Biology subjects

Varadarajan, R.

Publications and source records attributed to Varadarajan, R..

At least 19 recordsLinked to original sources

bioRxiv2025

Charged Scanning Mutagenesis as a High Throughput Approach for Epitope Mapping

Identifying neutralizing epitopes is important for developing vaccines and inhibitors against viral pathogens. We describe a rapid method for epitope mapping, employing barcoded charged scanning mutagenesis libraries displayed on the yeast surface, and screening using flow cytometry coupled with deep sequencing. Prior scanning mutagenesis data suggest that mutations to a charged residue, such as Aspartic acid or Arginine, will be well tolerated at exposed positions of an antigen, and minimally affect protein stability and expression. Yet such substitutions at epitope residues strongly perturb binding to a cognate partner. We constructed an Aspartate scanning library of SARS-CoV-2 RBD and linked every mutation in the library to a defined unique barcode. The approach was used to map epitopes targeted in polyclonal sera of mice immunized with different SARS-CoV-2 immunogens. In contrast to complete mutational scans, charged scanning mutagenesis with the introduced barcoding strategy employs libraries with >50-fold lower diversity, facilitating library construction, screening, and downstream analysis, and also allowing for further multiplexing of samples, thus accelerating interaction site identification, as well as vaccine and inhibitor development.

biophysics↗

bioRxiv2025

Development of a thermostable and broadly neutralizing pan-sarbecovirus vaccine candidate

Zoonotic spillover of sarbecoviruses to humans resulted in theSARS-CoV-1 outbreak in 2003 and the current COVID-19 pandemic caused by SARS-CoV-2. In both cases, the viral spike protein (S) is the principal target of neutralising antibodies that prevent infection. Within spike, the immunodominant receptor-binding domain (RBD) is the primary target of neutralising antibodies in COVID-19 convalescent sera and vaccine recipients. We have constructed stabilized RBD derivatives of different sarbecoviruses: SARS-CoV-1 (Clade 1a), WIV-1 (Clade 1a), RaTG13 (Clade 1b), RmYN02 (Clade 2) and BtKY72 (Clade 3). Stabilization enhanced yield by an 3-23-fold. The RBD derivatives were conformationally intact as assayed by binding to multiple broadly neutralizing antibodies. The stabilized RBDs show significant enhancement in apparent Tm, exhibit resistance to a 2-hour incubation at temperatures up to 60 in PBS in contrast to corresponding WT RBDs, and show prolonged stability of over 15 days at 37 after lyophilization. In mice immunizations, both stabilization and trimerization significantly enhanced elicited neutralization titers by [~]100 fold. The stabilized RBD cocktail elicited high neutralizing titers against both homologous and heterologous pseudoviruses. The immunogenicity of the vaccine formulation was assessed in both naive and SARS-CoV-2 pre-immunized mice, revealing an absence of immune imprinting, thus indicating its suitability for use in future sarbecovirus-origin epidemics or pandemics. Author summaryThe COVID-19 pandemic was caused by the sarbecovirus SARS-CoV-2. Phylogenetically, sarbecoviruses are divided into four clades: Clade 1a, Clade 1b, Clade 2 and Clade 3 and within these clades, are many other sarbecovirus strains with pandemic or epidemic potential. It is therefore important to develop a broadly protective, pan-sarbecovirus vaccine formulation that can be cheaply and rapidly produced. While mRNA vaccine formulations are efficacious, they have stringent low temperature storage requirements and there is limited manufacturing expertise in low and middle income countries for this modality. Neutralizing antibodies are important for protection and in the case of SARS-CoV-2 are primarily directed against the Receptor Binding Domain (RBD) of the surface spike protein. In this study, we have designed and developed an adjuvanted, protein subunit, pan sarbecovirus vaccine formulation using stabilized RBD derivatives from diverse sarbecoviruses as immunogens. We demonstrate that the stabilized RBD derivatives have considerably enhanced yield and thermal stability relative to corresponding WT proteins and that the formulation remains stable for up to two weeks at 37{degrees}C. The formulation was found to be highly immunogenic in both naive and pre-immunized mice, eliciting neutralizing titers well above the known protective threshold, indicating its suitability for use in future sarbecovirus-origin epidemics or pandemics.

immunology↗

bioRxiv2024

Ex Vivo Delivery of mRNA to Immune Cells Via a Non-Endosomal Route Obviates the Need for Nucleoside Modification

Base modification and the use of lipid nanoparticles (LNPs) are thought to be essential for efficient in vivo delivery and expression of mRNA. However, for ex vivo immune cell engineering, the need for either of the two is unclear. Previous reports have suggested that nucleic acids may be efficiently delivered to immune cells ex vivo, through a non-endosomal delivery route, but the need for base modification has not been determined. Herein, we demonstrate that when a non-endosomal delivery method is used, unmodified mRNA performs equally well to the commonly used base-modified mRNA, including the N1 methyl pseudo-uridine modification, in terms of protein expression and inflammatory response in cells. However, if an endosomal delivery route is used, then N1 methyl pseudo-uridine modification is necessary for high expression and low inflammatory response, as demonstrated by others as well. Overall, we show that non-endosomal mRNA delivery renders nucleoside modifications non-essential, and that unmodified mRNA combined with non-endosomal delivery route may be used for efficient ex vivo mRNA-based engineering of immune cells.

bioengineering↗

bioRxiv2024

CcdA chaperones CcdB against irreversible misfolding and aggregation via a cotranslational folding mechanism

Cotranslational subunit assembly is thought to be a prominent feature throughout the proteome, but in bacteria, there are only a limited number of experimentally confirmed examples and most involve addition of extraneous tag sequences for experimental convenience. CcdA and CcdB are the antitoxin and toxin components, respectively, of the ccdAB operon. They assemble in a hetero-multimeric complex in vivo. Mutant phenotypes in a saturation mutagenesis library of CcdB were inferred from deep sequencing in two contexts, one when CcdB is expressed alone, and the other when CcdB is present in an operonic context downstream of its cognate antitoxin, CcdA. When expressed in the absence of CcdA, charged and polar mutations in the CcdB core cause the protein to misfold. However, many such deleterious mutations are rescued when expressed along with CcdA in the native operon. CcdA thus acts as a chaperone and relieves the folding defect in CcdBcotranslationally. Assembly efficiency and efficacy decreases when CcdA and CcdB are expressed from separate mRNAs relative to when they are expressed from the same polycistronic mRNA. Gene organisation in operons in bacteria may thus reflect a fundamental cotranslational mechanism that is important for effective assembly of protein complexes and can potentially buffer substantial genetic variation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/618527v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@26e1ceorg.highwire.dtl.DTLVardef@b2a4b1org.highwire.dtl.DTLVardef@5a7c85org.highwire.dtl.DTLVardef@a50512_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefUsing the CcdAB Type II Toxin-Antitoxin system, the authors show that the antitoxin is able to suppress folding defects of the toxin, likely by processes involving cotranslational assembly. HighlightsO_LIDeep mutational scanning is used to assess and compare the foldability of buried-site CcdB mutants in the absence and presence of CcdA. C_LIO_LIFolding defects of CcdB mutants are rescued in the presence of CcdA when expressed in an operonic context. C_LIO_LICcdAB complex formation is enhanced when both the toxin and antitoxin components are synthesized from the same polycistronic mRNA, relative to synthesis from separate mRNAs. C_LIO_LICcdA acts as chaperone both to assist correct folding, and to buffer mutations in CcdB, likely via cotranslational assembly mechanisms. C_LI

biophysics↗

bioRxiv2024

Early 2022 breakthrough infection sera from India target the conserved cryptic class 5 epitope to counteract immune escape by SARS-CoV-2 variants

During the COVID-19 pandemic, the vast majority of epitope mapping studies have focused on sera from mRNA-vaccinated populations from high-income countries. In contrast, here we report an analysis of 164 serum samples isolated from breakthrough infection patients in India during early 2022 who received two doses of the ChAdOx viral vector vaccine. Sera were screened for neutralization breadth against wildtype, Kappa, Delta, and Omicron BA.1 viruses. Three sera with the highest neutralization breadth and potency were selected for epitope mapping using charged scanning mutagenesis coupled to yeast surface display and Next Generation Sequencing. All of these sera primarily targeted the recently identified class 5 cryptic epitope along with the class 1 and class 4 epitopes, targeted to a lesser extent. The class 5 epitope is completely conserved across all SARS-CoV-2 variants and for the majority of the Sarbecoviruses. In line with these observations, a major fraction of the serum samples, including the selected three, show broad neutralizing activity against recent Omicron subvariants including XBB.1.5. This is in contrast with the results obtained with the sera from individuals receiving multiple doses of original and updated mRNA vaccines, where impaired neutralization of XBB and later variant of concerns was observed. Our study demonstrates that two doses of the ChAdOx vaccine in a highly exposed population was sufficient to drive substantial neutralization breadth against emerging and upcoming variants of concern, thus paves the path towards the development of future vaccine candidates. ImportanceWorldwide implementation of COVID19 vaccine and parallel emergence of newer SARS-CoV-2 variants has shaped the humoral immune response in a population specific manner. While characterization of this immune response is important for monitoring the disease progression at population level, it is also imperative for the development of effective countermeasures in the form of novel vaccines and therapeutics. India has implemented the worlds second largest COVID19 vaccination drive and also encountered large number of post vaccination "breakthrough" infections. From a cohort of breakthrough infection patients, we identified individuals whose sera showed broadly cross-reactive immunity against different SARS-CoV-2 variants. Interestingly, these sera primarily target a novel cryptic epitope which was not identified in previous population level studies conducted in western countries. This rare cryptic epitope remains conserved across all SARS-CoV-2 variants including the recently emerged ones and also for the SARS-like coronaviruses that may cause future outbreaks, thus representing a potential target for future vaccines.

molecular biology↗

bioRxiv2024

Improved prediction of stabilizing mutations in proteins by incorporation of mutational effects on ligand binding

While many computational methods accurately predict destabilizing mutations, identifying stabilizing mutations has remained a challenge, due to their relative rarity. We tested {Delta}{Delta}G0 predictions from computational predictors such as Rosetta, ThermoMPNN, RaSP, and DeepDDG, using eighty-two mutants of the bacterial toxin CcdB as a test case. On this dataset, the best computational predictor is ThermoMPNN which identifies stabilizing mutations with a precision of 68%. However, the average increase in Tm for these predicted mutations was only 1{degrees}C for CcdB, and predictions were poorer for a more challenging target, influenza neuraminidase. Using data from multiple previously described yeast surface display libraries and in vitro thermal stability measurements, we trained logistic regression models to identify stabilizing mutations with a precision of 90% and an average increase in Tm of 3{degrees}C for CcdB. When such libraries contain a population of mutants with significantly enhanced binding relative to the corresponding wild type, there is no benefit in using computational predictors. It is then possible to predict stabilizing mutations without any training, simply by examining the distribution of mutational binding scores. This avoids laborious steps of in vitro expression, purification, and stability characterization. When this is not the case, combining data from computational predictors with high-throughput experimental binding data enhances the prediction of stabilizing mutations. However, this requires training on stability data measured in vitro with known stabilized mutants. It is thus feasible to predict stabilizing mutations rapidly and accurately for any system of interest that can be subjected to a binding selection or screen.

biophysics↗

bioRxiv2023

Enhanced protective efficacy of a novel, thermostable, RBD-S2 fusion immunogen against SARS-CoV-2 and its variants

With the rapid emergence of variants of concern (VOC), the efficacy of currently licensed vaccines has reduced drastically. VOC mutations largely occur in the S1 subunit of Spike. The S2 subunit of SARS-CoV-2 is conserved and thus more likely to elicit broadly protective immune responses. However, the contribution of the S2 subunit in improving the overall efficacy of vaccines remains unclear. Therefore, we designed, characterized, and evaluated the immunogenicity and protective potential of a stabilized SARS-CoV-2 Receptor Binding Domain (RBD) fused to a stabilized S2. Designed immunogens were expressed as soluble proteins with approximately fivefold higher purified yield than the Spike ectodomain and formulated along with Squalene-in-water emulsion (SWE) adjuvant. S2 immunization failed to elicit a neutralizing immune response but significantly reduced lung viral titers in mice challenged with the heterologous Beta variant. In hamsters, SWE-formulated RS2 showed enhanced immunogenicity and efficacy relative to corresponding RBD and Spike formulations. Despite being based on the ancestral Wuhan strain of SARS-CoV-2, RS2 exhibited broad neutralization, including against Omicron variants (BA.1, BA.5 and BF.7), as well as the clade 1a WIV-1 and SARS-CoV-1 strains. RS2 sera also showed enhanced competition with both S2 directed and RBD Class 4 directed broadly neutralizing antibodies, relative to RBD and Spike elicited sera. When lyophilized, RS2 retained antigenicity and immunogenicity even after incubation at 37 {degrees}C for a month. The data collectively suggest that the RS2 immunogen is a promising modality to combat SARS-CoV-2 variants.

biophysics↗

bioRxiv2023

Molecular bases for strong phenotypic effects of single-site synonymous substitutions in the E.coli ccdB toxin gene

Single-site synonymous mutations typically have only minor or no effects on gene function. Here, we estimate the effects on cell growth of ~200 single-site synonymous mutations in an operonic context by mutating each position of ccdB, the 101-residue long cytotoxin of the ccdAB Toxin-Antitoxin (TA) operon to all possible degenerate codons. Phenotypes were assayed by transforming the mutant library into CcdB sensitive and resistant E. coli strains, isolating plasmid pools, and subjecting them to deep sequencing. Since autoregulation is a hallmark of TA operons, phenotypes obtained for ccdB synonymous mutants after transformation in a RelE toxin reporter strain followed by deep sequencing provided information on the amount of CcdAB complex formed. Synonymous mutations in the N-terminal region involved in translation initiation showed the strongest non-neutral phenotypic effects. We observe an interplay of numerous factors, namely, location of the codon, codon usage, t-RNA abundance, formation of anti-Shine Dalgarno sequences, transcript secondary structure, and evolutionary conservation in determining phenotypic effects of ccdB synonymous mutations. Incorporation of an N-terminal, hyperactive synonymous mutation, in the background of the single-site synonymous mutant library sufficiently increased translation initiation, such that mutational effects on either folding or termination of translation became more apparent. Introduction of putative pause sites not only affects the translational rate but might also alter the folding kinetics of the protein in vivo. This information is useful in optimizing heterologous gene expression in E. coli and understanding the molecular bases for alteration in gene expression that arise due to synonymous mutations. SIGNIFICANCE STATEMENTSynonymous mutations which do not change amino-acid identity, typically have only minor or no effects on gene function. Using sensitive genetic screens in the context of the ccdAB bacterial toxin-antitoxin operon, we demonstrate that many single-site synonymous mutations of the ccdB toxin gene display significant phenotypic effects in an operonic context. The largest effects were seen for synonymous mutations in the N-terminal region involved in translation initiation. Synonymous mutations that affected either folding or translation termination were also identified. Lack of translational pausing due to synonymous mutations in hydrophobic residue stretches, was found to decrease the amount of properly folded CcdB protein. In summary the study provides novel insights into diverse mechanisms by which synonymous mutations modulate gene function.

molecular biology↗

bioRxiv2023

Mutations in S2 subunit of SARS-CoV-2 Omicron spike strongly influence its conformation, fusogenicity and neutralization sensitivity

SARS-CoV-2 has remarkable ability to respond to and evolve against the selection pressure by host immunity exemplified by emergence of Omicron lineage. Here, we characterized the functional significance of mutations in Omicron spike. By systematic transfer of mutations in WT spike we assessed neutralization sensitivity, fusogenicity, and TMPRSS2-dependence for entry. The data revealed that the mutations in both S1 and S2 complement to make Omicron highly resistant. Strikingly, the mutations in Omicron S2 modulated the neutralization sensitivity to NTD- and RBD-antibodies, but not to S2 specific neutralizing antibodies, suggesting that the mutations in S2 were primarily acquired to gain resistance to S1-antibodies. Although all six mutations in S2 appeared to act in concert, D796Y showed greatest impact on neutralization sensitivity and rendered WT virus >100-fold resistant to S309, COVA2-17, and 4A8. S2 mutations greatly reduced the antigenicity for NAbs due to reduced exposure of epitopes. In terms of the entry pathway, S1 or S2 mutations only partially altered the entry phenotype of WT and required both sets of mutations for complete switch to endosomal route and loss of syncytia formation. In particular, N856K and L981F in Omicron reduced fusion capacity and explain why subsequent Omicron variants lost them to regain fusogenicity.

microbiology↗

bioRxiv2022

High resolution cryo-EM structures of two potently SARS-CoV-2 neutralizing monoclonal antibodies of same donor origin that vary in neutralizing Omicron variants

While vaccines have by large been found to effective against the evolving SARS-CoV-2 variants, the profound and rapid effectivity of monoclonal antibodies (mAbs) in significantly reducing hospitalization to severe disease outcomes have also been demonstrated. In the present study, by high resolution cryo-electron microscopy (cryo-EM), we examined the structural insights of two trimeric spike (S) protein bound mAbs isolated from an Indian convalescent individual infected with ancestral SARS-CoV-2 which we recently reported to potently neutralize SARS-CoV-2 from its ancestral form through highly virulent Delta form however different in their ability to neutralize Omicron variants. Our findings showed binding and conformational heterogeneities of both the mAbs (THSC20.HVTR04 and THSC20.HVTR26) bound to S trimer in its apo and hACE-2 bound forms. Additionally, cryo-EM resolved structure assisted modeling highlighted key residues associated with the ability of these two mAbs to neutralize Omicron variants. Our findings highlighted key interacting features modulating antigen-antibody interacting that can further aid in structure guided antibody engineering to enhance their breadth and potency. HighlightsO_LITwo potent human mAbs obtained from a single donor differ binding to Omicron spikes C_LIO_LIPattern of binding and conformation of these mAbs bound to full length spike differs C_LIO_LIAntibody binding alters the conformational states of S trimer in its apo and hACE-2 bound forms. C_LIO_LICryo-EM structure guided modeling highlighted correlates of interacting residues associated with resistance and sensitivity of BA.1, BA.2, BA.4/BA.5 resistance and sensitivity against these mAbs. C_LI

biophysics↗

bioRxiv2022

Ter-Seq: A high-throughput method to stabilize transient ternary complexes and measure associated kinetics

Regulation of biological processes by proteins often involves the formation of transient, multimeric complexes whose characterisation is mechanistically important but challenging. The bacterial toxin CcdB binds and poisons DNA Gyrase. The corresponding antitoxin CcdA extracts CcdB from its complex with Gyrase through formation of a transient ternary complex, thus rejuvenating Gyrase. We describe a high throughput methodology called Ter-Seq to stabilize probable ternary complexes and measure associated kinetics using the CcdA-CcdB-GyrA14 ternary complex as a model system. The method involves screening a YSD saturation mutagenesis library of one partner (CcdB) for mutants that show enhanced ternary complex formation. We also isolated CcdB mutants that were either resistant or sensitive to rejuvenation, and used SPR with purified proteins to validate the kinetics measured using surface display. Positions where CcdB mutations lead to slower rejuvenation rates are largely involved in CcdA-binding, though there were several notable exceptions. Mutations at these positions reduce the affinity towards CcdA, thereby slowing down the rejuvenation process. Mutations at GyrA14-interacting positions significantly enhanced rejuvenation rates, either due to reduced affinity or complete loss of CcdB binding to GyrA14. We examined the effect of different parameters (CcdA affinity, GyrA14 affinity, surface accessibilities, evolutionary conservation) on the rate of rejuvenation. Finally, we further validated the Ter-Seq results by monitoring kinetics of ternary complex formation for individual CcdB mutants in solution by FRET studies.

biophysics↗

bioRxiv2022

Inferring bound structure and residue specific contributions to binding energetics in the Intrinsically Disordered Protein, CcdA

Unlike globular proteins, mutational effects on the function of Intrinsically Disordered Proteins (IDPs) are not well-studied. Deep Mutational Scanning of a yeast surface displayed mutant library yields insights into sequence-function relationships in the CcdA IDP. The approach enables facile prediction of interface residues and local structural signatures of the bound conformation. In contrast to previous titration-based approaches which use a number of ligand concentrations, we show that use of a single rationally chosen ligand concentration can provide quantitative estimates of relative binding constants for large numbers of protein variants. This is because the extended interface of IDP ensures that energetic effects of point mutations are spread over a much smaller range than for globular proteins. Our data also provides insights into the much-debated role of helicity and disorder in partner binding of IDPs. Based on this exhaustive mutational sensitivity dataset, a model was developed to predict mutational effects on binding affinity of IDPs that form alpha-helical structures upon binding.

biophysics↗

bioRxiv2022

Highly Thermotolerant SARS-CoV-2 Vaccine Elicits Neutralising Antibodies Against Delta and Omicron in Mice

As existing vaccines fail to completely prevent COVID-19 infections or community transmission, there is an unmet need for vaccines that can better combat SARS-CoV-2 variants of concern (VOC). We have previously developed highly thermo-tolerant monomeric and trimeric receptor binding domain derivatives that can withstand 100{degrees}C for 90 minutes and 37{degrees}C for four weeks, and help eliminate cold chain requirements. We show that mice immunised with these vaccine formulations elicit high titres of antibodies that neutralise SARS-CoV-2 variants VIC31 (with Spike: D614G mutation), Delta and Omicron (BA.1.1) VOC. Compared to VIC31, there was an average 14.4-fold reduction in neutralisation against BA.1.1 for the three monomeric antigen-adjuvant combinations, and 16.5-fold reduction for the three trimeric antigen-adjuvant combinations; the corresponding values against Delta were 2.5 and 3.0. Our findings suggest that monomeric formulations are suitable for the upcoming Phase I human clinical trials, and that there is potential for increasing efficacy with vaccine matching to improve responses against emerging variants. These findings are consistent with in silico modelling and AlphaFold predictions which show that while oligomeric presentation can be generally beneficial, it can make important epitopes inaccessible, and also carries the risk of eliciting unwanted antibodies against the oligomerisation domain.

immunology↗

bioRxiv2022

Structural and functional determinants inferred from deep mutational scans

Mutations that affect protein binding to a cognate partner primarily occur either at buried residues or at exposed residues directly involved in partner binding. Distinguishing between these two categories based solely on mutational phenotypes is challenging. The bacterial toxin CcdB kills cells by binding to DNA Gyrase. Cell death is prevented by binding to its cognate antitoxin CcdA, at an extended interface that partially overlaps with the GyrA binding site. Using the CcdAB toxin-antitoxin (TA) system as a model, a comprehensive site-saturation mutagenesis library of CcdB was generated in its native operonic context. The mutational sensitivity of each mutant was estimated by evaluating the relative abundance of each mutant in two strains, one resistant and the other sensitive to the toxic activity of the CcdB toxin, through deep sequencing. The ability to bind CcdA was inferred through a RelE reporter gene assay, since the CcdAB complex binds to its own promoter, repressing transcription. By analysing mutant phenotypes in the CcdB sensitive, CcdB resistant and RelE reporter strains, it was possible to assign residues to buried, CcdA interacting or GyrA interacting sites. A few mutants were individually constructed, expressed, and biophysically characterised to validate molecular mechanisms responsible for the observed phenotypes. Residues inferred to be important for antitoxin binding, are also likely to be important for rejuvenating CcdB from the CcdB-Gyrase complex. Therefore, even in the absence of structural information, when coupled to appropriate genetic screens, such high-throughput strategies can be deployed for predicting structural and functional determinants of proteins. Broader Impact StatementPartial loss-of-function mutations predominantly occur either at buried-site or exposed, active-site residues. We report a facile method to identify multiple binding sites for different interacting partners for a protein, and distinguish them from buried site and exposed non active-site residues, solely from mutational data.

biophysics↗

bioRxiv2022

The UBR box E3 ligases Poe and Hyd are required for efficient Pericentrin degradation

Centrosomes are the major microtubule organizing centers (MTOC) of many cells and are composed of centrioles and the pericentriolar material (PCM). Centrosomes are an essential part of diverse cellular processes and require precise regulation of their protein levels. One protein whose levels must be regulated is Pericentrin - PCNT in humans and PLP in Drosophila. Increased PCNT expression and its protein accumulation is linked to clinical conditions including cancer, mental disorders, and ciliopathies. However, the mechanisms by which PCNT levels are regulated remains underexplored. Our previous study (Galletta, 2020) demonstrated that PLP levels are sharply downregulated during early spermatogenesis and this regulation is essential to spatially position PLP on the proximal end of centrioles. We hypothesized that the sharp drop in PLP protein was a result of a rapid protein degradation during the male pre-meiotic G2 phase. In this study we show that the N-terminal region of PLP is required for regulating its levels, which when elevated, extends PLPs position distally on centrioles. Consequently, PCM was also mispositioned leading to defects in spermatids. We then performed a targeted screen where we identified the E2 ligases, Rad6 and UbcD1, and the UBR box family E3 ligases, Poe (UBR4) and Hyd (UBR5) as factors that promote PLP degradation in spermatocytes. Collectively, our study suggests that Poe and Hyd directly bind the N-terminus of PLP and target it for degradation, ensuring proper centriole/basal body assembly and male fertility.

developmental biology↗

bioRxiv2022

Codon optimality is the primary contributor to the exceptional mutational sensitivity of CcdA antitoxin in its operonic context

Deep mutational scanning studies suggest that synonymous mutations are typically silent and that most exposed, non active-site residues are tolerant to mutations. Here we show that the ccdA antitoxin component of the E.coli ccdAB toxin-antitoxin system is unusually sensitive to mutations when studied in the operonic context. A large fraction ([~]80%) of single-codon mutations, including many synonymous mutations in the ccdA gene shows inactive phenotype, but they retain native-like binding affinity towards cognate toxin, CcdB. Therefore, the observed phenotypic effects are largely not due to alterations in protein structure/stability, consistent with a large region of CcdA being intrinsically disordered. E. coli codon preference and strength of ribosome-binding associated with translation of downstream ccdB gene are found to be major contributors of the observed mutant phenotypes. In select cases, proteomics studies reveal altered ratios of CcdA:CcdB protein levels in vivo, suggesting that the ccdA mutations likely alter relative translation efficiencies of the two genes in the operon. We extend these results by studying single-site synonymous mutations that lead to loss of function phenotypes in the relBE operon upon introduction of rarer codons. Thus, in their operonic context, genes are likely to be more sensitive to both synonymous and non-synonymous point mutations than inferred previously.

molecular biology↗

bioRxiv2021

Mechanistic insights into global suppressors of protein folding defects

Most amino acid substitutions in a protein either lead to partial loss of function or are near neutral. Several studies have shown the existence of second-site mutations that can rescue defects caused by diverse loss of function mutations. Such global suppressor mutations are key drivers of protein evolution. However, the mechanisms responsible for such suppression remain poorly understood. To address this, we characterized multiple suppressor mutations both in isolation and in combination with inactive mutants. We examined six global suppressors of the bacterial toxin CcdB, the known M182T global suppressor of TEM-1 {beta}-lactamase, the N239Y global suppressor of p53-DBD and three suppressors of the SARS-CoV-2 spike Receptor Binding Domain. The suppressors both alone, and in conjunction with inactive mutants, stabilise the protein both thermodynamically and kinetically in-vitro, predominantly through acceleration of the refolding rate. When coupled to inactive mutants they promote increased in-vivo solubilities as well as regain-of-function phenotypes. In the case of CcdB, where novel suppressors were isolated, we determined the crystal structures of three such suppressors to obtain insight into the specific molecular interactions responsible for the observed effects. While most individual suppressors result in small stability enhancements relative to wildtype, which can be combined to yield significant stability increases, thermodynamic stabilisation is neither necessary nor sufficient for suppressor action.

biophysics↗

bioRxiv2021

Stabilizing proteins through saturation suppressor mutagenesis.

While there have been recent, transformative advances in the area of protein structure prediction, prediction of point mutations that improve protein stability remains challenging. It is possible to construct and screen large mutant libraries for improved activity or ligand binding, however reliable screens for mutants that improve protein stability do not exist, especially for proteins that are well folded and relatively stable. We demonstrate that incorporation of a single, specific destabilizing, (parent inactive) mutation into each member of a deep mutational scanning library followed by screening for suppressors, allows for robust and accurate identification of stabilizing mutations. When coupled to FACS sorting of a yeast surface display library of the bacterial toxin CcdB, followed by deep sequencing of sorted populations, multiple stabilizing mutations could be identified after a single round of sorting. Multiple libraries with different parent inactive mutations could be pooled and simultaneously screened to further enhance the accuracy of identification of stabilizing mutations. Individual stabilizing mutations could be combined to result in a multimutant with increase in thermal melting temperature of about 20 {degrees}C and enhanced tolerance to high temperature exposure. The method employs small library sizes and can be readily extended to other display and screening formats to rapidly isolate stabilized protein mutants.

biophysics↗