobic cores at a head-coiled coil junction region of cohesin facilitate its release of DNA during anaphase.

Cohesin holds sister chromatids together and is cleaved by separase/Cut1 to release DNA during the transition from mitotic metaphase to anaphase. The cohesin complex consists of heterodimeric structural maintenance of chromosomes (SMC) subunits (Psm1 and Psm3), which possess a head and a hinge, separated by long coiled coils. Non-SMC subunits (Rad21, Psc3 and Mis4) bind to the SMC heads. Kleisin/Rad21's N-terminal domain (Rad21-NTD) interacts with Psm3's head-coiled coil junction (Psm3-HCJ). Spontaneous mutations that rescued the cleavage defects in temperature-sensitive (ts) separase mutants were identified in the interaction interface, but the underlying mechanism is yet to be understood. Here, we performed site-directed random mutagenesis to introduce single amino acid substitutions in Psm3-HCJ and Rad21-NTD, and then identified 300 mutations that rescued the cohesin-releasing defects in a separase ts mutant. Mutational analysis indicated that the amino acids involved in hydrophobic cores (which may be in close contact) in Psm3-HCJ and Rad21-NTD are hotspots, since 80 mutations (approx. 27%) were mapped in these locations. Properties of these substitutions indicate that they destabilize the interaction between the Psm3 head and Rad21-NTD. Thus, they may facilitate sister chromatid separation in a cleavage-independent way through cohesin structural re-arrangement.

Programmed Supramolecular Assemblies Using Orthogonal Pairs of Heterodimeric Coiled Coil Peptides.

Despite recent progress, it remains challenging to program biomacromolecules to assemble into discrete nanostructures with pre-determined sizes and topologies. We report here a novel strategy to address this challenge. By using two orthogonal pairs of heterodimeric coiled coils as the building blocks, we constructed six discrete supramolecular assemblies, each composed of a prescribed number of coiled coil components. Within these assemblies, different coiled coils were connected via end-to-side covalent linkages strategically pre-installed between the non-complementary pairs. The overall topological features of two highly complex assemblies, a "barbell" and a "quadrilateral" form, were characterized experimentally and were in good agreement to the designs. This work expands the design paradigms for peptide-based discrete supramolecular assemblies and will provide a route for de novo fabrication of functional protein materials.

Design and Synthesis of Crosslinked Helix Dimers as Protein Tertiary Structure Mimics.

Crosslinked helix dimers (CHDs) are synthetic tertiary helical structure motifs designed to modulate interactions of proteins with binding partners. Helix dimers serve as mimics of coiled coils, which are known to be implicated in a multitude of protein complexes. Coiled coils are typically stable in long peptides (>21-28 residues), because sufficient intra- and interstrand contacts are not available in short peptides to coax strand assembly. To engineer conformationally stable CHDs in short sequences, we introduced a covalent linkage in place of an interhelical salt bridge and sculpted the helical interface with optimal hydrophobic packing. CHDs have shown efficacy for the disruption of targeted protein-protein interactions in biochemical, cellular, and animal models. This article describes our optimized approach to design and synthesize parallel and antiparallel helical tertiary structure mimics. Synthesis of CHDs involves conjugation of individual peptide segments, purification of the mono-conjugated strand, and alkylation of the two independent strands to yield crosslinked dimers. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Protocol for bis-triazole CHDs Basic Protocol 2: Protocol for dibenzyl ether CHDs.

Reconstruction of mechanical unfolding and refolding pathways of proteins with atomic force spectroscopy and computer simulations.

Most proteins in proteomes are large, typically consist of more than one domain and are structurally complex. This often makes studying their mechanical unfolding pathways challenging. Proteins composed of tandem repeat domains are a subgroup of multi-domain proteins that, when stretched, display a saw-tooth pattern in their mechanical unfolding force extension profiles due to their repetitive structure. However, the assignment of force peaks to specific repeats undergoing mechanical unraveling is complicated because all repeats are similar and they interact with their neighbors and form a contiguous tertiary structure. Here, we describe in detail a combination of experimental and computational single-molecule force spectroscopy methods that proved useful for examining the mechanical unfolding and refolding pathways of ankyrin repeat proteins. Specifically, we explain and delineate the use of atomic force microscope-based single molecule force spectroscopy (SMFS) to record the mechanical unfolding behavior of ankyrin repeat proteins and capture their unusually strong refolding propensity that is responsible for generating impressive refolding force peaks. We also describe Coarse Grain Steered Molecular Dynamic (CG-SMD) simulations which complement the experimental observations and provide insights in understanding the unfolding and refolding of these proteins. In addition, we advocate the use of novel coiled-coils-based mechanical polypeptide probes which we developed to demonstrate the vectorial character of folding and refolding of these repeat proteins. The combination of AFM-based SMFS on native and CC-equipped proteins with CG-SMD simulations is powerful not only for ankyrin repeat polypeptides, but also for other repeat proteins and more generally to various multidomain, non-repetitive proteins with complex topologies.

The coiled-coil domain of Escherichia coli FtsLB is a structurally detuned element critical for modulating its activation in bacterial cell division.

The FtsLB complex is a key regulator of bacterial cell division, existing in either an off state or an on state, which supports the activation of septal peptidoglycan synthesis. In Escherichia coli, residues known to be critical for this activation are located in a region near the C-terminal end of the periplasmic coiled-coil domain of FtsLB, raising questions about the precise role of this conserved domain in the activation mechanism. Here, we investigate an unusual cluster of polar amino acids found within the core of the FtsLB coiled coil. We hypothesized that these amino acids likely reduce the structural stability of the domain and thus may be important for governing conformational changes. We found that mutating these positions to hydrophobic residues increased the thermal stability of FtsLB but caused cell division defects, suggesting that the coiled-coil domain is a "detuned" structural element. In addition, we identified suppressor mutations within the polar cluster, indicating that the precise identity of the polar amino acids is important for fine-tuning the structural balance between the off and on states. We propose a revised structural model of the tetrameric FtsLB (named the "Y-model") in which the periplasmic domain splits into a pair of coiled-coil branches. In this configuration, the hydrophilic terminal moieties of the polar amino acids remain more favorably exposed to water than in the original four-helix bundle model ("I-model"). We propose that a shift in this architecture, dependent on its marginal stability, is involved in activating the FtsLB complex and triggering septal cell wall reconstruction.

Location-Dependent Lanthanide Selectivity Engineered into Structurally Characterized Designed Coiled Coils.

Herein we report unprecedented location-dependent, size-selective binding to designed lanthanide (Ln3+ ) sites within miniature protein coiled coil scaffolds. Not only do these engineered sites display unusual Ln3+ selectivity for moderately large Ln3+ ions (Nd to Tb), for the first time we demonstrate that selectivity can be location-dependent and can be programmed into the sequence. A 1 nm linear translation of the binding site towards the N-terminus can convert a selective site into a highly promiscuous one. An X-ray crystal structure, the first of a lanthanide binding site within a coiled coil to be reported, coupled with CD studies, reveal the existence of an optimal radius that likely stems from the structural constraints of the coiled coil scaffold. To the best of our knowledge this is the first report of location-dependent metal selectivity within a coiled coil scaffold, as well as the first report of location-dependent Ln3+ selectivity within a protein.

How do I get the most out of my protein sequence using bioinformatics tools?

Biochemical and biophysical experiments are essential for uncovering the three-dimensional structure and biological role of a protein of interest. However, meaningful predictions can frequently also be made using bioinformatics resources that transfer knowledge from a well studied protein to an uncharacterized protein based on their evolutionary relatedness. These predictions are helpful in developing specific hypotheses to guide wet-laboratory experiments. Commonly used bioinformatics resources include methods to identify and predict conserved sequence motifs, protein domains, transmembrane segments, signal sequences, and secondary as well as tertiary structure. Here, several such methods available through the MPI Bioinformatics Toolkit (https://toolkit.tuebingen.mpg.de) are described and how their combined use can provide meaningful information on a protein of unknown function is demonstrated. In particular, the identification of homologs of known structure using HHpred, internal repeats using HHrepID, coiled coils using PCOILS and DeepCoil, and transmembrane segments using Quick2D are focused on.

Decoding the role of coiled-coil motifs in human prion-like proteins.

Prions are self-propagating proteins that cause fatal neurodegenerative diseases in humans. However, increasing evidence suggests that eukaryotic cells exploit prion conformational conversion for functional purposes. A recent study delineated a group of twenty prion-like proteins in humans, characterized by the presence of low-complexity glutamine-rich sequences with overlapping coiled-coil (CCs) motifs. This is the case of Mediator complex subunit 15 (MED15), which is overexpressed in a wide range of human cancers. Biophysical studies demonstrated that the prion-like domain (PrLD) of MED15 forms homodimers in solution, sustained by CCs interactions. Furthermore, the same coiled-coil (CC) region plays a crucial role in the PrLD structural transition to a transmissible ß-sheet amyloid state. In this review, we discuss the role of CCs motifs and their contribution to amyloid transitions in human prion-like domains (PrLDs), while providing a comprehensive overview of six predicted human prion-like proteins involved in transcription, gene expression, or DNA damage response and associated with human disease, whose PrLDs contain or overlap with CCs sequences. Finally, we try to rationalize how these molecular signatures might relate to both their function and involvement in disease.