Computational Estimation of Residues Involving Resistance to the SARS-CoV-2 Main Protease Inhibitor Ensitrelvir Based on Virtual Alanine Scan of the Active Site.

Ensitrelvir is a noncovalent inhibitor of the main protease (Mpro) of severe acute respiratory syndrome coronavirus 2. Acquisition of drug resistance in virus-derived proteins is a serious therapeutic concern, and drug resistance occurs due to amino acid mutations. In this study, we computationally constructed 24 mutants, in which one residue around the active site was replaced with alanine and performed molecular dynamics simulations to the complex of Mpro and ensitrelvir to predict the residues involved in drug resistance. We evaluated the changes in the entire protein structure and ligand configuration in each of these mutants and estimated which residues were involved in ensitrelvir recognition. This method is called a virtual alanine scan. In nine mutants (S1A, T26A, H41A, M49A, L141A, H163A, E166A, V186A, and R188A), although the entire protein structure and catalytic dyad (cysteine (Cys)145 and histidine (His)41) were not significantly moved, the ensitrelvir configuration changed. Thus, it is considered that these mutants did not recognize ensitrelvir while maintaining Mpro enzymatic activities, and Ser1, Thr26, His41, Met49, Leu141, His163, Glu166, Val186, and Arg188 may be related to ensitrelvir resistance. The ligand shift noted in M49A was similar to that observed in M49I, which has been shown to be experimentally ensitrelvir resistant. These findings suggest that our research approach can predict mutations that incite drug resistance.

The Role of Heparan Sulfate in CCL26-Induced Eosinophil Chemotaxis.

Proinflammatory chemokine ligand 26 (CCL26, eotaxin-3) mediates transendothelial cell migration of eosinophils by binding and activating the G-protein-coupled (GPC) chemokine receptor 3 on the surface of eosinophilic cells. Here we have investigated the role of glycosaminoglycans (GAGs) as potential co-receptors in the process of CCL26-induced eosinophil chemotaxis. For this purpose, we have first identified the GAG-binding site of CCL26 by a site-directed mutagenesis approach in the form of an alanine screening. A panel of GAG-binding-deficient mutants has been designed, generated, and analyzed with respect to their binding affinities to heparan sulphate (HS) by isothermal fluorescence titration studies. This showed that basic amino acids in the α-helical part of CCL26 are strongly involved in GAG-binding. In chemotaxis experiments, we found that decreased GAG-binding affinity correlated with decreased chemotactic activity, which indicates an involvement of GAGs in eosinophil migration. This was further proven by the negative impact of heparinase III treatment and, independently, by the incubation of eosinophils with an anti heparan sulfate antibody. We finally investigated eosinophils' proteoglycan (PG) expression patterns by real-time PCR, which revealed the highest expression level for serglycin. Including an anti-serglycin antibody in CCL26-induced eosinophil migration experiments reduced the chemotaxis of these immune cells, thereby proving the dependence of eosinophil mobilization on the proteoglycan serglycin.

High-resolution crystal structures of two prototypical ß- and γ-herpesviral nuclear egress complexes unravel the determinants of subfamily specificity.

Herpesviruses uniquely express two essential nuclear egress-regulating proteins forming a heterodimeric basic structure of the nuclear egress complex (core NEC). These core NECs serve as a hexameric lattice-structured platform for capsid docking and recruit viral and cellular NEC-associated factors that jointly exert nuclear lamina- and membrane-rearranging functions (multicomponent NEC). Here, we report the X-ray structures of ß- and γ-herpesvirus core NECs obtained through an innovative recombinant expression strategy based on NEC-hook::NEC-groove protein fusion constructs. This approach yielded the first structure of γ-herpesviral core NEC, namely the 1.56 Å structure of Epstein-Barr virus (EBV) BFRF1-BFLF2, as well as an increased resolution 1.48 Å structure of human cytomegalovirus (HCMV) pUL50-pUL53. Detailed analysis of these structures revealed that the prominent hook segment is absolutely required for core NEC formation and contributes approximately 80% of the interaction surface of the globular domains of NEC proteins. Moreover, using HCMV::EBV hook domain swap constructs, computational prediction of the roles of individual hook residues for binding, and quantitative binding assays with synthetic peptides presenting the HCMV- and EBV-specific NEC hook sequences, we characterized the unique hook-into-groove NEC interaction at various levels. Although the overall physicochemical characteristics of the protein interfaces differ considerably in these ß- and γ-herpesvirus NECs, the binding free energy contributions of residues displayed from identical positions are similar. In summary, the results of our study reveal critical details of the molecular mechanism of herpesviral NEC interactions and highlight their potential as an antiviral drug target.

Virtual Alanine Scan of the Main Protease Active Site in Severe Acute Respiratory Syndrome Coronavirus 2.

Recently, inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) have been proposed as potential therapeutic agents for COVID-19. Studying effects of amino acid mutations in the conformation of drug targets is necessary for anticipating drug resistance. In this study, with the structure of the SARS-CoV-2 Mpro complexed with a non-covalent inhibitor, we performed molecular dynamics (MD) simulations to determine the conformation of the complex when single amino acid residue in the active site is mutated. As a model of amino acid mutation, we constructed mutant proteins with one residue in the active site mutated to alanine. This method is called virtual alanine scan. The results of the MD simulations showed that the conformation and configuration of the ligand was changed for mutants H163A and E166A, although the structure of the whole protein and of the catalytic dyad did not change significantly, suggesting that mutations in His163 and Glu166 may be linked to drug resistance.

Total synthesis and a systematic structure-activity relationship study of WAP-8294A2.

WAP-8294A2 is a cyclic peptide antibiotic with novel structure and excellent activity against Gram-positive pathogens. Herein, we report the total synthesis of complex macrocyclic peptide WAP-8294A2 (W1), ent-analogue W2, deoxy analogue W3 and de-methyl analogue W4 using a solid-phase synthetic route followed by a final stage solution-phase cyclization reaction. Exploitation of this process allowed the synthesis of eleven alanine-scanning analogues and eight lysine-scanning analogues. The antimicrobial activity of these analogues was evaluated in vitro against Gram-positive bacteria. Based on the MIC results, a primary systematic structure-activity relationship has been established.

Structural implications of hERG K+ channel block by a high-affinity minimally structured blocker.

Cardiac potassium channels encoded by human ether-à-go-go-related gene (hERG) are major targets for structurally diverse drugs associated with acquired long QT syndrome. This study characterized hERG channel inhibition by a minimally structured high-affinity hERG inhibitor, Cavalli-2, composed of three phenyl groups linked by polymethylene spacers around a central amino group, chosen to probe the spatial arrangement of side chain groups in the high-affinity drug-binding site of the hERG pore. hERG current (IhERG) recorded at physiological temperature from HEK293 cells was inhibited with an IC50 of 35.6 nm with time and voltage dependence characteristic of blockade contingent upon channel gating. Potency of Cavalli-2 action was markedly reduced for attenuated inactivation mutants located near (S620T; 54-fold) and remote from (N588K; 15-fold) the channel pore. The S6 Y652A and F656A mutations decreased inhibitory potency 17- and 75-fold, respectively, whereas T623A and S624A at the base of the selectivity filter also decreased potency (16- and 7-fold, respectively). The S5 helix F557L mutation decreased potency 10-fold, and both F557L and Y652A mutations eliminated voltage dependence of inhibition. Computational docking using the recent cryo-EM structure of an open channel hERG construct could only partially recapitulate experimental data, and the high dependence of Cavalli-2 block on Phe-656 is not readily explainable in that structure. A small clockwise rotation of the inner (S6) helix of the hERG pore from its configuration in the cryo-EM structure may be required to optimize Phe-656 side chain orientations compatible with high-affinity block.

Binding conformation and determinants of a single-chain peptide antagonist at the relaxin-3 receptor RXFP3.

The neuropeptide relaxin-3 and its receptor relaxin family peptide receptor-3 (RXFP3) play key roles in modulating behavior such as memory and learning, food intake, and reward seeking. A linear relaxin-3 antagonist (R3 B1-22R) based on a modified and truncated relaxin-3 B-chain was recently developed. R3 B1-22R is unstructured in solution; thus, the binding conformation and determinants of receptor binding are unclear. Here, we have designed, chemically synthesized, and pharmacologically characterized more than 60 analogues of R3 B1-22R to develop an extensive understanding of its structure-activity relationships. We show that the key driver for affinity is the nonnative C-terminal Arg23 Additional contributors to binding include amino acid residues that are important also for relaxin-3 binding, including Arg12, Ile15, and Ile19 Intriguingly, amino acid residues that are not exposed in native relaxin-3, including Phe14 and Ala17, also interact with RXFP3. We show that R3 B1-22R has a propensity to form a helical structure, and modifications that support a helical conformation are functionally well-tolerated, whereas helix breakers such as proline residues disrupt binding. These data suggest that the peptide adopts a helical conformation, like relaxin-3, upon binding to RXFP3, but that its smaller size allows it to penetrate deeper into the orthosteric binding site, creating more extensive contacts with the receptor.

Identification of Antimicrobial Peptides from the Microalgae Tetraselmis suecica (Kylin) Butcher and Bactericidal Activity Improvement.

The outburst of microbial resistance to antibiotics creates the need for new sources of active compounds for the treatment of pathogenic microorganisms. Marine microalgae are of particular interest in this context because they have developed tolerance and defense strategies to resist the exposure to pathogenic bacteria, viruses, and fungi in the aquatic environment. Although antimicrobial activities have been reported for some microalgae, natural algal bioactive peptides have not been described yet. In this work, acid extracts from the microalga Tetraselmis suecica with antibacterial activity were analyzed, and de novo sequences of peptides were determined. Synthetic peptides and their alanine and lysine analogs allowed identifying key residues and increasing their antibacterial activity. Additionally, it was determined that the localization of positive charges within the peptide sequence influences the secondary structure with tendency to form an alpha helical structure.

Rigorous Computational and Experimental Investigations on MDM2/MDMX-Targeted Linear and Macrocyclic Peptides.

There is interest in peptide drug design, especially for targeting intracellular protein-protein interactions. Therefore, the experimental validation of a computational platform for enabling peptide drug design is of interest. Here, we describe our peptide drug design platform (CMDInventus) and demonstrate its use in modeling and predicting the structural and binding aspects of diverse peptides that interact with oncology targets MDM2/MDMX in comparison to both retrospective (pre-prediction) and prospective (post-prediction) data. In the retrospective study, CMDInventus modules (CMDpeptide, CMDboltzmann, CMDescore and CMDyscore) were used to accurately reproduce structural and binding data across multiple MDM2/MDMX data sets. In the prospective study, CMDescore, CMDyscore and CMDboltzmann were used to accurately predict binding affinities for an Ala-scan of the stapled α-helical peptide ATSP-7041. Remarkably, CMDboltzmann was used to accurately predict the results of a novel D-amino acid scan of ATSP-7041. Our investigations rigorously validate CMDInventus and support its utility for enabling peptide drug design.

An entirely fmoc solid phase approach to the synthesis of daptomycin analogs.

Daptomycin is an important Ca2+ -dependent cyclic lipodepsipeptide antibiotic used to treat serious gram-positive infections. The search for daptomycin analogs with improved activity and their application as tools for studying its mechanism of action has prompted us to develop an entirely Fmoc solid phase approach to the synthesis of daptomycin analogs. Key to the success of this approach was the development of conditions that allowed for the formation of the ester bond on resin-bound peptides consisting of residues 1-10 and the decanoyl lipid tail. The esterification reaction proceeded more efficiently on Tentagel resin as opposed to standard polystyrene resin. This approach was used to synthesize a series of analogs in which each position of Dap-E12-W13, a relatively active daptomycin analog, was individually substituted by alanine. Only positions 2, 6, and 11 were found to be amenable to substitution by alanine in that the corresponding alanine analogs were only 1.5- to 4-fold less active than Dap-E12-W13. We also found that the daptomycin analog, Dap-K6-E12-W13, exhibits in vitro activity approaching that of daptomycin at physiological Ca2+ concentration. Studies with Dap-K6-E12-W13 and model liposomes indicate that this analog interacts with membranes by the same mechanism as daptomycin. This analog is currently being used as a lead for the development daptomycin analogs with improved activity.

Alanine scan and (2)H NMR analysis of the membrane-active peptide BP100 point to a distinct carpet mechanism of action.

The short membrane-active peptide BP100 [KKLFKKILKYL-NH2] is known as an effective antimicrobial and cell penetrating agent. For a functional alanine scan each of the 11 amino acids was replaced with deuterated Ala-d3, one at a time. MIC assays showed that a substitution of Lys did not affect the antimicrobial activity, but it decreased when a hydrophobic residue was replaced. In most cases, a reduction in hydrophobicity led to a decrease in hemolysis, and some peptide analogues had an improved therapeutic index. Circular dichroism showed that BP100 folds as an amphiphilic α-helix in a bilayer. Its alignment was determined from (2)H NMR in oriented membranes of different composition. The azimuthal rotation angle was the same under all conditions, but the average helix tilt angle and the dynamical behavior of the peptide varied in a systematic manner. In POPC/POPG bilayers, with a negative spontaneous curvature, the peptide was found to lie flat on the bilayer surface, and with little wobble. In DMPC/DMPG, with a positive spontaneous curvature, BP100 at higher concentrations became tilted obliquely into the membrane, with the uncharged C-terminus inserted more deeply into the lipid bilayer, experiencing significant fluctuations in tilt angle. In DMPC/DMPG/lyso-MPC, with a pronounced positive spontaneous curvature, the helix tilted even further and became even more mobile. The 11-mer BP100 is obviously too short to form transmembrane pores. We conclude that BP100 operates via a carpet mechanism, whereby the C-terminus gets inserted into the hydrophobic core of the bilayer, which leads to membrane perturbation and induces transient permeability.

Alanine scan of the opioid peptide dynorphin B amide.

To date structure-activity relationship (SAR) studies of the dynorphins (Dyn), endogenous peptides for kappa opioid receptors (KOR), have focused almost exclusively on Dyn A with minimal studies on Dyn B. While both Dyn A and Dyn B have identical N-terminal sequences, their C-terminal sequences differ, which could result in differences in pharmacological activity. We performed an alanine scan of the non-glycine residues up through residue 11 of Dyn B amide to explore the roles of these side chains in the activity of Dyn B. The analogs were synthesized by fluorenylmethyloxycarbonyl (Fmoc)-based solid phase peptide synthesis and evaluated for their opioid receptor affinities and opioid potency and efficacy at KOR. Similar to Dyn A the N-terminal Tyr1 and Phe4 residues of Dyn B amide are critical for opioid receptor affinity and KOR agonist potency. The basic residues Arg6 and Arg7 contribute to the KOR affinity and agonist potency of Dyn B amide, while Lys10 contributes to the opioid receptor affinity, but not KOR agonist potency, of this peptide. Comparison to the Ala analogs of Dyn A (1-13) suggests that the basic residues in the C-terminus of both peptides contribute to KOR binding, but differences in their relative positions may contribute to the different pharmacological profiles of Dyn A and Dyn B. The other unique C-terminal residues in Dyn B amide also appear to influence the relative affinity of this peptide for KOR versus mu and delta opioid receptors. This SAR information may be applied in the design of new Dyn B analogs that could be useful pharmacological tools.

Naturally Inspired Peptide Leads: Alanine Scanning Reveals an Actin-Targeting Thiazole Analogue of Bisebromoamide.

Systematic alanine scanning of the linear peptide bisebromoamide (BBA), isolated from a marine cyanobacterium, was enabled by solid-phase peptide synthesis of thiazole analogues. The analogues have comparable cytotoxicity (nanomolar) to that of BBA, and cellular morphology assays indicated that they target the actin cytoskeleton. Pathway inhibition in human colon tumour (HCT116) cells was explored by reverse phase protein array (RPPA) analysis, which showed a dose-dependent response in IRS-1 expression. Alanine scanning reveals a structural dependence to the cytotoxicity, actin targeting and pathway inhibition, and allows a new readily synthesised lead to be proposed.

Efficient calculation of relative binding free energies by umbrella sampling perturbation.

An important task of biomolecular simulation is the calculation of relative binding free energies upon chemical modification of partner molecules in a biomolecular complex. The potential of mean force (PMF) along a reaction coordinate for association or dissociation of the complex can be used to estimate binding affinities. A free energy perturbation approach, termed umbrella sampling (US) perturbation, has been designed that allows an efficient calculation of the change of the PMF upon modification of a binding partner based on the trajectories obtained for the wild type reference complex. The approach was tested on the interaction of modified water molecules in aqueous solution and applied to in silico alanine scanning of a peptide-protein complex. For the water interaction test case, excellent agreement with an explicit PMF calculation for each modification was obtained as long as no long range electrostatic perturbations were considered. For the alanine scanning, the experimentally determined ranking and binding affinity changes upon alanine substitutions could be reproduced within 0.1-2.0 kcal/mol. In addition, good agreement with explicitly calculated PMFs was obtained mostly within the sampling uncertainty. The combined US and perturbation approach yields, under the condition of sufficiently small system modifications, rigorously derived changes in free energy and is applicable to any PMF calculation.

Key residues in octyl-tridecaptin A1 analogues linked to stable secondary structures in the membrane.

Tridecaptin A1 is a linear antimicrobial lipopeptide comprised of 13 amino acids, including three diaminobutyric acid (Dab) residues. It displays potent activity against Gram-negative bacteria, including multidrug-resistant strains. Using solid-phase peptide synthesis, we performed an alanine scan of a fully active analogue, octyl-tridecaptin A1 , to determine key residues responsible for activity. The synthetic analogues were tested against ten organisms, both Gram-positive and Gram-negative bacteria. Modification of D-Dab8 abolished activity, and marked decreases were observed with substitution of D-allo-Ile12 and D-Trp5. Circular dichroism showed that octyl-tridecaptin A1 adopts a secondary structure in the presence of model phospholipid membranes, which was weakened by D-Dab8-D-Ala, D-allo-Ile12-D-Ala, and D-Trp5-D-Ala substitutions. The antimicrobial activity of the analogues is directly correlated to their ability to adopt a stable secondary structure in a membrane environment.

Dissecting a role of a charge and conformation of Tat2 peptide in allosteric regulation of 20S proteasome.

Proteasome is a 'proteolytic factory' that constitutes an essential part of the ubiquitin-proteasome pathway. The involvement of proteasome in regulation of all major aspects of cellular physiology makes it an attractive drug target. So far, only inhibitors of the proteasome entered the clinic as anti-cancer drugs. However, proteasome regulators may also be useful for treatment of inflammatory and neurodegenerative diseases. We established in our previous studies that the peptide Tat2, comprising the basic domain of HIV-1 Tat protein: R(49) KKRRQRR(56) , supplemented with Q(66) DPI(69) fragment, inhibits the 20S proteasome in a noncompetitive manner. Mechanism of Tat2 likely involves allosteric regulation because it competes with the proteasome natural 11S activator for binding to the enzyme noncatalytic subunits. In this study, we performed alanine walking coupled with biological activity measurements and FTIR and CD spectroscopy to dissect contribution of a charge and conformation of Tat2 to its capability to influence peptidase activity of the proteasome. In solution, Tat2 and most of its analogs with a single Ala substitution preferentially adopted a conformation containing PPII/turn structural motifs. Replacing either Asp10 or two or more adjacent Arg/Lys residues induced a random coil conformation, probably by disrupting ionic interactions responsible for stabilization of the peptides ordered structure. The random coil Tat2 analogs lost their capability to activate the latent 20S proteasome. In contrast, inhibitory properties of the peptides more significantly depended on their positive charge. The data provide valuable clues for the future optimization of the Tat2-based proteasome regulators.

Improving enzyme functional annotation by integrating in vitro and in silico approaches: The example of histidinol phosphate phosphatases.

Advances in sequencing technologies have led to a rapid growth of public protein sequence databases, whereby the fraction of proteins with experimentally verified function continuously decreases. This problem is currently addressed by automated functional annotations with computational tools, which however lack the accuracy of experimental approaches and are susceptible to error propagation. Here, we present an approach that combines the efficiency of functional annotation by in silico methods with the rigor of enzyme characterization in vitro. First, a thorough experimental analysis of a representative enzyme of a group of homologues is performed which includes a focused alanine scan of the active site to determine a fingerprint of function-determining residues. In a second step, this fingerprint is used in combination with a sequence similarity network to identify putative isofunctional enzymes among the homologues. Using this approach in a proof-of-principle study, homologues of the histidinol phosphate phosphatase (HolPase) from Pseudomonas aeruginosa, many of which were annotated as phosphoserine phosphatases, were predicted to be HolPases. This functional annotation of the homologues was verified by in vitro testing of several representatives and an analysis of the occurrence of annotated HolPases in the corresponding phylogenetic groups. Moreover, the application of the same approach to the homologues of the HolPase from the archaeon Nitrosopumilus maritimus, which is not related to the HolPase from P. aeruginosa and was newly discovered in the course of this work, led to the annotation of the putative HolPase from various archaeal species.

Through virtual saturation mutagenesis and rational design for superior substrate conversion in engineered d-amino acid oxidase.

The d-amino acid oxidase (DAAO) is pivotal in obtaining optically pure l-glufosinate (l-PPT) by converting d-glufosinate (d-PPT) to its deamination product. We screened and designed a Rasamsonia emersonii DAAO (ReDAAO), making it more suitable for oxidizing d-PPT. Using Caver 3.0, we delineated three substrate binding pockets and, via alanine scanning, identified nearby key residues. Pinpointing key residues influencing activity, we applied virtual saturation mutagenesis (VSM), and experimentally validated mutants which reduced substrate binding energy. Analysis of positive mutants revealed elongated side-chain prevalence in substrate binding pocket periphery. Although computer-aided approaches can rapidly identify advantageous mutants and guide further design, the mutations obtained in the first round may not be suitable for combination with other advantageous mutations. Therefore, each round of combination requires reasonable iteration. Employing VSM-assisted screening multiple times and after four rounds of combining mutations, we ultimately obtained a mutant, N53V/F57Q/V94R/V242R, resulting in a mutant with a 5097% increase in enzyme activity compared to the wild type. It provides valuable insights into the structural determinants of enzyme activity and introduces a novel rational design procedure.

Epitope mapping of monoclonal antibodies: a comprehensive comparison of different technologies.

Monoclonal antibodies have become an important class of therapeutics in the last 30 years. Because the mechanism of action of therapeutic antibodies is intimately linked to their binding epitopes, identification of the epitope of an antibody to the antigen plays a central role during antibody drug development. The gold standard of epitope mapping, X-ray crystallography, requires a high degree of proficiency with no guarantee of success. Here, we evaluated six widely used alternative methods for epitope identification (peptide array, alanine scan, domain exchange, hydrogen-deuterium exchange, chemical cross-linking, and hydroxyl radical footprinting) in five antibody-antigen combinations (pembrolizumab+PD1, nivolumab+PD1, ipilimumab+CTLA4, tremelimumab+CTLA4, and MK-5890+CD27). The advantages and disadvantages of each technique are demonstrated by our data and practical advice on when and how to apply specific epitope mapping techniques during the drug development process is provided. Our results suggest chemical cross-linking most accurately identifies the epitope as defined by crystallography.

Determination of key residues in tospoviral NSm required for Sw-5b recognition, their potential ability to overcome resistance, and the effective resistance provided by improved Sw-5b mutants.

Sw-5b is an effective resistance gene used widely in tomato to control tomato spotted wilt virus (TSWV), which causes severe losses in crops worldwide. Sw-5b confers resistance by recognizing a 21-amino-acid peptide region of the viral movement protein NSm (NSm21, amino acids 115-135). However, C118Y or T120N mutation within this peptide region of NSm has given rise to field resistance-breaking (RB) TSWV isolates. To investigate the potential ability of TSWV to break Sw-5b-mediated resistance, we mutagenized each amino acid on NSm21 and determined which amino acid mutations would evade Sw-5b recognition. Among all alanine-scan mutants, NSmP119A , NSmW121A , NSmD122A , NSmR124A , and NSmQ126A failed to induce a hypersensitive response (HR) when coexpressed with Sw-5b in Nicotiana benthamiana leaves. TSWV with the NSmP119A , NSmW121A , or NSmQ126A mutation was defective in viral cell-to-cell movement and systemic infection, while TSWV carrying the NSmD122A or NSmR124A mutation was not only able to infect wild-type N. benthamiana plants systemically but also able to break Sw-5b-mediated resistance and establish systemic infection on Sw-5b-transgenic N. benthamiana plants. Two improved mutants, Sw-5bL33P/K319E/R927A and Sw-5bL33P/K319E/R927Q , which we recently engineered and which provide effective resistance against field RB isolates carrying NSmC118Y or NSmT120N mutations, recognized all NSm21 alanine-substitution mutants and conferred effective resistance against new experimental RB TSWV with the NSmD122A or NSmR124A mutation. Collectively, we determined the key residues of NSm for Sw-5b recognition, investigated their potential RB ability, and demonstrated that the improved Sw-5b mutants could provide effective resistance to both field and potential RB TSWV isolates.