ACE2 (19-740) Human

What is ACE2 (19-740) Human and what distinguishes it from other ACE2 variants?

ACE2 (19-740) Human refers to the full-length ectodomain of the angiotensin-converting enzyme 2, encompassing amino acids 19-740. This version includes both the catalytic domain (approximately aa 19-615) and the collectrin domain (approximately aa 615-740). The full-length ectodomain differs from truncated versions (such as trACE2, aa 19-615) that contain only the catalytic portion. In research contexts, the full-length ectodomain is significant because it maintains the complete extracellular structure of native ACE2, including the collectrin domain which contributes to proper protein folding and stability .

How does the structure-function relationship of ACE2 (19-740) Human influence experimental design?

The structure-function relationship of ACE2 (19-740) Human is critical for experimental design in several ways. The catalytic domain (aa 19-615) contains the binding interface for the SARS-CoV-2 spike protein RBD, while the collectrin domain (aa 615-740) contributes to structural stability. Understanding this relationship informs the design of experiments targeting specific ACE2 functions.

When designing ACE2-Fc fusion proteins, researchers must consider how to preserve the native structure while adding functional domains. For example, in constructing full-length ACE2-Fc (flACE2-Fc), the fusion point between the last residue of ACE2 (S740) and the IgG1-Fc domain requires careful linker design. Researchers have employed linker sequences such as "S740-GGGGS-T223" to maintain proper protein folding and function .

What methodologies are used to produce recombinant ACE2 (19-740) Human for research applications?

Production of recombinant ACE2 (19-740) Human typically involves mammalian expression systems to ensure proper folding and post-translational modifications. The methodological approach includes:

• Vector construction: The ACE2 (19-740) sequence is cloned into mammalian expression vectors such as pcDNA3.4 or pHLsec. For fusion proteins, synthetic DNA encoding the desired domains (e.g., IgG1-Fc) is incorporated with appropriate linker sequences .

• Cell line selection: Human embryonic kidney (HEK) cells, particularly HEK293T or HEK293F, are commonly used for expression due to their ability to produce properly folded human proteins with appropriate glycosylation patterns.

• Transfection optimization: Transient transfection protocols are optimized for high-yield protein production, utilizing reagents such as polyethylenimine (PEI) or commercial transfection agents.

• Protein purification: For ACE2-Fc fusion proteins, purification typically employs affinity chromatography using Protein A or Protein G columns, followed by size exclusion chromatography to ensure purity and proper dimerization .

• Quality control: Analytical methods including SDS-PAGE, Western blot, and functional binding assays are used to verify protein integrity and activity.

These methodological considerations are essential for producing research-grade ACE2 proteins with consistent quality and biological activity.

How can ACE2 (19-740) Human be engineered to create therapeutic receptor traps?

Engineering ACE2 (19-740) Human into therapeutic receptor traps involves several sophisticated strategies:

• Affinity optimization: Computational design followed by experimental affinity maturation can significantly enhance binding to viral spike proteins. Researchers have achieved up to 170-fold higher affinity than wild-type ACE2 through this approach . This process involves:

  • Computational redesign of the ACE2-RBD interface using flexible protein backbone design

  • Random mutagenesis and selection using techniques like yeast surface display

  • Validation of improved binding through biophysical assays

• Fusion to Fc domains: Conjugating ACE2 to human IgG1 Fc domains creates dimeric molecules with enhanced avidity and extended half-life. The strategic placement of the Fc domain and appropriate linker design are critical for maintaining proper structure and function .

• Domain optimization: Different ACE2 constructs can be engineered for specific applications:

  • Truncated ACE2 (trACE2, aa 19-615): Contains only the catalytic domain responsible for RBD binding

  • Full-length ACE2 ectodomain (flACE2, aa 19-740): Includes both catalytic and collectrin domains for improved stability

  • Enhanced-affinity variants (EflACE2): Incorporates specific mutations (T27Y, L79T, N330Y) that improve binding to SARS-CoV-2 spike RBD

• Fc engineering: Additional mutations in the Fc region (H429F, H429Y, E430G) can be introduced to modulate effector functions like antibody-dependent cellular cytotoxicity (ADCC) or complement activation .

This multifaceted engineering approach yields receptor traps that can potently neutralize viral infection by blocking the spike protein's ability to engage cellular ACE2.

What methodologies are used to assess binding kinetics between engineered ACE2 variants and viral spike proteins?

Assessing binding kinetics between engineered ACE2 variants and viral spike proteins employs several sophisticated methodological approaches:

• Biolayer Interferometry (BLI): This label-free technique measures real-time binding kinetics by detecting changes in the interference pattern of white light reflected from a biosensor surface. BLI has been used to determine association (kon) and dissociation (koff) rate constants and equilibrium dissociation constants (KD) for interactions between ACE2 variants and RBDs . Validation experiments show that mutations like K493Y and T498W in the RBD of African sarbecovirus BtKY72 enable binding to human ACE2, with improved affinity for double mutants .

• Flow Cytometry-Based Titration Assays: Researchers use this approach to systematically measure binding affinities for large libraries of ACE2 variants. The methodology involves:

  • Display of ACE2 variants on yeast or mammalian cell surfaces

  • Labeling with fluorescently tagged RBDs at varying concentrations

  • FACS-based separation of cells based on binding signal

  • Determination of apparent KD values using the Hill equation :

  The binding affinity is calculated using the standard non-cooperative Hill equation:
  Mean bin value = b + (a × [ACE2 concentration]/(KD,app + [ACE2 concentration]))

  Where:

  • a is the titration response range (constrained between 2 and 3)

  • b is the titration curve baseline (constrained between 1 and 1.5)

  • KD,app is the apparent binding constant (constrained between 1×10^-15 and 1×10^-5)

• Pseudovirus Entry Assays: These functional assays evaluate whether observed binding translates to inhibition of viral entry. Vesicular stomatitis virus (VSV) particles pseudotyped with wild-type or mutant spike proteins are tested for entry into ACE2-expressing cells. Quantification of entry provides a functional correlate to binding measurements .

These methodologies collectively provide comprehensive characterization of the binding parameters between engineered ACE2 variants and viral spike proteins, critical for therapeutic development.

How do epistatically constrained mutations in ACE2 (19-740) Human affect binding to different sarbecovirus receptor binding domains?

The impact of epistatically constrained mutations in ACE2 (19-740) Human on binding to different sarbecovirus RBDs reveals complex evolutionary dynamics in virus-host interactions. Research findings demonstrate that:

• Mutation effects are context-dependent: The same mutation can have dramatically different effects depending on the genetic background. For example, the N501Y mutation increases human ACE2-binding affinity for SARS-CoV-2 where it appears in variants of concern, but the homologous mutation in the SARS-CoV-1 RBD (position 487) is highly deleterious for human ACE2 binding .

• Epistatic turnover increases with sequence divergence: The variation in mutational effects increases as RBD sequences diverge from each other, indicating that the genetic context becomes increasingly important as sequences evolve (Fig. 3g) . This pattern of epistatic turnover follows a predictable trajectory that correlates with sequence distance.

• Position-specific variability: Some positions show relatively consistent effects across different RBD backgrounds (positions 486 and 494), while others display substantial variability in mutation effects as RBDs diverge (positions 498 and 501) . This position-specific pattern provides insight into evolutionary constraints at the ACE2-RBD interface.

• Single mutations can confer binding: In most cases where an RBD does not naturally bind to a particular ACE2 ortholog, single amino acid substitutions can confer low to moderate binding affinity. For example, the K493Y mutation in AncSarbecovirus enables binding to human ACE2, illustrating the evolutionary accessibility of host adaptation .

• Combinatorial effects: Multiple mutations can have synergistic effects on binding. The K493Y/T498W double mutant in the African sarbecovirus BtKY72 RBD shows enhanced binding to human ACE2 compared to single mutations, sufficient to enable pseudovirus entry into human ACE2-expressing cells .

These findings highlight the evolvability of ACE2 binding across diverse sarbecovirus lineages and provide insights into the molecular determinants of host range expansion potential.

What structural modeling approaches are used to study ACE2 (19-740) Human fusion proteins?

Structural modeling of ACE2 (19-740) Human fusion proteins utilizes several sophisticated computational approaches to predict and analyze their three-dimensional configurations:

• AlphaFold-based modeling: AlphaFold v2.2 has been employed to predict the structure of complex ACE2-Fc fusion proteins. The methodology involves:

  • Running AlphaFold on the complete sequence specifying homo-oligomerization

  • Generating multiple models (typically five) and selecting the most biologically plausible arrangement

  • Validating structural predictions by comparing to known crystal structures (e.g., PDB ID: 6M17 for ACE2 homodimer)

  For EflACE2-Fc, AlphaFold successfully recapitulated the observed structure of the ACE2 homodimer with an RMSD of 1.378 Å, although IgG1-Fc domains required additional modeling .

• Hybrid modeling approaches: For complex fusion proteins, researchers often employ a hybrid approach:

  • Separately modeling individual domains (e.g., ACE2 catalytic domain, collectrin domain, IgG1-Fc)

  • Superimposing domains based on known structures

  • Manually modeling linker regions based on reference structures (e.g., human B12 IgG crystal structure, PDB ID: 1HZH)

• Protein-protein docking: HADDOCK v2.4 has been used to model the interaction between ACE2-Fc constructs and SARS-CoV-2 spike RBD. The docking approach generates multiple clusters of potential binding modes, ranked by HADDOCK score. In published studies, the best models achieved HADDOCK scores of -151 ± 4.2 with RMSD from the lowest energy structure of 0.7 Å ± 0.5 .

• Validation against experimental structures: Modeled structures are validated by comparing with experimental structures of similar complexes. For example, SARS-CoV-2 spike RBD docked to ACE2 models has been compared with crystal structures (PDB ID: 6M0J), showing good alignment with RMSDs of 0.400 Å and 1.400 Å for the SARS-CoV-2 and ACE2 chains, respectively .

These computational approaches provide valuable structural insights that guide the design and optimization of ACE2-based therapeutic proteins.

How can ACE2 (19-740) Human be used in combination therapies against SARS-CoV-2?

ACE2 (19-740) Human can serve as a key component in combination therapeutic strategies against SARS-CoV-2, with several methodological approaches showing promise:

• Combination with antiviral drugs: Recombinant soluble ACE2 (hrsACE2) has been investigated in combination with remdesivir, showing synergistic effects against SARS-CoV-2 in both Vero E6 cells and kidney organoids . This combination approach allows for dose reduction of both agents while maintaining antiviral efficacy, potentially reducing side effects while enhancing therapeutic outcomes.

• Pharmacokinetic considerations: Previous clinical trials with hrsACE2 in healthy volunteers demonstrated that administering 800 μg/kg achieves plasma concentrations of 5–10 μg/ml between 2 and 8 hours post-administration . These pharmacokinetic parameters inform dosing strategies for combination therapies to achieve effective antiviral concentrations in vivo.

• Dual-action mechanisms: ACE2-Fc fusion proteins offer multiple mechanisms of action in combination settings:

  • Direct viral neutralization by competing with cellular ACE2 for spike protein binding

  • Potential Fc-mediated effector functions depending on the Fc variant used

  • Counteracting ACE2 downregulation induced by viral infection, potentially restoring physiological RAS balance

• Multi-sarbecovirus activity: Full-length ACE2-Fc fusion proteins display broad activity against diverse sarbecoviruses beyond SARS-CoV-2, potentially providing coverage against emerging variants and related coronaviruses in combination treatment regimens .

• Resistance mitigation: ACE2-based therapeutics present a high barrier to resistance development since they target the virus's essential entry mechanism. Mutations that reduce binding to ACE2-based therapeutics would likely also reduce binding to cellular ACE2, compromising viral fitness. When combined with antivirals targeting other viral components (like RNA-dependent RNA polymerase inhibitors), the risk of resistance development is further minimized .

These methodological approaches highlight the potential of ACE2 (19-740) Human as a versatile component in combination therapies against SARS-CoV-2 and related coronaviruses.

What are the functional differences between truncated ACE2 (trACE2) and full-length ACE2 (19-740) Human in research applications?

The functional differences between truncated ACE2 (trACE2, aa 19-615) and full-length ACE2 (19-740) Human in research applications are substantial and impact experimental outcomes. A comparative analysis reveals:

The truncated version (trACE2) contains only the catalytic domain responsible for viral binding, while the full-length version (flACE2) includes both the catalytic and collectrin domains. The collectrin domain contributes to protein stability and provides a more physiologically relevant context for studying ACE2 interactions .

For Fc fusion proteins, the design of linker sequences differs between trACE2-Fc (D615-GSGSGSG-T223) and flACE2-Fc (S740-GGGGS-T223), reflecting the different fusion points necessitated by the presence or absence of the collectrin domain . These structural differences can impact the orientation and flexibility of the fusion protein, potentially affecting binding avidity and neutralization potency.

In structural studies, modeling the full-length ACE2-Fc requires more complex approaches, often involving separate modeling of domains followed by superimposition and linker modeling, whereas truncated constructs are somewhat simpler to model .

These differences highlight the importance of selecting the appropriate ACE2 construct based on specific research objectives and experimental requirements.

How does the evolutionary history of ACE2 binding inform therapeutic development strategies?

The evolutionary history of ACE2 binding among sarbecoviruses provides critical insights that inform therapeutic development strategies in several important ways:

• Ancestral binding properties: Research has revealed that ACE2 binding is an ancestral trait of sarbecovirus receptor-binding domains that has been subsequently lost in some clades . This finding suggests that ACE2-targeting therapeutics may have broad potential against diverse sarbecoviruses, including those that have not yet spilled over to humans.

• Evolvability of human ACE2 binding: Multiple evolutionary paths can lead to human ACE2 binding capability. Single mutations at positions 493, 498, and 501 can enable RBDs from non-human-infecting sarbecoviruses to acquire human ACE2 binding . This evolutionary accessibility indicates that ACE2-based therapeutics should ideally target conserved binding interfaces to counter potential emergent strains.

• Geographic diversity considerations: Sarbecoviruses from Africa and Europe, not just Asia, show potential for human ACE2 binding. For example, the Khosta-2 RBD from Russia binds to human ACE2 even without adaptive mutations . This geographic diversity suggests that therapeutic development should consider globally diverse sarbecovirus strains rather than focusing solely on Asian lineages.

• Epistatic constraints: The effects of specific mutations vary across different RBD backgrounds, with the variability increasing as sequences diverge . This pattern of epistatic turnover provides insights into which positions might be more evolutionarily constrained and therefore better targets for therapeutic intervention.

• Broad-spectrum design strategy: The finding that binding to bat ACE2 (particularly from Rhinolophus affinis) is widespread among sarbecoviruses suggests that therapeutics designed to mimic conserved binding interfaces could have broad spectrum activity . This approach could lead to countermeasures effective against both current and future sarbecovirus threats.

These evolutionary insights guide the development of therapeutics with broad-spectrum activity and high barriers to resistance, potentially offering protection against future pandemic threats from the sarbecovirus lineage.

What are the key experimental controls needed when evaluating ACE2 (19-740) Human binding to viral spike proteins?

When evaluating ACE2 (19-740) Human binding to viral spike proteins, several critical experimental controls must be implemented to ensure reliable, reproducible, and interpretable results:

• Protein quality controls:

  • Purity assessment via SDS-PAGE and size exclusion chromatography to ensure homogeneity

  • Confirmation of proper folding through circular dichroism or thermal stability assays

  • Verification of expected molecular weight by mass spectrometry

  • Endotoxin testing for experiments involving cellular systems

• Binding assay controls:

  • Positive control: Known high-affinity ACE2-spike interaction (e.g., wild-type human ACE2 with SARS-CoV-2 RBD)

  • Negative control: Non-binding protein with similar size/structure to ACE2

  • Background binding control: Target surfaces without immobilized protein

  • Concentration-matched controls: Ensuring equivalent molar concentrations across compared samples

• Specificity controls:

  • Competition assays with soluble ACE2 or neutralizing antibodies

  • Testing against non-related viral proteins to confirm binding specificity

  • Mutant controls with known binding-disrupting mutations in either ACE2 or spike

  • Cross-reactivity assessment with related coronaviruses

• Methodological validation:

  • Internal replicate controls with multiple independently barcoded libraries

  • Technical replicates to assess measurement variation

  • Barcode collapsing procedures to minimize noise, such as discarding outliers (top and bottom 5% for expression measurements or 2.5% for titration affinities)

  • Curve fitting quality metrics, including normalized mean square residual thresholds

• Functional correlation controls:

  • Parallel assessment of binding and functional outcomes (e.g., pseudovirus neutralization)

  • Comparison of binding affinity (KD,app) with functional potency (IC50)

  • Dose-response relationships to establish physiological relevance

How should researchers address potential artifacts in structural modeling of ACE2 (19-740) Human complexes?

Addressing potential artifacts in structural modeling of ACE2 (19-740) Human complexes requires a systematic approach that combines computational validation with experimental verification:

By implementing these approaches, researchers can minimize artifacts in structural models of ACE2 (19-740) Human complexes and develop reliable structural understanding to guide experimental design and therapeutic development.

What emerging technologies could enhance the therapeutic potential of ACE2 (19-740) Human against future coronavirus threats?

Several emerging technologies hold promise for enhancing the therapeutic potential of ACE2 (19-740) Human against future coronavirus threats:

• Advanced protein engineering platforms:

  • Deep mutational scanning combined with machine learning to predict optimal ACE2 variants

  • Directed evolution in cell-free systems for rapid selection of improved binding properties

  • Computational design of "super-binders" with optimized interfaces targeting conserved epitopes across diverse sarbecoviruses

  • Engineering ACE2 variants that selectively bind viral spike proteins without affecting physiological ACE2 substrates

• Novel delivery technologies:

  • Lipid nanoparticle formulations for improved biodistribution and extended half-life

  • Inhalation delivery systems to target respiratory epithelium directly

  • Engineered extracellular vesicles expressing ACE2-based decoys on their surface

  • mRNA-based approaches for in vivo production of optimized ACE2 decoys

• Multi-specific fusion constructs:

  • Tri-specific constructs combining ACE2 with two different neutralizing antibody domains

  • ACE2-cytokine fusions that combine viral neutralization with immunomodulatory effects

  • Mosaic designs incorporating binding domains from multiple ACE2 orthologs to broaden coverage

  • Integration with immune checkpoint modulators to enhance innate immune responses

• Combination therapeutic approaches:

  • Further development of ACE2-based therapeutics in combination with antivirals like remdesivir

  • Strategic combinations with broadly neutralizing antibodies targeting non-overlapping epitopes

  • Integration into multi-mechanism therapeutic cocktails targeting different stages of the viral life cycle

  • Companion diagnostic development to personalize combination therapy based on viral variants

• Pandemic preparedness applications:

  • Development of broadly active ACE2-based biologics as ready-to-deploy countermeasures

  • Creation of ACE2 decoy libraries targeting predicted future coronavirus threats

  • Integration of ACE2-based therapeutics into rapid response platforms for emerging viruses

  • Development of extended room-temperature stability formulations for global distribution

These emerging technologies could collectively transform ACE2-based therapeutics into powerful tools for addressing future coronavirus pandemic threats, potentially providing broad-spectrum protection against both known and yet-to-emerge sarbecoviruses.

How might methodological advances improve the characterization of ACE2 (19-740) Human interactions with diverse sarbecoviruses?

Methodological advances offer significant potential to enhance our understanding of ACE2 (19-740) Human interactions with diverse sarbecoviruses:

• High-throughput binding characterization:

  • Deep mutational scanning of the ACE2-RBD interface across multiple sarbecovirus lineages

  • Massively parallel yeast display systems for simultaneous assessment of thousands of ACE2-RBD interactions

  • Microfluidic systems for rapid, low-volume binding kinetics measurements

  • Advanced computational approaches to predict binding affinities between ACE2 and novel RBDs

• Structural biology innovations:

  • Cryo-electron microscopy (cryo-EM) approaches for capturing dynamic binding events

  • AlphaFold2 and RoseTTAFold integration with experimental data for highly accurate modeling

  • Time-resolved structural studies to capture conformational changes during ACE2-RBD binding

  • Hydrogen-deuterium exchange mass spectrometry for mapping binding interfaces of difficult-to-crystallize complexes

• Single-molecule techniques:

  • Single-molecule FRET to observe real-time binding dynamics

  • Optical tweezers measurements of ACE2-RBD bond strength across sarbecovirus lineages

  • Advanced atomic force microscopy approaches to quantify binding forces at the single-molecule level

  • Super-resolution microscopy to visualize ACE2-spike interactions in cellular contexts

• Advanced cellular models:

  • Organoid systems representing diverse host tissues for studying ACE2-mediated viral entry

  • CRISPR-engineered cell lines expressing ACE2 orthologs from different potential host species

  • Tissue chips incorporating primary human respiratory epithelium for physiologically relevant entry studies

  • Multi-cellular systems modeling the immune microenvironment during viral entry

• In silico prediction tools:

  • Machine learning algorithms to predict zoonotic potential based on ACE2-RBD binding parameters

  • Network analysis approaches to identify key evolutionary constraints in the ACE2-RBD interaction

  • Molecular dynamics simulations to model binding energetics and conformational changes

  • Phylogenetic analysis tools integrating binding data to trace the evolution of ACE2 usage

These methodological advances would enable more comprehensive characterization of ACE2-sarbecovirus interactions, facilitating better prediction of zoonotic potential and more effective design of broad-spectrum therapeutics and preventive measures against future coronavirus threats.