SARS MERS RBD, Active

How does the MERS-CoV RBD interact with its cellular receptor?

The MERS-CoV RBD interacts specifically with the β-propeller domain of dipeptidyl peptidase 4 (DPP4), not with its intrinsic hydrolase domain . This interaction involves multiple key residues in the receptor-binding subdomain. Mutagenesis studies have identified critical residues in both patch 1 and other regions. For example, the Y499A substitution, which eliminates hydrogen bonding between Y499 of the RBD and R336 of DPP4, significantly disrupts binding and hinders viral entry. Similarly, combined substitutions involving E536R, D537K, and D539K, which disrupt native interactions with K267 on DPP4, profoundly reduce binding and viral entry efficiency .

What is the significance of the RBM in coronavirus research?

The Receptor Binding Motif (RBM) is the main neutralizing epitope for beta-coronaviruses and thus represents a critical target for vaccine development . The RBM is a specific segment within the RBD that directly interacts with host cell receptors. For MERS-CoV, the RBM spans residues K493 to E565 . Functional reconstitution of the MERS-CoV RBM has been achieved by connecting segments of the natural MERS-CoV RBM via combinatorial linkers, yielding diverse conformers that can be expressed and displayed on bacteriophage for further study. This approach provides valuable immunogens for potential MERS-CoV epitope-based vaccines that could be developed for both humans and camels .

What methodologies are effective for studying conformational changes in coronavirus RBDs?

For studying conformational dynamics, site-directed mutagenesis combined with binding assays (such as surface plasmon resonance) can identify residues critical for maintaining proper conformation. For example, mutations at Leu507, Leu545, Ser546, Pro547, and Glu549 in the MERS-CoV RBD significantly decreased binding affinity to neutralizing antibodies MERS-4 and MERS-4V2 by more than 10-fold . Additionally, cryo-electron microscopy has proven valuable for studying the spike protein trimer structure, though challenges with incomplete modeling of the RBM have been noted .

How can researchers functionally reconstruct and express coronavirus RBMs for immunological studies?

Functional reconstitution of coronavirus RBMs can be achieved through a systematic approach as demonstrated with the MERS-CoV RBM :

• Segment the natural RBM sequence into discrete parts (e.g., Segment A: residues K493-P515 of the MERS-CoV RBM)

• Connect these segments via combinatorial linkers of varying lengths (3-7 random amino acids)

• Express the constructs as phage-display libraries using modified vectors such as fth1

• Screen the libraries against neutralizing antibodies or receptor proteins

• Validate binding specificity through assays with relevant antibodies and receptors

This methodology generates diverse RBM conformers that can be evaluated for proper folding and immunogenicity. The approach allows researchers to identify optimal constructs that maintain the critical neutralizing epitopes while potentially enhancing stability or expression levels .

What techniques are most effective for identifying and characterizing neutralizing epitopes on coronavirus RBDs?

A multi-technique approach yields the most comprehensive characterization of neutralizing epitopes:

• Structural analysis: Crystal structures of RBD-antibody complexes provide atomic-level details of binding interfaces. In cases where high-resolution structures are challenging to obtain, molecular docking and homology modeling can provide preliminary insights .

• Alanine scanning mutagenesis: Systematically replacing key residues with alanine helps identify critical contact points. For example, mutations at Leu507, Leu545, Ser546, Pro547, and Glu549 in the MERS-CoV RBD significantly affected binding to neutralizing antibodies .

• Competition binding assays: These determine whether antibodies compete with the receptor for RBD binding, revealing the mechanism of neutralization. MERS-4 and MERS-4V2 antibodies exhibit a unique mechanism by approaching the RBD from outside the RBD-DPP4 binding interface, inducing conformational changes that indirectly interfere with receptor binding .

• Gel filtration analysis: This technique can examine combined binding of multiple antibodies to determine if they recognize distinct or overlapping epitopes, as demonstrated with MERS-4 and MERS-27 antibodies .

How do neutralizing antibodies that target coronavirus RBDs differ in their mechanisms of action?

Neutralizing antibodies targeting coronavirus RBDs exhibit diverse mechanisms of action:

• Direct receptor competition: Most RBD-targeting neutralizing antibodies directly compete with the receptor for binding to the RBD. This is the predominant mechanism observed for many SARS-CoV and MERS-CoV neutralizing antibodies .

• Conformational change induction: A unique mechanism demonstrated by antibodies like MERS-4 and MERS-4V2, which approach the RBD from outside the RBD-DPP4 binding interface. These antibodies induce significant conformational changes in the RBD, indirectly interfering with receptor binding .

• Cross-reactive epitope targeting: Some antibodies target conserved epitopes across different coronaviruses. For example, antibody CR3022 can bind to both SARS-CoV and SARS-CoV-2 RBDs with nanomolar affinity, though its precise epitope location remains under investigation .

The efficacy of these mechanisms varies depending on the specific antibody-RBD interaction, with binding affinity ranging from nanomolar to picomolar levels. Understanding these diverse mechanisms provides valuable insights for therapeutic antibody development and vaccine design .

What factors determine cross-reactivity of antibodies between different coronavirus RBDs?

Cross-reactivity of antibodies between different coronavirus RBDs depends on several key factors:

• Epitope conservation: The degree of sequence and structural conservation at the epitope is critical. Analysis of antibodies m396 and 80R, which bind SARS-CoV RBD but not SARS-CoV-2 RBD, revealed 7/21 and 16/25 residue changes in their respective epitopes, explaining their lack of cross-reactivity .

• Binding interface adaptability: Antibodies that can accommodate variations in side chains at the binding interface have higher potential for cross-reactivity. Structural analysis shows that even in the variable RBM regions, some residues remain identical between SARS-CoV and SARS-CoV-2, providing potential targets for cross-reactive antibodies .

• Conformational epitopes vs. linear epitopes: Antibodies targeting conformational epitopes preserved across different coronaviruses show greater potential for cross-reactivity than those targeting variable linear epitopes .

• Target subdomain: Antibodies targeting the more conserved core subdomain rather than the variable receptor-binding subdomain have higher potential for cross-reactivity, as demonstrated by the structural similarity between MERS-CoV and SARS-CoV core subdomains despite low sequence homology .

This understanding guides the identification and development of broadly neutralizing antibodies with therapeutic potential against multiple coronavirus species .

What experimental approaches can enhance antibody production for structural studies of RBD-antibody complexes?

Several strategies can improve antibody production for structural studies:

• CDR modification: Targeting specific complementarity-determining regions (CDRs) can significantly enhance expression levels. For example, researchers generated a library of mutant MERS-4 antibodies with random replacements in the 5-residue-long CDR3 region and identified variant MERS-4V2 with >10-fold improvement in productivity while maintaining neutralization potency. Sequence analysis revealed that the original CDR3 residues Ala-Gly-Asn-Asp (AGND) were replaced by Thr-Asn-Thr-Tyr (TNTY) in the improved variant .

• Expression system optimization: Using optimized expression vectors and host cells can improve antibody yields. HEK293F cells have been successfully employed to express antibodies like MERS-4 and MERS-4V2 for structural studies .

• Fab fragment generation: Working with Fab fragments rather than full antibodies can facilitate crystallization and structural determination. This approach was used successfully for obtaining MERS-4 Fab/RBD complex structures, though challenges with crystal quality remained .

• Crystallization condition screening: Extensive optimization and screening (>200 crystals in some cases) may be necessary to obtain diffraction-quality crystals suitable for structural studies .

These approaches have proven valuable for overcoming production challenges that have hampered structural analyses of important neutralizing antibodies against coronavirus RBDs.

How can structural insights into coronavirus RBDs guide epitope-based vaccine development?

Structural insights into coronavirus RBDs provide valuable guidance for epitope-based vaccine development through several approaches:

• Identifying conserved neutralizing epitopes: Crystal structures of RBD-antibody complexes reveal critical epitopes that can be conserved across coronavirus variants or species. For example, the core subdomain of MERS-CoV and SARS-CoV RBDs shows high structural similarity despite sequence divergence, suggesting selection pressure for structural conservation in this region . Targeting these conserved regions could lead to broadly protective vaccines.

• Epitope stabilization strategies: Understanding the conformational states of the RBD enables stabilization of neutralizing epitopes in their native conformation. The functional reconstitution of the MERS-CoV RBM through combinatorial linkers creates diverse conformers that can serve as effective immunogens for developing epitope-based vaccines .

• Rational immunogen design: Detailed knowledge of receptor-binding interfaces allows for precise engineering of immunogens that present critical epitopes while eliminating non-neutralizing or potentially harmful epitopes. For MERS-CoV, the receptor-binding subdomain that interacts with DPP4 β-propeller contains key residues (Y499, E536, D537, D539) that are essential for viral entry and represent important targets for neutralizing antibodies .

• Cross-protective vaccine strategies: Structural comparisons between SARS-CoV, MERS-CoV, and SARS-CoV-2 RBDs reveal both unique and shared features that can inform the development of vaccines with broader protection against multiple coronaviruses or future emergent strains .

What methodological approaches can identify potential therapeutic targets within the RBD-receptor interface?

Identification of therapeutic targets within RBD-receptor interfaces relies on several complementary methodologies:

• High-resolution structural analysis: Crystal structures at 3.0 Å resolution or better can reveal atomic details of the interface between viral RBDs and host receptors. For the MERS-CoV RBD-DPP4 complex, this approach identified specific interaction "patches" that are critical for binding .

• Systematic mutagenesis: Site-directed mutagenesis of interface residues followed by binding and functional assays can validate the importance of specific interactions. For example, the Y499A mutation in MERS-CoV RBD disrupts hydrogen bonding with R336 on DPP4, significantly reducing binding affinity and viral entry efficiency . This systematic approach identifies residues that could be targeted by therapeutics.

• Computational interface analysis: Bioinformatic analysis of binding interfaces can predict hotspots and conserved regions that represent promising therapeutic targets. This approach complements experimental data and can guide the design of focused experiments .

• Conformational change analysis: Comparing RBD structures in unbound, receptor-bound, and antibody-bound states reveals conformational changes that could be exploited therapeutically. Antibodies like MERS-4 that indirectly interfere with receptor binding through induced conformational changes highlight the potential of targeting regions outside the direct receptor-binding interface .

• Cross-species conservation analysis: Examining conservation of interface residues across coronavirus species or variants can identify targets with reduced potential for escape mutations. Regions under evolutionary constraint represent particularly valuable therapeutic targets .

How do researchers assess the potential for receptor binding domain (RBD) mutations to facilitate zoonotic transmission?

Researchers employ multiple approaches to evaluate the potential for RBD mutations to facilitate zoonotic transmission:

• Comparative structural analysis: By comparing RBD structures from different coronaviruses that have successfully crossed species barriers (like SARS-CoV and MERS-CoV) with those that remain species-restricted, researchers can identify structural features associated with zoonotic potential. The structural conservation in the core subdomain despite sequence diversity suggests possible convergent evolution in this region under selection pressure .

• Receptor binding affinity studies: Quantitative measurement of binding affinity between variant RBDs and receptors from different species using techniques like surface plasmon resonance (SPR) can identify mutations that enhance cross-species receptor recognition. Higher binding affinity to human receptors often correlates with increased zoonotic potential .

• Pseudovirus entry assays: Testing the ability of pseudoviruses bearing mutant spike proteins to enter cells expressing receptors from different species provides functional validation of zoonotic potential. Mutations that enhance entry into human cells while maintaining entry into the original host species cells are particularly concerning .

• Epidemic surveillance and genomic analysis: Monitoring circulating coronaviruses in animal populations, particularly in regions with high human-animal contact, allows for identification of RBD mutations that might indicate adaptation toward human receptor binding. This is especially relevant in countries like Saudi Arabia and UAE, which have both MERS-CoV-infected camel populations and high SARS-CoV-2 prevalence, creating conditions for potential recombination or co-evolution .

• In silico modeling: Computational prediction of how specific mutations might affect receptor binding across species can prioritize variants for experimental verification and surveillance. This approach is particularly valuable for rapid assessment of emerging variants .

What are the main difficulties in expressing and purifying functional coronavirus RBDs for structural studies?

Researchers face several significant challenges when expressing and purifying functional coronavirus RBDs:

• Proper folding and disulfide bond formation: Coronavirus RBDs contain multiple disulfide bonds that are crucial for maintaining their native conformation. The SARS-CoV RBD, for example, contains a disulfide bond between C467 and C474 that is essential for its structural integrity . Ensuring proper formation of these disulfide bonds during recombinant expression requires careful optimization of expression systems.

• Glycosylation requirements: RBDs contain N-linked glycosylation sites that can affect folding, stability, and functionality. Expression systems must support appropriate glycosylation patterns, which often necessitates the use of mammalian or insect cell expression systems rather than bacterial systems .

• Conformational heterogeneity: RBDs can exist in multiple conformational states, complicating structural studies that require homogeneous samples. This is evidenced by the observed conformational changes in RBDs when bound to antibodies like MERS-4 compared to their receptor-bound states .

• Expression yield variability: Expression levels can vary dramatically based on the specific RBD construct. For example, some antibody variants like MERS-4 showed poor expression (< 1 mg/L), while the optimized variant MERS-4V2 demonstrated >10-fold improvement in productivity .

• Purification complexity: Obtaining highly pure, monodisperse RBD samples suitable for crystallization or cryo-EM studies often requires multiple chromatography steps and careful quality control to remove aggregates and contaminants .

These challenges necessitate systematic optimization of expression constructs, host cells, and purification protocols tailored to each specific RBD variant.

What crystallization challenges are commonly encountered when studying RBD-antibody or RBD-receptor complexes?

Crystallization of RBD-antibody or RBD-receptor complexes presents several significant challenges:

• Complex size and flexibility: These complexes typically have high molecular weights and contain flexible regions that can hinder crystal formation or lead to poor diffraction quality. Despite extensive efforts optimizing and screening more than 200 crystals, researchers were only able to obtain X-ray diffraction data of MERS-CoV RBD in complex with MERS-4 Fab at 4.5 Å and with MERS-4V2 Fab at 7 Å resolution .

• Glycan heterogeneity: N-linked glycans on RBDs and receptor proteins introduce heterogeneity that can interfere with crystal packing. Strategies to address this include using glycosidase treatments, expressing in cells with restricted glycosylation capabilities, or engineering glycan-free variants while maintaining proper folding .

• Conformational dynamics: RBDs can undergo significant conformational changes upon binding to antibodies or receptors, potentially introducing sample heterogeneity that complicates crystallization. The observed conformational changes in the RBD when bound to MERS-4 or MERS-4V2 illustrate this challenge .

• Complex stability: Some RBD-antibody or RBD-receptor complexes may have moderate binding affinities or fast dissociation rates, resulting in dynamic equilibrium that hinders crystallization. Stabilizing these complexes through chemical crosslinking or engineering higher-affinity variants may be necessary .

• Reproducibility issues: Even successful crystallization conditions may yield crystals with variable diffraction quality due to subtle differences in protein batches or crystallization setup. This necessitates extensive screening and optimization .

These challenges often require innovative approaches, including Fab fragment usage instead of whole antibodies, construct optimization to remove flexible regions, and extensive crystallization condition screening.

How can researchers address the challenge of incomplete modeling of receptor binding motifs (RBMs) in structural studies?

Incomplete modeling of receptor binding motifs (RBMs) in structural studies presents a significant challenge, as evidenced by the issues noted in cryo-EM studies of the SARS-CoV-2 spike trimer . Researchers can address this challenge through several complementary approaches:

• Multi-technique integration: Combining data from different structural techniques can provide more complete information. For example, while cryo-EM studies of the spike trimer may struggle to resolve the RBM, targeted X-ray crystallography studies of the isolated RBD-receptor complex can provide high-resolution details of the RBM, as demonstrated in the 3.0 Å structure of the MERS-CoV RBD-DPP4 complex .

• Molecular dynamics simulations: Computational modeling can help predict the conformational dynamics of poorly resolved regions based on the well-defined portions of the structure and physicochemical principles. These simulations can generate hypotheses about RBM conformations that can be tested experimentally .

• Antibody-assisted structural determination: Using antibodies that bind specifically to the RBM can stabilize this flexible region and facilitate its structural determination. The structure of antibody-RBD complexes can reveal the conformation of the RBM in its antibody-bound state, providing valuable insights into its structural properties .

• Functional validation through mutagenesis: Even with incomplete structural models, systematic mutagenesis can validate the functional importance of specific residues within the RBM. This approach identified key residues in the MERS-CoV RBD (Y499, E536, D537, D539) that are critical for receptor binding and viral entry .

• Construct optimization: Engineering stabilized constructs of the RBM through techniques like disulfide bond introduction or systematic linker screening can reduce flexibility and improve structural resolution. The functional reconstitution approach used for the MERS-CoV RBM, which connected segments via combinatorial linkers, represents a strategy to generate more stable RBM conformers .

By combining these approaches, researchers can overcome the limitations of individual structural techniques and develop more complete models of receptor binding motifs.

How might structural insights into coronavirus RBDs inform strategies for developing broadly neutralizing antibodies against future emerging coronaviruses?

Structural insights into coronavirus RBDs offer several promising strategies for developing broadly neutralizing antibodies against future emerging coronaviruses:

• Targeting conserved structural epitopes: Despite sequence variability, coronavirus RBDs show remarkable structural conservation in their core subdomains. The high structural similarity between MERS-CoV and SARS-CoV RBD core subdomains (r.m.s.d. of 2.0 Å for 95 aligned Cα atoms) despite low sequence homology suggests evolutionary pressure to maintain certain structural features . Antibodies targeting these conserved structural elements rather than specific sequences could provide broader protection.

• Identifying cross-reactive epitopes: Structural comparisons have revealed that identical residues exist between SARS-CoV-2 and SARS-CoV RBDs, even within the more variable RBM regions . Antibodies like CR3022 can bind to both SARS-CoV and SARS-CoV-2 RBDs with nanomolar affinity, demonstrating the feasibility of cross-reactive antibodies . Detailed mapping of these conserved epitopes across multiple coronavirus species can guide the development of broadly neutralizing antibodies.

• Exploiting indirect neutralization mechanisms: The discovery that antibodies like MERS-4 can neutralize virus by inducing conformational changes in the RBD rather than directly competing with receptor binding represents an alternative strategy . Such indirect mechanisms might target regions under less selective pressure and therefore more conserved across coronavirus species.

• Structure-guided antibody engineering: Understanding the structural basis for antibody cross-reactivity allows for rational engineering to enhance breadth without sacrificing potency. For instance, analyzing why certain antibodies like m396 and 80R fail to cross-neutralize related coronaviruses (due to 7/21 and 16/25 residue changes in their epitopes, respectively) provides valuable information for engineering more broadly reactive variants .

• Combinatorial antibody approaches: Structural mapping of non-overlapping epitopes can inform the development of antibody cocktails targeting multiple conserved regions simultaneously, reducing the likelihood of escape mutations and increasing protection breadth across coronavirus species .

These strategies, informed by detailed structural comparisons across multiple coronavirus RBDs, offer promising approaches for developing therapeutics with potential efficacy against both known coronaviruses and future emerging strains.

What are the potential applications of RBD conformer libraries in coronavirus research and therapeutic development?

RBD conformer libraries offer numerous valuable applications in coronavirus research and therapeutic development:

• Epitope mapping and antibody discovery: Phage-display of RBD conformer libraries facilitates rapid screening against serum antibodies or monoclonal antibodies to identify specific epitopes and novel neutralizing antibodies. The MERS-CoV RBM conformer library constructed by connecting segments via combinatorial linkers provides a diverse array of conformers for such screening applications .

• Vaccine immunogen development: Different RBD conformers may present distinct epitopes, allowing for the identification of constructs that optimally display neutralizing epitopes while minimizing exposure of non-neutralizing or potentially harmful epitopes. These selected conformers can serve as rationally designed immunogens for vaccines with enhanced protective efficacy .

• Structure-function relationship studies: Systematic variation in RBD conformers through different linker combinations allows researchers to correlate structural features with functional properties such as receptor binding affinity, antibody recognition, and stability. This provides fundamental insights into the molecular determinants of coronavirus-host interactions .

• Cross-reactivity assessment: RBD conformer libraries can be screened against receptors and antibodies from different species to identify variants with broad recognition patterns, informing our understanding of zoonotic potential and cross-species transmission barriers .

• Therapeutic protein engineering: Insights gained from RBD conformer libraries can guide the engineering of optimized therapeutic proteins, including receptor decoys, antibody-mimetic proteins, and other biologics designed to block virus-receptor interactions with enhanced specificity and potency .

The functional reconstitution approach demonstrated with the MERS-CoV RBM, which generated diverse conformers through combinatorial linkers, represents a powerful methodology for creating such libraries and exploring their applications in coronavirus research .

How can comparative structural analysis of RBDs from different coronaviruses inform predictions about emerging pandemic threats?

Comparative structural analysis of RBDs from different coronaviruses provides valuable frameworks for predicting and preparing for emerging pandemic threats:

• Identifying structural adaptations for human receptor binding: By analyzing the structural differences between RBDs that efficiently bind human receptors versus those that don't, researchers can identify key structural adaptations that facilitate human infection. The distinct receptor-binding subdomains of MERS-CoV (84 residues forming a four-stranded antiparallel β sheet) and SARS-CoV (68 amino acids forming a long extended loop with two short antiparallel β strands) illustrate how different structural solutions can evolve to target human receptors .

• Recognizing convergent evolution patterns: Despite sequence diversity, the core subdomains of MERS-CoV and SARS-CoV RBDs show striking structural similarity, suggesting convergent evolution under selection pressure . Identifying such patterns can help predict which circulating animal coronaviruses might be predisposed to human adaptation.

• Mapping adaptability hotspots: Structural comparisons can reveal regions within RBDs that tolerate mutations while maintaining receptor binding capability. These adaptability hotspots represent potential sites for mutations that could facilitate species jumping or enhance transmissibility in humans .

• Monitoring for high-risk recombination scenarios: Understanding the structural compatibility of different coronavirus RBDs can help identify scenarios where recombination events might generate particularly dangerous new viruses. This is especially concerning in regions like Saudi Arabia and UAE with both MERS-CoV-infected camel populations and high SARS-CoV-2 prevalence, creating conditions for potential recombination events .

• Establishing structure-based surveillance priorities: By identifying structural features associated with pandemic potential, surveillance efforts can be prioritized toward coronavirus lineages exhibiting these high-risk characteristics, allowing for earlier detection and intervention before widespread human transmission occurs .