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Long interspersed nuclear element-1 (L1) is a retrotransposable element that autonomously replicates in the human genome, resulting in DNA damage and genomic instability. Activation of L1 in senescent cells triggers a type I interferon response and age-associated inflammation. Two open reading frames encode an ORF1 protein functioning as messenger RNA chaperone and an ORF2 protein providing catalytic activities necessary for retrotransposition. No function has been identified for the conserved, disordered N-terminal region of ORF1. Using microscopy and NMR spectroscopy, we demonstrate that ORF1 forms liquid droplets in vitro in a salt-dependent manner and that interactions between its N-terminal region and coiled-coil domain are necessary for phase separation. Mutations disrupting blocks of charged residues within the N-terminus impair phase separation, whereas some mutations within the coiled-coil domain enhance phase separation. Demixing of the L1 particle from the cytosol may provide a mechanism to protect the L1 transcript from degradation.

Hexameric structure formation through packing of three C-terminal helices and an N-terminal trimeric coiled-coil core has been proposed as a general mechanism of class I enveloped virus entry. In this process, the C-terminal helical repeat (HR2) region of viral membrane fusion proteins becomes transiently exposed and accessible to N-terminal helical repeat (HR1) trimer-based fusion inhibitors. Herein, we describe a mimetic of the HIV-1 gp41 HR1 trimer, N3G, as a promising therapeutic against HIV-1 infection. Surprisingly, we found that in addition to protection against HIV-1 infection, N3G was also highly effective in inhibiting infection of human β-coronaviruses, including MERS-CoV, HCoV-OC43, and SARS-CoV-2, possibly by binding the HR2 region in the spike protein of β-coronaviruses to block their hexameric structure formation. These studies demonstrate the potential utility of anti-HIV-1 HR1 peptides in inhibiting human β-coronavirus infection. Moreover, this strategy could be extended to the design of broad-spectrum antivirals based on the supercoiling structure of peptides.

Hydrogenase enzymes produce H(2) gas, which can be a potential source of alternative energy. Inspired by the [NiFe] hydrogenases, we report the construction of a de novo designed artificial hydrogenase (ArH). The ArH is a dimeric coiled coil where two cysteine (Cys) residues are introduced at tandem a/d positions of a heptad to create a tetrathiolato Ni binding site. Spectroscopic studies show that Ni binding significantly stabilizes the peptide producing electronic transitions characteristic of Ni-thiolate proteins. The ArH produces H(2) photocatalytically, demonstrating a bell-shaped pH-dependence on activity. Fluorescence lifetimes and transient absorption spectroscopic studies are undertaken to elucidate the nature of pH-dependence, and to monitor the reaction kinetics of the photochemical processes. pH titrations are employed to determine the role of protonated Cys on reactivity. Combined, we find that a fine balance between solution acidity and the electron transfer steps need to be maintained such that the yield of reduced photosensitizer can be maximized to produce the Ni(I)-peptide and the Ni(II)-H(−) intermediate (Ni-R) is protonated by a Cys (pK(a)~6.4) to produce H(2).

In bacteria, the co-chaperone GrpE acts as a nucleotide exchange factor and plays an important role in controlling the chaperone cycle of DnaK. The functional form of GrpE is an asymmetric dimer, consisting of a non-ideal coiled coil. Partial unfolding of this region during heat stress results in reduced nucleotide exchange and disrupts protein folding by DnaK. In this study, we elucidate the role of non-ideality in the coiled-coil domain of Escherichia coli GrpE in controlling its co-chaperone activity. The presence of a four-residue stutter introduces nonheptad periodicity in the GrpE coiled coil, resulting in global structural changes in GrpE and regulating its interaction with DnaK. Introduction of hydrophobic residues at the stutter core increased the structural stability of the protein. Using an in vitro FRET assay, we show that the enhanced stability of GrpE resulted in an increased affinity for DnaK. However, these mutants were unable to support bacterial growth at 42°C in a grpE-deleted E. coli strain. This work provides valuable insights into the functional role of a stutter in GrpE in regulating the DnaK-chaperone cycle during heat stress. More generally, our findings illustrate how stutters in a coiled-coil domain regulate structure-function trade-off in proteins.

Swallow, a 62 kDa multidomain protein, is required for the proper localization of several mRNAs involved in the development of Drosophila oocytes. The dimerization of Swallow depends on a 71-residue self-association domain in the center of the protein sequence, and is significantly stabilized by a binding interaction with dynein light chain (LC8). Here, we detail the use of solution-state nuclear magnetic resonance spectroscopy to characterize the structure of this self-association domain, thereby establishing that this domain forms a parallel coiled-coil and providing insight into how the stability of the dimerization interaction is regulated.

Coiled coils are characterized by an arrangement of two or more alpha-helices into a superhelix and one of few protein motifs where the sequence-to-structure relationship to a large extent have been decoded and understood. The abundance of both natural and de novo designed coil coils provides a rich molecular toolbox for self assembly of elaborate bespoke molecular architectures, nanostructures, and materials. Leveraging on the numerous possibilities to tune both affinities and preferences for polypeptide oligomerization, coiled coils offer unique possibilities to design modular and dynamic assemblies that can respond in a predictable manner to biomolecular interactions and subtle physicochemical cues. In this review, strategies to use coiled coils in design of novel therapeutics and advanced drug delivery systems are discussed. The applications of coiled coils for generating drug carriers and vaccines, and various aspects of using coiled coils for controlling and triggering drug release, and for improving drug targeting and drug uptake are described. The plethora of innovative coiled coil-based molecular systems provide new knowledge and techniques for improving efficacy of existing drugs and can facilitate development of novel therapeutic strategies. (c) 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/). 1. Introduction 27 2. The coiled coil motif 27 3. Coiled coils and coiled coil-hybrids for drug delivery and therapeutics 30 3.1. Coiled coils in liposome drug delivery systems 30 3.2. Lipidated coiled coils for assembly of virus-like particles 31 3.3. Coiled coil nanoparticles 31 3.4. Coiled coil nanocarriers 33 3.5. Coiled coil polymer-hybrids 33 3.6. Coiled coil-based hydrogels 36 3.7. Coiled coil inorganic nanoparticle hybrids 37 3.8. Coiled coils combined with cell penetrating peptides 37 3.9. Coiled coils for improved targeting 38 Funding Agencies|Swedish Foundation for Strategic Research (SFF)Swedish Foundation for Strategic Research [FFL15-0026, RMX18-0039]; Swedish Government Strategic Research Area inMaterials Science on Functional Materials at Linkoping University [2009-00971]; Knut and Alice Wallenberg FoundationKnut & Alice Wallenberg Foundation [KAW 2016.0231]; Swedish Research CouncilSwedish Research CouncilEuropean Commission [2017-04475]; Cancer Foundation [17 0534]

The coiled-coil domains of the putative yeast cargo receptors Emp46p and Emp47p (Emp46pcc and Emp47pcc) assemble into heterocomplexes at neutral pH. Upon lowering the pH, the complex dissociates and reassembles into homo-oligomers. A glutamate residue (E303) located on the hydrophobic surface of Emp46pcc serves as the pH-sensing switch for assembly and segregation, and we have suggested that its side chains are protonated in the heterocomplex, even at neutral pH. To examine this hypothesis, we constructed two structural models in which the side chains of E303 were negatively charged or protonated and analyzed the effects of these charged states on the structure of the heterocomplex using molecular dynamics (MD) simulations. The calculated structures suggested the side chains of E303 to be protonated in the heterocomplex, even at neutral pH. Based on these computational results, the pH dependence of Emp47pcc homo-oligomer assembly was experimentally modified by a glutamate mutation on its hydrophobic surface. The Q306E mutant of Emp47pcc underwent a structural transition at physiological pH. Our results suggest a method for modifying pH-dependent protein-protein interactions.

Computational design of fully artificial peptides is extensively researched by material scientists and engineers for the construction of novel nanostructures and biomaterials. Such design has yielded a peptide-based building block or bundlemer, a coiled coil peptide assembly that undergoes further physical-covalent interactions to form 1D, 2D and, potentially, 3D hierarchical assemblies and displays targeted and biomimetic material properties. Recombinant expression is a convenient, flexible tool to synthesize such artificial and modified peptides in large quantities while also enabling economical synthesis of isotopically labeled peptides and longer protein-like artificial peptides. This report describes the protocol for recombinant expression of a 31-amino acid, computationally designed bundlemer-forming peptide in Escherichia coli. Peptide yields of 10 mgs per liter of media were achieved which highlights complementary advantages of recombinant expression technique relative to conventional laboratory-scale synthesis, such as solid-phase peptide synthesis.

We previously reported a family of hydrocarbon-stapled peptides designed to interact with the epidermal growth factor receptor (EGFR) juxtamembrane (JM) segment, blocking its ability to form a coiled coil dimer that is essential for receptor activation. These hydrocarbon-stapled peptides, most notably E1S, decreased the proliferation of cell lines that express wild-type EGFR (H2030 and A431) as well as those expressing the oncogenic mutants EGFR L858R (H3255) and L858R/T790M (H1975). Although our previous investigations provided evidence that E1S interacted with EGFR directly, the location and details of these interactions were not established. Here we apply biochemical and cross-linking mass spectrometry tools to better define the interactions between E1S and EGFR. Taken with previously reported structure-activity relationships, our results support a model in which E1S interacts simultaneously with both the JM and the C-lobe of the activator kinase, effectively displacing the JM of the receiver kinase. Our results also reveal potential interactions between E1S and the N-terminal region of the C-terminal tail. We propose a model in which E1S inhibits EGFR by both mimicking and inhibiting JM coiled coil formation. This model could be used to design novel, allosteric (and perhaps nonpeptidic) EGFR inhibitors.

Epidermal growth factor receptor (EGFR) is a clinically validated target for a multitude of human cancers. The receptor is activated upon ligand binding through a critical dimerization step. Dimerization can be replicated in vitro by locally concentrating the receptor kinase domains on the surface of lipid-based vesicles. In this study we investigated the use of coiled coils to induce spontaneous receptor kinase domain dimerization in vitro to form non-membrane-bound artificial receptor mimics in solution. Two engineered forms of EGFR kinase domain fused to coiled coil complementary peptides were designed to self-associate upon mixing. Two fusion protein species (P3-EGFR and P4-EGFR) independently showed the same activity and polymerization profile known to exist with EGFR kinase domains. Upon mixing the two species, coiled coil heterodimers were formed that induced EGFR association to form dimers of the kinase domains. This was accompanied by 11.5-fold increase in the phosphorylation rate indicative of kinase domain activation equivalent to the levels achieved using vesicle localization and mimicking in vivo ligand-induced activation. This study presents a soluble tyrosine kinase receptor mimic capable of spontaneous in vitro activation that can facilitate functional and drug discovery studies for this clinically important receptor class.

Plant nucleotide-binding, leucine-rich repeat receptors (NLRs) perceive pathogen avirulence effectors and activate defense responses. Nucleotide-binding, leucine-rich repeat receptors are classified into coiled-coil (CC)-containing and Toll/interleukin-1 receptor (TIR)-containing NLRs. Recent advances suggest that NLR CC domains often function in signaling activation, especially for induction of cell death. In this review, we outline our current understanding of NLR CC domains, including their diversity/classification and structure, their roles in cell death induction, disease resistance, and interaction with other proteins. Furthermore, we provide possible directions for future work.

Kinesin-1 is a motor protein moving along a microtubule with its two identical motor heads dimerized by two neck linkers and a coiled-coil stalk. When both motor heads bind the microtubule, an internal strain is built up between the two heads, which is indispensable to ensure proper coordination of the two motor heads during kinesin-1's mechanochemical cycle. The internal strain forms a tensile force along the neck linker that tends to unwind the neck coiled coil (NCC). Experiments showed that the kinesin-1's NCC has a high antiunwinding ability compared with conventional coiled coils, which was mainly attributed to the enhanced hydrophobic pressure arising from the unconventional sequence of kinesin-1's NCC. However, hydrophobic pressure cannot provide the shearing force which is needed to balance the tensile force on the interface between two helices. To find out the true origin of the mechanical stability of kinesin-1's NCC, we perform a novel and detailed mechanical analysis for the system based on molecular dynamics simulation at an atomic level. We find that the needed shearing force is provided by a buckle structure formed by two tyrosines which form effective steric hindrance in the presence of tensile forces. The tensile force is balanced by the tensile direction component of the contact force between the two tyrosines which forms the shearing force. The hydrophobic pressure balances the other component of the contact force perpendicular to the tensile direction. The antiunwinding strength of NCC is defined by the maximum shearing force, which is finally determined by the hydrophobic pressure. Kinesin-1 uses residues with plane side chains, tryptophans and tyrosines, to form the hydrophobic center and to shorten the interhelix distance so that a high antiunwinding strength is obtained. The special design of NCC ensures exquisite cooperation of steric hindrance and hydrophobic pressure that results in the surprising mechanical stability of NCC.

Coiled-coil protein origami (CCPO) polyhedra are designed self-assembling nanostructures constructed from coiled coil (CC)-forming modules connected into a single chain. For testing new CCPO building modules, simpler polyhedra could be used that should maintain most features relevant to larger scaffolds. We show the design and characterization of nanoscale single-chain triangles, composed of six concatenated parallel CC dimer-forming segments connected by flexible linker peptides. The polypeptides self-assembled in bacteria in agreement with the design, and the shape of the polypeptides was confirmed with small-angle X-ray scattering. Fusion with split-fluorescent protein domains was used as a functional assay in bacteria, based on the discrimination between the correctly folded and misfolded nanoscale triangles comprising correct, mismatched, or truncated modules. This strategy was used to evaluate the optimal size of linkers between CC segments which comprised eight amino acid residues.

The coiled coil is a common protein tertiary structure intimately involved in mediating protein recognition and function. Due to their structural simplicity, coiled coils have served as attractive scaffolds for the development of functional biomaterials. Herein we describe the design of conformationally-defined coiled coil photoswitches as potential environmentally-sensitive biomaterials.

The ability to spatiotemporally control the presentation of relevant biomolecules in synthetic culture systems has gained significant attention as researchers strive to recapitulate the endogenous extracellular matrix (ECM) in vitro. With the biochemical composition of the ECM constantly in flux, the development of platforms that allow for user-defined control of bioactivity is desired. Here, we reversibly conjugate bioactive molecules to hydrogel-based substrates through supramolecular coiled coil complexes that form between complementary peptides. Our system employs a thiolated peptide for tethering to hydrogel surfaces (T-peptide) through a spatially-controlled photomediated click reaction. The complementary association peptide (A-peptide), containing the bioactive domain, forms a heterodimeric coiled coil complex with the T-peptide. Addition of a disruptor peptide (D-peptide) engineered specifically to target the A-peptide outcompetes the T-peptide for binding, and removes the A-peptide and the attached bioactive motif from the scaffold. We use this platform to demonstrate spatiotemporal control of biomolecule presentation within hydrogel systems in a repeatable process that can be extended to adhesive motifs for cell culture. NIH 3T3 fibroblasts seeded on hyaluronic acid hydrogels and polyethylene glycol-based fibrous substrates supramolecularly functionalized with an RGD motif demonstrated significant cell spreading over their nonfunctionalized counterparts. Upon displacement of the RGD motif, fibroblasts occupied less area and clustured on the substrates. Taken together, this platform enables facile user-defined incorporation and removal of biomolecules in a repeatable process for controlled presentation of bioactivity in engineered culture systems.

Here, we report a rapid and efficient method to fabricate Quenchbodies (Q-bodies) that can detect targets with antigen-dependent fluorescence augmentation using a stable coiled-coil peptide pair, E4 and K4 (coiled Q-body, CQ-body). The CQ-body allowed antigen detection not only in buffer but also in 50% plasma. Furthermore, we describe FRET-type CQ-bodies using a dual-coloured K4 peptide, which allowed a more precise antigen quantification. Lastly, successful fabrication of nanobody-based CQ-body shows its applicability to a range of antibody fragments.

Biological membrane fusion is a highly specific and coordinated process as a multitude of vesicular fusion events proceed simultaneously in a complex environment with minimal off-target delivery. In this study, we develop a liposomal fusion model system with specific recognition using lipidated derivatives of a set of four de novo designed heterodimeric coiled coil (CC) peptide pairs. Content mixing was only obtained between liposomes functionalized with complementary peptides, demonstrating both fusogenic activity of CC peptides and the specificity of this model system. The diverse peptide fusogens revealed important relationships between the fusogenic efficacy and the peptide characteristics. The fusion efficiency increased from 20% to 70% as affinity between complementary peptides decreased, (from KF ≈ 108 to 104 M−1), and fusion efficiency also increased due to more pronounced asymmetric role-playing of membrane interacting ‘K’ peptides and homodimer-forming ‘E’ peptides. Furthermore, a new and highly fusogenic CC pair (E3/P1K) was discovered, providing an orthogonal peptide triad with the fusogenic CC pairs P2E/P2K and P3E/P3K. This E3/P1k pair was revealed, via molecular dynamics simulations, to have a shifted heptad repeat that can accommodate mismatched asparagine residues. These results will have broad implications not only for the fundamental understanding of CC design and how asparagine residues can be accommodated within the hydrophobic core, but also for drug delivery systems by revealing the necessary interplay of efficient peptide fusogens and enabling the targeted delivery of different carrier vesicles at various peptide-functionalized locations., We developed a liposomal fusion model system with specific recognition using a set of heterodimeric coiled coil peptide pairs. This study unravels important structure–fusogenic efficacy relationships of peptide fusogens.

For much of their history, lanthanides were thought to be biologically inert. However, the last decade has seen the discovery and development of the field of native lanthanide biochemistry. Lanthanides exhibit a variety of interesting photophysical properties from which many useful applications derive. The development of effective functional lanthanide complexes requires control of their coordination sphere; something proteins manage very effectively through their 3D metal-binding sites. α-Helical coiled coil peptides are miniature scaffolds which can be designed de novo and can retain the favourable properties of larger proteins within a much simplified system. Metal binding sites, including those which bind lanthanides can be engineered into the coiled coil sequence. This review will highlight the opportunities presented by the use of coiled coil peptides as scaffolds for lanthanide binding and the potential to control the coordination environment by simple modifications to peptide sequence. Designed lanthanide coiled coils offer opportunities to gain greater insight into native lanthanide biochemistry as well as to develop new functional complexes, including imaging agents.

Background DNA binding KfrA-type proteins of broad-host-range bacterial plasmids belonging to IncP-1 and IncU incompatibility groups are characterized by globular N-terminal head domains and long alpha-helical coiled-coil tails. They have been shown to act as transcriptional auto-regulators. Results This study was focused on two members of the growing family of KfrA-type proteins encoded by the broad-host-range plasmids, R751 of IncP-1β and RA3 of IncU groups. Comparative in vitro and in silico studies on KfrAR751 and KfrARA3 confirmed their similar biophysical properties despite low conservation of the amino acid sequences. They form a wide range of oligomeric forms in vitro and, in the presence of their cognate DNA binding sites, they polymerize into the higher order filaments visualized as “threads” by negative staining electron microscopy. The studies revealed also temperature-dependent changes in the coiled-coil segment of KfrA proteins that is involved in the stabilization of dimers required for DNA interactions. Conclusion KfrAR751 and KfrARA3 are structural homologues. We postulate that KfrA type proteins have moonlighting activity. They not only act as transcriptional auto-regulators but form cytoskeletal structures, which might facilitate plasmid DNA delivery and positioning in the cells before cell division, involving thermal energy.

To date, most research into the inhibition of oncogenic transcriptional regulator, Activator Protein 1 (AP-1), has focused on heterodimers of cJun and cFos. However, the Fra1 homologue remains an important cancer target. Here we describe library design coupled with computational and intracellular screening as an effective methodology to derive an antagonist that is selective for Fra1 relative to Jun counterparts. To do so the isCAN computational tool was used to rapidly screen >75 million peptide library members, narrowing the library size by >99.8% to one accessible to intracellular PCA selection. The resulting 131 072-member library was predicted to contain high quality binders with both a high likelihood of target engagement, while simultaneously avoiding homodimerization and off-target interaction with Jun homologues. PCA screening was next performed to enrich those members that meet these criteria. In particular, optimization was achieved via inclusion of options designed to generate the potential for compromised intermolecular contacts in both desired and non-desired species. This is an often-overlooked prerequisite in the conflicting design requirement of libraries that must be selective for their target in the context of a range of alternative potential interactions. Here we demonstrate that specificity is achieved via a combination of both hydrophobic and electrostatic contacts as exhibited by the selected peptide (Fra1W). In vitro analysis of the desired Fra1–Fra1W interaction further validates high Fra1 affinity (917 nM) yet selective binding relative to Fra1W homodimers or affinity for cJun. The isCAN → PCA based multidisciplinary approach provides a robust screening pipeline in generating target-specific hits, as well as new insight into rational peptide design in the search for novel bZIP family inhibitors., Here we describe library design coupled with computational and intracellular screening as an effective methodology to derive an antagonist that is selective for Fra1 relative to Jun counterparts.

Proteins are fundamentally the most important macromolecules for biochemical, mechanical, and structural functions in living organisms. Therefore, they provide us with diverse structural building blocks for constructing various types of biomaterials, including an important class of such materials, hydrogels. Since natural peptides and proteins are biocompatible and biodegradable, they have features advantageous for their use as the building blocks of hydrogels for biomedical applications. They display constitutional and mechanical similarities with the native extracellular matrix (ECM), and can be easily bio-functionalized via genetic and chemical engineering with features such as bio-recognition, specific stimulus-reactivity, and controlled degradation. This review aims to give an overview of hydrogels made up of recombinant proteins or synthetic peptides as the structural elements building the polymer network. A wide variety of hydrogels composed of protein or peptide building blocks with different origins and compositions - including β-hairpin peptides, α-helical coiled coil peptides, elastin-like peptides, silk fibroin, and resilin - have been designed to date. In this review, the structures and characteristics of these natural proteins and peptides, with each of their gelation mechanisms, and the physical, chemical, and mechanical properties as well as biocompatibility of the resulting hydrogels are described. In addition, this review discusses the potential of using protein- or peptide-based hydrogels in the field of biomedical sciences, especially tissue engineering.

Liposomes are widely investigated as vaccine delivery systems, but antigen loading efficiency can be low. Moreover, adsorbed antigen may rapidly desorb under physiological conditions. Encapsulation of antigens overcomes the latter problem but results in significant antigen loss during preparation and purification of the liposomes. Here, we propose an alternative attachment method, based on a complementary heterodimeric coiled coil peptide pair pepK and pepE. PepK was conjugated to cholesterol (yielding CPK) and pepE was covalently linked to model antigen OVA323 (yielding pepE-OVA323). CPK was incorporated in the lipid bilayer of cationic liposomes (180 nm in size). Antigen was associated more efficiently to functionalized liposomes (Kd 166 nM) than to cationic liposomes (Kd not detectable). In vivo co-localization of antigen and liposomes was strongly increased upon CPK-functionalization (35% -> 80%). CPK-functionalized liposomes induced 5-fold stronger CD4+ T-cell proliferation than non-functionalized liposomes in vitro. Both formulations were able to induce strong CD4+ T-cell expansion in mice, but more IFN-y and IL-10 production was observed after immunization with functionalized liposomes. In conclusion, antigen association via coiled coil peptide pair increased co-localization of antigen and liposomes, increased CD4+ T-cell proliferation in vitro and induced a stronger CD4+ T-cell response in vivo.

The envelope glycoprotein, termed the spike protein, of severe acute respiratory syndrome coronavirus (SARS-CoV) is known to mediate viral entry. Similar to other class 1 viral fusion proteins, the heptad repeat regions of SARS-CoV spike are thought to undergo conformational changes from a prefusion form to a subsequent post-fusion form that enables fusion of the viral and host membranes. Recently, the structure of a post-fusion form of SARS-CoV spike, which consists of isolated domains of heptad repeats 1 and 2 (HR1 and HR2), has been determined by x-ray crystallography. To date there is no structural information for the prefusion conformations of SARS-CoV HR1 and HR2. In this work we present the NMR structure of the HR2 domain (residues 1141-1193) from SARS-CoV (termed S2-HR2) in the presence of the co-solvent trifluoroethanol. We find that in the absence of HR1, S2-HR2 forms a coiled coil symmetric trimer with a complex molecular mass of 18 kDa. The S2-HR2 structure, which is the first example of the prefusion form of coronavirus envelope, supports the current model of viral membrane fusion and gives insight into the design of structure-based antagonists of SARS.

[Image: see text] Antibiotic-resistant microbes have become a global health threat. New delivery systems that enhance the efficacy of antibiotics and/or overcome the resistances can help combat them. In this context, we present FF03, a fibril-forming α-helical coiled-coil peptide that functions as an efficient scaffold for the multivalent presentation of the weakly cationic antimicrobial peptide (AMP) IN4. The resulting IN4-decorated FF03 coiled-coil fibrils (FF03 + IN4) are nonhemolytic and noncytotoxic and show enhanced antimicrobial activity relative to unconjugated IN4 and standard antibiotics against several bacterial strains. Scanning electron microscopy analysis shows that FF03 + IN4 nanofibers disrupt methicillin-resistant Staphylococcus aureus membranes, indicating a surface-level mode of action. Furthermore, transmission electron microscopy and circular dichroism studies indicate that decoration of the FF03 scaffold with IN4 does not alter the secondary-structure propensity or fibril-forming properties of FF03. Thus, the approach reported herein provides a new peptidic scaffold for the multivalent presentation of AMPs to obtain nonhemolytic and noncytotoxic antimicrobial systems with improved efficacy relative to the unconjugated AMP analogues.