Investigating vulnerability of the conserved SARS-CoV-2 spike's heptad repeat 2 as target for fusion inhibitors using chimeric miniproteins.

Inhibition of SARS-CoV-2 membrane fusion is a highly desired target to combat COVID-19. The interaction between the spike's heptad repeat (HR) regions 1 (HR1) and 2 (HR2) is a crucial step during the fusion process and these highly conserved HR regions constitute attractive targets for fusion inhibitors. However, the relative importance of each subregion of the long HR1-HR2 interface for viral inhibition remains unclear. Here, we designed, produced, and characterized a series of chimeric miniproteins that mimic two different half subdomains of HR1. The proteins were designed as single polypeptide chains that spontaneously fold into antiparallel trimeric helical bundles aimed at structurally imitate the molecular surface of each HR1 half subregion. All the miniproteins folded stably as helical structures and could bind complementary HR2 peptides with moderate affinity. However, only the miniproteins mimicking the N-terminal HR1 half subdomain, but not those imitating C-terminal one, could inhibit cell infection by SARS-COV-2 real viruses in cell cultures. Most interestingly, the inhibitory activity of the miniproteins correlated with their structural stability, but not with their relative binding affinity for HR2 peptides. These results are highly relevant for designing more focused and active fusion inhibitors targeting the highly conserved HR2 region of the Spike.

Selective Formation of Pd-DNA Hybrids Using Tailored Palladium-Mediated Base Pairs: Towards Heteroleptic Pd-DNA Systems.

The formation of highly organized metal-DNA structures has significant implications in bioinorganic chemistry, molecular biology and material science due to their unique properties and potential applications. In this study, we report on the conversion of single-stranded polydeoxycytidine (dC15 ) into a Pd-DNA supramolecular structure using the [Pd(Aqa)] complex (Aqa=8-amino-4-hydroxyquinoline-2-carboxylic acid) through a self-assembly process. The resulting Pd-DNA assembly closely resembles a natural double helix, with continuous [Pd(Aqa)(C)] (C=cytosine) units serving as palladium-mediated base pairs, forming interbase hydrogen bonds and intrastrand stacking interactions. Notably, the design of the [Pd(Aqa)] complex favours the interaction with cytosine, distinguishing it from our previously reported [Pd(Cheld)] complex (Cheld=chelidamic acid). This finding opens possibilities for creating heteroleptic Pd-DNA hybrids where different complexes specifically bind to nucleobases. We confirmed the Pd-DNA supramolecular structural assembly and selective binding of the complexes using NMR spectroscopy, circular dichroism, mass spectrometry, isothermal titration calorimetry, and DFT calculations.

Preparation and Investigation of Crucial Oligomers in the Early Stages of Aß40 and Aß42 Aggregation.

Amyloid aggregation is a hallmark in many neuropathologies and other diseases of tremendous impact. It is increasingly evident that neuronal death associated with Alzheimer's disease (AD) is mainly produced by oligomers of the amyloid-ß (Aß) peptide. Yet little is known about the detailed structural and biophysical mechanisms of their formation. This lack of complete understanding comes from the labile nature and handling complexity of the oligomers. Consequently, providing reproducible and robust protocols for oligomer preparation is of particular importance.In this study, we describe detailed methods for the preparation and isolation of micellar oligomers of Aß that evolve towards larger and more stable oligomers enriched in beta-sheet structure and able to acquire a higher capacity to fibrillate. We also describe briefly some biophysical experiments allowing oligomer characterization.

Exploring Highly Conserved Regions of SARS-CoV-2 Spike S2 Subunit as Targets for Fusion Inhibition Using Chimeric Proteins.

Since the beginning of the COVID-19 pandemic, considerable efforts have been made to develop protective vaccines against SARS-CoV-2 infection. However, immunity tends to decline within a few months, and new virus variants are emerging with increased transmissibility and capacity to evade natural or vaccine-acquired immunity. Therefore, new robust strategies are needed to combat SARS-CoV-2 infection. The viral spike composed of S1 and S2 subunits mediates viral attachment and membrane fusion to infect the host cell. In this process, interaction between the highly conserved heptad repeat 1 and 2 regions (HR1 and HR2) of S2 is crucial and for this reason; these regions are promising targets to fight SARS-CoV-2. Here, we describe the design and characterization of chimeric proteins that structurally imitate the S2 HR1 region in a trimeric coiled-coil conformation. We biophysically characterized the proteins and determined their capacity to bind the HR2 region, as well as their inhibitory activity of SARS-CoV-2 infection in vitro. HR1 mimetic proteins showed conformational heterogeneity and a propensity to form oligomers. Moreover, their structure is composed of subdomains with varied stability. Interestingly, the full HR1 proteins showed high affinity for HR2-derived peptides and SARS-CoV-2 inhibitory activity, whereas smaller proteins mimicking HR1 subdomains had a decreased affinity for their complementary HR2 region and did not inhibit the virus. The results provide insight into effective strategies to create mimetic proteins with broad inhibitory activity and therapeutic potential against SARS-CoV-2.

Novel chimeric proteins mimicking SARS-CoV-2 spike epitopes with broad inhibitory activity.

SARS-CoV-2 spike (S) protein mediates virus attachment to the cells and fusion between viral and cell membranes. Membrane fusion is driven by mutual interaction between the highly conserved heptad-repeat regions 1 and 2 (HR1 and HR2) of the S2 subunit of the spike. For this reason, these S2 regions are interesting therapeutic targets for COVID-19. Although HR1 and HR2 have been described as transiently exposed during the fusion process, no significant antibody responses against these S2 regions have been reported. Here we designed chimeric proteins that imitate highly stable HR1 helical trimers and strongly bind to HR2. The proteins have broad inhibitory activity against WT B.1 and BA.1 viruses. Sera from COVID-19 convalescent donors showed significant levels of reactive antibodies (IgG and IgA) against the HR1 mimetic proteins, whereas these antibody responses were absent in sera from uninfected donors. Moreover, both inhibitory activity and antigenicity of the proteins correlate positively with their structural stability but not with the number of amino acid changes in their HR1 sequences, indicating a conformational and conserved nature of the involved epitopes. Our results reveal previously undetected spike epitopes that may guide the design of new robust COVID-19 vaccines and therapies.

Conformational Stabilization of Gp41-Mimetic Miniproteins Opens Up New Ways of Inhibiting HIV-1 Fusion.

Inhibition of the HIV-1 fusion process constitutes a promising strategy to neutralize the virus at an early stage before it enters the cell. In this process, the envelope glycoprotein (Env) plays a central role by promoting membrane fusion. We previously identified a vulnerability at the flexible C-terminal end of the gp41 C-terminal heptad repeat (CHR) region to inhibition by a single-chain miniprotein (named covNHR-N) that mimics the first half of the gp41 N-terminal heptad repeat (NHR). The miniprotein exhibited low stability, moderate binding to its complementary CHR region, both as an isolated peptide and in native trimeric Envs, and low inhibitory activity against a panel of pseudoviruses. The addition of a disulfide bond stabilizing the miniprotein increased its inhibitory activity, without altering the binding affinity. Here, to further study the effect of conformational stability on binding and inhibitory potency, we additionally stabilized these miniproteins by engineering a second disulfide bond stapling their N-terminal end, The new disulfide-bond strongly stabilizes the protein, increases binding affinity for the CHR target and strongly improves inhibitory activity against several HIV-1 strains. Moreover, high inhibitory activity could be achieved without targeting the preserved hydrophobic pocket motif of gp41. These results may have implications in the discovery of new strategies to inhibit HIV targeting the gp41 CHR region.

Conformational flexibility of the conserved hydrophobic pocket of HIV-1 gp41. Implications for the discovery of small-molecule fusion inhibitors.

During HIV-1 infection, the envelope glycoprotein subunit gp41 folds into a six-helix bundle structure (6HB) formed by the interaction between its N-terminal (NHR) and C-terminal (CHR) heptad-repeats, promoting viral and cell membranes fusion. A highly preserved, hydrophobic pocket (HP) on the NHR surface is crucial in 6HB formation and, therefore, HP-binding compounds constitute promising therapeutics against HIV-1. Here, we investigated the conformational and dynamic properties of the HP using a rationally designed single-chain protein (named covNHR) that mimics the gp41 NHR structure. We found that the fluorescent dye 8-anilino-naphtalene-1-sulfonic acid (ANS) binds specifically to the HP, suggesting that ANS derivatives may constitute lead compounds to inhibit 6HB formation. ANS shows different binding modes to the HP, depending on the occupancy of other NHR pockets. Moreover, in presence of a CHR peptide bound to the N-terminal pockets in gp41, two ANS molecules can occupy the HP showing cooperative behavior. This binding mode was assessed using molecular docking and molecular dynamics simulations. The results show that the HP is conformationally flexible and connected allosterically to other NHR regions, which strongly influence the binding of potential ligands. These findings could guide the development of small-molecule HIV-1 inhibitors targeting the HP.

Rapid Conversion of Amyloid-Beta 1-40 Oligomers to Mature Fibrils through a Self-Catalytic Bimolecular Process.

The formation of fibrillar aggregates of the amyloid beta peptide (Aß) in the brain is one of the hallmarks of Alzheimer's disease (AD). A clear understanding of the different aggregation steps leading to fibrils formation is a keystone in therapeutics discovery. In a recent study, we showed that Aß40 and Aß42 form dynamic micellar aggregates above certain critical concentrations, which mediate a fast formation of more stable oligomers, which in the case of Aß40 are able to evolve towards amyloid fibrils. Here, using different biophysical techniques we investigated the role of different fractions of the Aß aggregation mixture in the nucleation and fibrillation steps. We show that both processes occur through bimolecular interplay between low molecular weight species (monomer and/or dimer) and larger oligomers. Moreover, we report here a novel self-catalytic mechanism of fibrillation of Aß40, in which early oligomers generate and deliver low molecular weight amyloid nuclei, which then catalyze the rapid conversion of the oligomers to mature amyloid fibrils. This fibrillation catalytic activity is not present in freshly disaggregated low-molecular weight Aß40 and is, therefore, a property acquired during the aggregation process. In contrast to Aß40, we did not observe the same self-catalytic fibrillation in Aß42 spheroidal oligomers, which could neither be induced to fibrillate by the Aß40 nuclei. Our results reveal clearly that amyloid fibrillation is a multi-component process, in which dynamic collisions between different interacting species favor the kinetics of amyloid nucleation and growth.

Extremely Thermostabilizing Core Mutations in Coiled-Coil Mimetic Proteins of HIV-1 gp41 Produce Diverse Effects on Target Binding but Do Not Affect Their Inhibitory Activity.

A promising strategy to neutralize HIV-1 is to target the gp41 spike subunit to block membrane fusion with the cell. We previously designed a series of single-chain proteins (named covNHR) that mimic the trimeric coiled-coil structure of the gp41 N-terminal heptad repeat (NHR) region and potently inhibit HIV-1 cell infection by avidly binding the complementary C-terminal heptad repeat (CHR) region. These proteins constitute excellent tools to understand the structural and thermodynamic features of this therapeutically important interaction. Gp41, as with many coiled-coil proteins, contains in core positions of the NHR trimer several highly conserved, buried polar residues, the role of which in gp41 structure and function is unclear. Here we produced three covNHR mutants by substituting each triad of polar residues for the canonical isoleucine. The mutants preserve their helical structure and show an extremely increased thermal stability. However, increased hydrophobicity enhances their self-association. Calorimetric analyses show a marked influence of mutations on the binding thermodynamics of CHR-derived peptides. The mutations do not affect however the in vitro HIV-1 inhibitory activity of the proteins. The results support a role of buried core polar residues in maintaining structural uniqueness and promoting an energetic coupling between conformational stability and NHR-CHR binding.

Insulin Crystals Grown in Short-Peptide Supramolecular Hydrogels Show Enhanced Thermal Stability and Slower Release Profile.

Protein therapeutics have a major role in medicine in that they are used to treat diverse pathologies. Their three-dimensional structures not only offer higher specificity and lower toxicity than small organic compounds but also make them less stable, limiting their in vivo half-life. Protein analogues obtained by recombinant DNA technology or by chemical modification and/or the use of drug delivery vehicles has been adopted to improve or modulate the in vivo pharmacological activity of proteins. Nevertheless, strategies to improve the shelf-life of protein pharmaceuticals have been less explored, which has challenged the preservation of their activity. Herein, we present a methodology that simultaneously increases the stability of proteins and modulates the release profile, and implement it with human insulin as a proof of concept. Two novel thermally stable insulin composite crystal formulations intended for the therapeutic treatment of diabetes are reported. These composite crystals have been obtained by crystallizing insulin in agarose and fluorenylmethoxycarbonyl-dialanine (Fmoc-AA) hydrogels. This process affords composite crystals, in which hydrogel fibers are occluded. The insulin in both crystalline formulations remains unaltered at 50 °C for 7 days. Differential scanning calorimetry, high-performance liquid chromatography, mass spectrometry, and in vivo studies have shown that insulin does not degrade after the heat treatment. The nature of the hydrogel modifies the physicochemical properties of the crystals. Crystals grown in Fmoc-AA hydrogel are more stable and have a slower dissolution rate than crystals grown in agarose. This methodology paves the way for the development of more stable protein pharmaceuticals overcoming some of the existing limitations.

Probing Vulnerability of the gp41 C-Terminal Heptad Repeat as Target for Miniprotein HIV Inhibitors.

One of the therapeutic strategies in HIV neutralization is blocking membrane fusion. In this process, tight interaction between the N-terminal and C-terminal heptad-repeat (NHR and CHR) regions of gp41 is essential to promote membranes apposition and merging. We have previously developed single-chain proteins (named covNHR) that accurately mimic the complete gp41 NHR region in its trimeric conformation. They tightly bind CHR-derived peptides and show a potent and broad HIV inhibitory activity in vitro. However, the extremely high binding affinity (sub-picomolar) is not in consonance with their inhibitory activity (nanomolar), likely due to partial or temporal accessibility of their target in the virus. Here, we have designed and characterized two single-chain covNHR miniproteins each encompassing one of the two halves of the NHR region and containing two of the four sub-pockets of the NHR crevice. The two miniproteins fold as trimeric helical bundles as expected but while the C-terminal covNHR (covNHR-C) miniprotein is highly stable, the N-terminal counterpart (covNHR-N) shows only marginal stability that could be improved by engineering an internal disulfide bond. Both miniproteins bind their respective complementary CHR peptides with moderate (micromolar) affinity. Moreover, the covNHR-N miniproteins can access their target in the context of trimeric native envelope proteins and show significant inhibitory activity for several HIV pseudoviruses. In contrast, covNHR-C cannot bind its target sequence and neither inhibits HIV, indicating a higher vulnerability of C-terminal part of CHR. These results may guide the development of novel HIV inhibitors targeting the gp41 CHR region.

Thermodynamic dissection of the interface between HIV-1 gp41 heptad repeats reveals cooperative interactions and allosteric effects.

HIV-1 glycoprotein 41 (gp41) mediates fusion between virus and target cells by folding into a fusion active state, in which the C-terminal heptad repeat (CHR) regions associate externally to the N-terminal heptad repeat (NHR) trimer and form a very stable six-helix bundle coiled-coil structure. Therefore, interfering with the NHR-CHR interaction of gp41 is a promising therapeutic approach against HIV-1. However, a full understanding of the molecular and mechanistic details of this interaction is still incomplete. Here, we use single-chain, chimeric proteins (named covNHR) that reproduce accurately the CHR-NHR interactions to analyze the binding thermodynamics of several peptides with different length from the CHR region. The results indicate that cooperative binding involving two or more pockets of the NHR groove is necessary to obtain relevant affinities and that the binding energy is broadly distributed along the interface, underlining a crucial role of a middle pocket to achieve tight binding. In contrast, targeting only the deep hydrophobic pocket is insufficient to achieve significant affinity. Moreover, calorimetry experiments in combination with limited proteolysis performed using a mutant with occluded binding in the N-terminal pocket reveal the existence of an allosteric communication between the different regions. This study is the first detailed thermodynamic dissection of the NHR-CHR interaction in gp41 and contributes therefore to a better understanding of HIV fusion. These results are relevant for the development of potential fusion inhibitors.

Early mechanisms of amyloid fibril nucleation in model and disease-related proteins.

Protein amyloid aggregation is a hallmark in neuropathologies and other diseases of tremendous impact such as Alzheimer's or Parkinson's diseases. During the last decade, it has become increasingly evident that neuronal death is mainly induced by proteinaceous oligomers rather than the mature amyloid fibrils. Therefore, the earliest molecular events occurring during the amyloid aggregation cascade represent a growing interest of study. Important breakthroughs have been achieved using experimental data from different proteins, used as models, as well as systems related to diseases. Here, we summarize the structural properties of amyloid oligomeric and fibrillar aggregates and review the recent advances on how biophysical techniques can be combined with quantitative kinetic analysis and theoretical models to study the detailed mechanism of oligomer formation and nucleation of fibrils. These insights into the mechanism of early oligomerization and amyloid nucleation are of relevant interest in drug discovery and in the design of preventive strategies against neurodegenerative diseases.

Structural and Thermodynamic Analysis of HIV-1 Fusion Inhibition Using Small gp41 Mimetic Proteins.

Development of effective inhibitors of the fusion between HIV-1 and the host cell membrane mediated by gp41 continues to be a grand challenge due to an incomplete understanding of the molecular and mechanistic details of the fusion process. We previously developed single-chain, chimeric proteins (named covNHR) that accurately mimic the N-heptad repeat (NHR) region of gp41 in a highly stable coiled-coil conformation. These molecules bind strongly to peptides derived from the gp41 C-heptad repeat (CHR) and are potent and broad HIV-1 inhibitors. Here, we investigated two covNHR variants differing in two mutations, V10E and Q123R (equivalent to V38E and Q40R in gp41 sequence) that reproduce the effect of HIV-1 mutations associated with resistance to fusion inhibitors, such as T20 (enfuvirtide). A detailed calorimetric analysis of the binding between the covNHR proteins and CHR peptides (C34 and T20) reveals drastic changes in affinity due to the mutations as a result of local changes in interactions at the site of T20 resistance. The crystallographic structure of the covNHR:C34 complex shows a virtually identical CHR-NHR binding interface to that of the post-fusion structure of gp41 and underlines an important role of buried interfacial water molecules in binding affinity and in development of resistance against CHR peptides. Despite the great difference in affinity, both covNHR variants demonstrate strong inhibitory activity for a wide variety of HIV-1 strains. These properties support the high potential of these covNHR proteins as new potent HIV-1 inhibitors. Our results may guide future inhibition approaches.

Global Protein Stabilization Does Not Suffice to Prevent Amyloid Fibril Formation.

Mutations or cellular conditions that destabilize the native protein conformation promote the population of partially unfolded conformations, which in many cases assemble into insoluble amyloid fibrils, a process associated with multiple human pathologies. Therefore, stabilization of protein structures is seen as an efficient way to prevent misfolding and subsequent aggregation. This has been suggested to be the underlying reason why proteins living in harsh environments, such as the extracellular space, have evolved disulfide bonds. The effect of protein disulfides on the thermodynamics and kinetics of folding has been extensively studied, but much less is known on its effect on aggregation reactions. Here, we designed a single point mutation that introduces a disulfide bond in the all-α FF domain, a protein that, despite being devoid of preformed ß-sheets, forms ß-sheet-rich amyloid fibrils. The novel and unique covalent bond in the FF domain dramatically increases its thermodynamic stability and folding speed. Nevertheless, these optimized properties cannot counteract the inherent aggregation propensity of the protein, thus indicating that a high global protein stabilization does not suffice to prevent amyloid formation unless it contributes to hide from exposure the specific regions that nucleate the aggregation reaction.

Dynamic micellar oligomers of amyloid beta peptides play a crucial role in their aggregation mechanisms.

A deep understanding of the early molecular mechanism of amyloid beta peptides (Aß) is crucial to develop therapeutic and preventive approaches for Alzheimer's disease (AD). Using a variety of biophysical techniques, we have found that micelle-like dynamic oligomers are rapidly formed by Aß40 and Aß42 above specific critical concentrations. Analysis of the initial aggregation rates at 37 °C measured by thioflavin T and Bis-ANS fluorescence using a mass-action micellization model revealed a concentration-dependent switch in the nucleation mechanism. Bimolecular nucleation appears to occur at low peptide concentration while above the critical micellar concentration, the nucleation takes place more efficiently in the micelles. Upon incubation, these micelles mediate a rapid formation of larger, more stable oligomers enriched in beta-sheet structure. These oligomers formed from Aß40, enriched in amyloid nuclei, acquire a higher capacity to fibrillate than their micellar precursors. Aß42 can also form similar oligomers but they have lower beta-sheet structure content and lower capacity to fibrillate. On the other hand, a considerable fraction of the Aß42 peptide forms morphologically distinct oligomers that are unable to fibrillate and show significant effect on SH-SY5Y cell viability. Overall, our results highlight the importance of micellar structures as mediators of amyloid nucleation and contribute to the understanding of the differences between the aggregation pathways of Aß40 and Aß42.

The influence of N-terminal acetylation on micelle-induced conformational changes and aggregation of α-Synuclein.

The biological function of α-Synuclein has been related to binding to lipids and membranes but these interactions can also mediate α-Synuclein aggregation, which is associated to Parkinson's disease and other neuropathologies. In brain tissue α-Synuclein is constitutively N-acetylated, a modification that plays an important role in its conformational propensity, lipid and membrane binding, and aggregation propensity. We studied the interactions of the lipid-mimetic SDS with N-acetylated and non-acetylated α-Synuclein, as well as their early-onset Parkinson's disease variants A30P, E46K and A53T. At low SDS/protein ratios α-Synuclein forms oligomeric complexes with SDS micelles with relatively low α-helical structure. These micellar oligomers can efficiently nucleate aggregation of monomeric α-Synuclein, with successive formation of oligomers, protofibrils, curly fibrils and mature amyloid fibrils. N-acetylation reduces considerably the rate of aggregation of WT α-Synuclein. However, in presence of any of the early-onset Parkinson's disease mutations the protective effect of N-acetylation against micelle-induced aggregation becomes impaired. At higher SDS/protein ratios, N-acetylation favors another conformational transition, in which a second type of α-helix-rich, non-aggregating oligomers become stabilized. Once again, the Parkinson's disease mutations disconnect the influence of N-acetylation in promoting this transition. These results suggest a cooperative link between the N-terminus and the region of the mutations that may be important for α-Synuclein function.

Sublingual Priming with a HIV gp41-Based Subunit Vaccine Elicits Mucosal Antibodies and Persistent B Memory Responses in Non-Human Primates.

Persistent B cell responses in mucosal tissues are crucial to control infection against sexually transmitted pathogens like human immunodeficiency virus 1 (HIV-1). The genital tract is a major site of infection by HIV. Sublingual (SL) immunization in mice was previously shown to generate HIV-specific B cell immunity that disseminates to the genital tract. We report here the immunogenicity in female cynomolgus macaques of a SL vaccine based on a modified gp41 polypeptide coupled to the cholera toxin B subunit designed to expose hidden epitopes and to improve mucosal retention. Combined SL/intramuscular (IM) immunization with such mucoadhesive gp41-based vaccine elicited mucosal HIV-specific IgG and IgA antibodies more efficiently than IM immunization alone. This strategy increased the number and duration of gp41-specific IgA secreting cells. Importantly, combined immunization improved the generation of functional antibodies 3 months after vaccination as detected in HIV-neutralizing assays. Therefore, SL immunization represents a promising vaccine strategy to block HIV-1 transmission.

Molecular and Physicochemical Factors Governing Solubility of the HIV gp41 Ectodomain.

The HIV gp41 ectodomain (e-gp41) is an attractive target for the development of vaccines and drugs against HIV because of its crucial role in viral fusion to the host cell. However, because of the high insolubility of e-gp41, most biophysical and structural analyses have relied on the production of truncated versions removing the loop region of gp41 or the utilization of nonphysiological solubilizing conditions. The loop region of gp41 is also known as principal immunodominant domain (PID) because of its high immunogenicity, and it is essential for gp41-mediated HIV fusion. In this study we identify the aggregation-prone regions of the amino acid sequence of the PID and engineer a highly soluble mutant that preserves the trimeric structure of the wild-type e-gp41 under physiological pH. Furthermore, using a reverse mutagenesis approach, we analyze the role of mutated amino acids upon the physicochemical factors that govern solubility of e-gp41. On this basis, we propose a molecular model for e-gp41 self-association, which can guide the production of soluble e-gp41 mutants for future biophysical analyses and biotechnological applications.

Single-chain protein mimetics of the N-terminal heptad-repeat region of gp41 with potential as anti-HIV-1 drugs.

During HIV-1 fusion to the host cell membrane, the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) of the envelope subunit gp41 become transiently exposed and accessible to fusion inhibitors or Abs. In this process, the NHR region adopts a trimeric coiled-coil conformation that can be a target for therapeutic intervention. Here, we present an approach to rationally design single-chain protein constructs that mimic the NHR coiled-coil surface. The proteins were built by connecting with short loops two parallel NHR helices and an antiparallel one with the inverse sequence followed by engineering of stabilizing interactions. The constructs were expressed in Escherichia coli, purified with high yield, and folded as highly stable helical coiled coils. The crystal structure of one of the constructs confirmed the predicted fold and its ability to accurately mimic an exposed gp41 NHR surface. These single-chain proteins bound to synthetic CHR peptides with very high affinity, and furthermore, they showed broad inhibitory activity of HIV-1 fusion on various pseudoviruses and primary isolates.