ACE2 (18-615) Human

What is the structural composition of ACE2 (18-615) Human?

ACE2 (18-615) represents the extracellular peptidase domain of human Angiotensin-Converting Enzyme 2. This soluble fragment contains the SARS-CoV-2 spike binding interface while excluding the transmembrane and cytoplasmic domains. Structurally, it has been characterized through cryo-EM studies with several structures available in the Protein Data Bank .

The construct typically contains two key domains:

• The N-terminal peptidase domain (PD) which includes the viral binding interface

• Two subdomains within the PD: residues 18-102 (PD1) and 272-409 (PD2)

For research applications, mutations H376N and H380N are often introduced to abolish zinc binding and proteolytic activity, particularly when studying binding interactions rather than enzymatic functions .

How is ACE2 (18-615) expressed and purified for research applications?

The standard methodology for expressing and purifying this construct involves:

• Synthetic gene generation for codon-optimized human ACE2 (residues 1-615)

• Cloning into expression vectors, typically pCMV-IRES-puro for mammalian expression

• Addition of C-terminal tags (commonly 6xHis tag) for purification

• Transient transfection in HEK293F cells using Opti-MEM

• Incubation for 4 days at 37°C with 5.5% CO₂

• Harvesting by centrifugation at 2,524 ×g for 30 minutes

• Purification via affinity chromatography

For specialized applications such as enhanced binding or stability studies, additional modifications may include:

• Introduction of a foldon trimerization tag between ACE2 and the His tag

• Fusion with Fc segment of human IgG1 to create ACE2-Fc constructs

• Site-specific mutations guided by structural data

How do ACE2 (18-615) variants affect SARS-CoV-2 binding?

Deep mutational scanning experiments have identified over one hundred human single-nucleotide variants (SNVs) that significantly alter SARS-CoV-2 spike recognition . These studies revealed:

• Mutations at the direct binding interface can either enhance or diminish binding through alterations in:

  • Hydrophobic packing

  • Hydrogen-bonding geometry

  • Electrostatic interactions

• Unexpectedly, mutations in residues distal to the spike-binding interface can also significantly influence binding dynamics, suggesting allosteric effects that propagate structural changes .

• The effect of mutations can be quantified through "mutation effect" coefficients, with a p-value threshold of <0.05 indicating significant impact on spike protein binding .

Based on population genetic data, approximately 320-365 individuals per 100,000 in the general population carry SNVs predicted to decrease spike binding, while 4-12 per 100,000 possess variants that may enhance binding .

What methodologies are used to study ACE2-spike protein interactions?

Several complementary approaches are employed to characterize ACE2-spike interactions:

• Structural analysis:

  • Cryo-electron microscopy of ACE2-spike complexes

  • X-ray crystallography of ACE2-RBD complexes

  • Computational modeling and molecular dynamics simulations

• Binding kinetics:

  • Surface plasmon resonance (SPR)

  • Bio-layer interferometry (BLI)

  • Enzyme-linked immunosorbent assays (ELISA)

• Cooperativity studies:

  • Hill coefficient calculations to determine the degree of cooperativity between ACE2 and membrane-bound spike trimers

  • Comparison of binding parameters between SARS-CoV-1 and SARS-CoV-2 spike proteins

• Cell-based assays:

  • Flow cytometry to measure binding of fluorescently-labeled RBD to cell-surface ACE2

  • Pseudovirus neutralization assays to evaluate functional consequences of binding

What are the key differences between ACE2 interaction with SARS-CoV-1 versus SARS-CoV-2?

Despite the high genomic similarity between SARS-CoV-1 and SARS-CoV-2 (~80% genomic sequence identity), there are notable differences in their ACE2 interactions :

• Structural similarities:

  • Both viruses utilize similar contact interfaces on ACE2

  • Both require the "up" orientation and slight rotation of the RBD for effective receptor engagement

  • ACE2 binding epitopes on both RBDs are inaccessible in the fully closed spike conformation

• Key differences:

  • The amino acid sequence identity between their spike proteins is approximately 76%

  • Their RBDs share 74% amino acid sequence identity

  • Different cooperativity of ACE2 within their respective spike trimers, measurable through Hill coefficient calculations

  • SARS-CoV-2, particularly the D614G variant, shows altered binding kinetics compared to SARS-CoV-1

These differences may contribute to the distinct transmission patterns and pathogenicity observed between the two coronaviruses.

How can directed evolution enhance ACE2 binding affinity to SARS-CoV-2?

Directed evolution has been successfully employed to generate high-affinity ACE2 variants through several methodological approaches:

Key considerations in system selection include:

• Human cell-based systems better reflect native glycosylation patterns

• Experimental validation of each mutation at the soluble protein level is critical, as high affinity in surface display doesn't always correlate with RBD-competing activity

This approach has yielded ACE2 variants with 100-fold improved neutralization potency against SARS-CoV-2 .

What approaches are used to analyze human ACE2 genetic variants and their impact on viral susceptibility?

A comprehensive methodology for analyzing ACE2 genetic variants includes:

• Population genetics analysis:

  • Data retrieval from Genome Aggregation Database (gnomAD)

  • Integration of exome (gnomAD v2.1.1) and genome (gnomAD v3) data

  • Calculation of allele frequencies across populations (>250,000 individuals)

  • Subpopulation analysis across Admixed American, African, European, East Asian, and South Asian groups

• Experimental characterization:

  • Deep mutational scanning using yeast surface display

  • Expression of ACE2 variants fused to aga2 protein

  • Screening using fluorescently labeled RBD

  • Cell sorting by FACS based on binding signal

  • Deep sequencing to identify enriched or depleted variants

• Computational analysis:

  • Positive-unlabeled (PU) learning methods to estimate mutation effects

  • Calculation of mutation effect coefficients with associated p-values

  • Site importance evaluation by aggregating mutation effects at each position

  • Structure-based analysis to understand mechanism of altered binding

This integrated approach enables prediction of how natural human genetic variation impacts SARS-CoV-2 susceptibility and potential disease severity .

How can engineered ACE2 variants overcome viral mutational escape?

Engineering ACE2 variants resistant to viral escape involves multiple strategic approaches:

• Affinity enhancement strategies:

  • Random mutagenesis and selection for higher binding affinity

  • Structure-guided mutations targeting the binding interface

  • Introduction of stabilizing mutations such as disulfide bonds to fix ACE2 in an optimal binding conformation

• Validation methodologies:

  • In vitro viral neutralization assays comparing wild-type and engineered ACE2

  • Viral escape studies involving co-incubation of virus with engineered ACE2 over multiple passages

  • Structural characterization of engineered ACE2-RBD complexes

Key research findings demonstrate:

• Engineered ACE2 variants neutralize SARS-CoV-2 at 100-fold lower concentrations than wild-type

• No escape mutants emerged in co-incubation experiments after 15 passages, suggesting a high barrier to resistance

• Enhanced affinity typically results from improved molecular interactions at the binding interface

• Additional disulfide mutations improve both structural stability and therapeutic potential

These approaches provide advantages over antibody-based therapeutics, which are more susceptible to escape mutations.

What are the key considerations for designing ACE2-based therapeutic strategies?

Development of ACE2-based therapeutics requires careful attention to multiple parameters:

• Design considerations:

  • Format selection: soluble ACE2 (residues 1-615) can be fused with various tags

  • Half-life extension: fusion with Fc segment (human IgG1-Fc)

  • Avidity enhancement: multimerization strategies such as foldon trimerization tags

  • Stability optimization: introduction of disulfide bonds to fix closed conformation

• Potential challenges:

  • Immunogenicity of modified ACE2 potentially inducing anti-drug antibodies

  • Balancing improved binding with maintenance of proper folding and stability

  • Potential off-target effects related to ACE2's natural enzymatic activity

• Essential validation studies:

  • In vitro neutralization against diverse viral strains

  • In vivo protective efficacy in animal models (hamsters show decreased lung viral titers and pathology)

  • Pharmacokinetic and biodistribution studies

  • Safety assessments regarding impact on the renin-angiotensin system

Engineered ACE2 therapeutics offer potential advantages over antibodies, particularly in addressing viral variants, as they target the evolutionarily constrained receptor-binding interface of the spike protein .

How do residues distal from the binding interface influence ACE2-spike interactions?

One of the most intriguing discoveries in ACE2 research is that mutations in residues not directly at the spike-binding interface can significantly alter viral recognition :

• Experimental evidence:

  • Deep mutational scanning reveals "hotspots" of binding influence outside the direct contact interface

  • Site importance analysis across the entire extracellular domain identifies unexpected regions of functional significance

• Proposed mechanisms:

  • Allosteric effects that propagate structural changes to the binding interface

  • Alterations in protein dynamics affecting the conformational ensemble

  • Changes in protein stability influencing the presentation of binding epitopes

  • Modifications to glycosylation patterns indirectly impacting binding

• Research implications:

  • Computational studies should expand beyond the direct binding interface

  • Human genetic variation across the entire ACE2 gene may influence COVID-19 susceptibility

  • Therapeutic design strategies should consider the entire ACE2 extracellular domain

  • Comprehensive mutational analysis is necessary to fully understand ACE2-spike interactions

This finding has significant implications for understanding differential susceptibility to infection and may inform broader approaches to therapeutic design targeting distal regulatory sites .