A CcdB toxin-derived peptide acts as a broad-spectrum antibacterial therapeutic in infected mice.

The bacterial toxin CcdB (Controller of Cell death or division B) targets DNA Gyrase, an essential bacterial topoisomerase, which is also the molecular target for fluoroquinolones. Here, we present a short cell-penetrating 24-mer peptide, CP1-WT, derived from the Gyrase-binding region of CcdB and examine its effect on growth of Escherichia coli, Salmonella Typhimurium, Staphylococcus aureus and a carbapenem- and tigecycline-resistant strain of Acinetobacter baumannii in both axenic cultures and mouse models of infection. The CP1-WT peptide shows significant improvement over ciprofloxacin in terms of its in vivo therapeutic efficacy in treating established infections of S. Typhimurium, S. aureus and A. baumannii. The molecular mechanism likely involves inhibition of Gyrase or Topoisomerase IV, depending on the strain used. The study validates the CcdB binding site on bacterial DNA Gyrase as a viable and alternative target to the fluoroquinolone binding site.

Combining cysteine scanning with chemical labeling to map protein-protein interactions and infer bound structure in an intrinsically disordered region.

The Mycobacterium tuberculosis genome harbours nine toxin-antitoxin (TA) systems of the mazEF family. These consist of two proteins, a toxin and an antitoxin, encoded in an operon. While the toxin has a conserved fold, the antitoxins are structurally diverse and the toxin binding region is typically intrinsically disordered before binding. We describe high throughput methodology for accurate mapping of interfacial residues and apply it to three MazEF complexes. The method involves screening one partner protein against a panel of chemically masked single cysteine mutants of its interacting partner, displayed on the surface of yeast cells. Such libraries have much lower diversity than those generated by saturation mutagenesis, simplifying library generation and data analysis. Further, because of the steric bulk of the masking reagent, labeling of virtually all exposed epitope residues should result in loss of binding, and buried residues are inaccessible to the labeling reagent. The binding residues are deciphered by probing the loss of binding to the labeled cognate partner by flow cytometry. Using this methodology, we have identified the interfacial residues for MazEF3, MazEF6 and MazEF9 TA systems of M. tuberculosis. In the case of MazEF9, where a crystal structure was available, there was excellent agreement between our predictions and the crystal structure, superior to those with AlphaFold2. We also report detailed biophysical characterization of the MazEF3 and MazEF9 TA systems and measured the relative affinities between cognate and non-cognate toxin-antitoxin partners in order to probe possible cross-talk between these systems.

A clade C HIV-1 vaccine protects against heterologous SHIV infection by modulating IgG glycosylation and T helper response in macaques.

The rising global HIV-1 burden urgently requires vaccines capable of providing heterologous protection. Here, we developed a clade C HIV-1 vaccine consisting of priming with modified vaccinia Ankara (MVA) and boosting with cyclically permuted trimeric gp120 (CycP-gp120) protein, delivered either orally using a needle-free injector or through parenteral injection. We tested protective efficacy of the vaccine against intrarectal challenges with a pathogenic heterologous clade C SHIV infection in rhesus macaques. Both routes of vaccination induced a strong envelope-specific IgG in serum and rectal secretions directed against V1V2 scaffolds from a global panel of viruses with polyfunctional activities. Envelope-specific IgG showed lower fucosylation compared with total IgG at baseline, and most of the vaccine-induced proliferating blood CD4+ T cells did not express CCR5 and α4ß7, markers associated with HIV target cells. After SHIV challenge, both routes of vaccination conferred significant and equivalent protection, with 40% of animals remaining uninfected at the end of six weekly repeated challenges with an estimated efficacy of 68% per exposure. Induction of envelope-specific IgG correlated positively with G1FB glycosylation, and G2S2F glycosylation correlated negatively with protection. Vaccine-induced TNF-α+ IFN-γ+ CD8+ T cells and TNF-α+ CD4+ T cells expressing low levels of CCR5 in the rectum at prechallenge were associated with decreased risk of SHIV acquisition. These results demonstrate that the clade C MVA/CycP-gp120 vaccine provides heterologous protection against a tier2 SHIV rectal challenge by inducing a polyfunctional antibody response with distinct Fc glycosylation profile, as well as cytotoxic CD8 T cell response and CCR5-negative T helper response in the rectum.

A dimeric proteomimetic prevents SARS-CoV-2 infection by dimerizing the spike protein.

Protein tertiary structure mimetics are valuable tools to target large protein-protein interaction interfaces. Here, we demonstrate a strategy for designing dimeric helix-hairpin motifs from a previously reported three-helix-bundle miniprotein that targets the receptor-binding domain (RBD) of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Through truncation of the third helix and optimization of the interhelical loop residues of the miniprotein, we developed a thermostable dimeric helix-hairpin. The dimeric four-helix bundle competes with the human angiotensin-converting enzyme 2 (ACE2) in binding to RBD with 2:2 stoichiometry. Cryogenic-electron microscopy revealed the formation of dimeric spike ectodomain trimer by the four-helix bundle, where all the three RBDs from either spike protein are attached head-to-head in an open conformation, revealing a novel mechanism for virus neutralization. The proteomimetic protects hamsters from high dose viral challenge with replicative SARS-CoV-2 viruses, demonstrating the promise of this class of peptides that inhibit protein-protein interaction through target dimerization.

A facile method of mapping HIV-1 neutralizing epitopes using chemically masked cysteines and deep sequencing.

Identification of specific epitopes targeted by neutralizing antibodies is essential to advance epitope-based vaccine design strategies. We report a facile methodology for rapid epitope mapping of neutralizing antibodies (NAbs) against HIV-1 Envelope (Env) at single-residue resolution, using Cys labeling, viral neutralization assays, and deep sequencing. This was achieved by the generation of a library of Cys mutations in Env glycoprotein on the viral surface, covalent labeling of the Cys residues using a Cys-reactive label that masks epitope residues, followed by infection of the labeled mutant virions in mammalian cells in the presence of NAbs. Env gene sequencing from NAb-resistant viruses was used to accurately delineate epitopes for the NAbs VRC01, PGT128, and PGT151. These agreed well with corresponding experimentally determined structural epitopes previously inferred from NAb:Env structures. HIV-1 infection is associated with complex and polyclonal antibody responses, typically composed of multiple antibody specificities. Deconvoluting the epitope specificities in a polyclonal response is a challenging task. We therefore extended our methodology to map multiple specificities of epitopes targeted in polyclonal sera, elicited in immunized animals as well as in an HIV-1-infected elite neutralizer capable of neutralizing tier 3 pseudoviruses with high titers. The method can be readily extended to other viruses for which convenient reverse genetics or lentiviral surface display systems are available.

Mechanism of CcdA-Mediated Rejuvenation of DNA Gyrase.

Most biological processes involve formation of transient complexes where binding of a ligand allosterically modulates function. The ccd toxin-antitoxin system is involved in plasmid maintenance and bacterial persistence. The CcdA antitoxin accelerates dissociation of CcdB from its complex with DNA gyrase, binds and neutralizes CcdB, but the mechanistic details are unclear. Using a series of experimental and computational approaches, we demonstrate the formation of transient ternary and quaternary CcdA:CcdB:gyrase complexes and delineate the molecular steps involved in the rejuvenation process. Binding of region 61-72 of CcdA to CcdB induces the vital structural and dynamic changes required to facilitate dissociation from gyrase, region 50-60 enhances the dissociation process through additional allosteric effects, and segment 37-49 prevents gyrase rebinding. This study provides insights into molecular mechanisms responsible for recovery of CcdB-poisoned cells from a persister-like state. Similar methodology can be used to characterize other important transient, macromolecular complexes.

Rapid Mapping of Protein Binding Sites and Conformational Epitopes by Coupling Yeast Surface Display to Chemical Labeling and Deep Sequencing.

Delineating the precise regions on an antigen that are targeted by antibodies is important for the development of vaccines and antibody therapeutics. X-ray crystallography and NMR are considered the gold standard for providing precise information about these binding sites at atomic resolution. However, these are labor-intensive and require purified protein at high concentration. We have recently described [1] a rapid and reliable method that overcomes these constraints, using a panel of single cysteine mutants of the protein of interest and now provide protocols to facilitate its adoption. Mutants are displayed on the yeast cell surface either individually or as a pool, and labeled covalently with a cysteine specific probe. Binding site residues are inferred by monitoring loss of ligand or antibody binding by flow cytometry coupled to deep sequencing of sorted populations, or Sanger sequencing of individual clones. Buried cysteine residues are not labeled and library sizes are small, facilitating rapid identification of binding-site residues. The methodology was used to identify epitopes on the bacterial toxin CcdB targeted by twenty-four different monoclonal antibodies as well as by polyclonal sera. The method does not require purified protein or protein structural information and can be applied to a variety of display formats.

Mapping Protein Binding Sites and Conformational Epitopes Using Cysteine Labeling and Yeast Surface Display.

We describe a facile method for mapping protein:ligand binding sites and conformational epitopes. The method uses a combination of Cys scanning mutagenesis, chemical labeling, and yeast surface display. While Ala scanning is widely used for similar purposes, often mutation to Ala (or other amino acids) has little effect on binding, except at hotspot residues. Many residues in physical contact with a binding partner are insensitive to substitution with Ala. In contrast, we show that labeling of Cys residues in a binding site consistently abrogates binding. We couple this methodology to yeast surface display and deep sequencing to map conformational epitopes targeted by both monoclonal antibodies and polyclonal sera as well as a protein:ligand binding site. The method does not require purified protein, can distinguish buried and exposed residues, and can be extended to other display formats, including mammalian cells and viruses, emphasizing its wide applicability.

Stalking influenza by vaccination with pre-fusion headless HA mini-stem.

Inaccuracies in prediction of circulating viral strain genotypes and the possibility of novel reassortants causing a pandemic outbreak necessitate the development of an anti-influenza vaccine with increased breadth of protection and potential for rapid production and deployment. The hemagglutinin (HA) stem is a promising target for universal influenza vaccine as stem-specific antibodies have the potential to be broadly cross-reactive towards different HA subtypes. Here, we report the design of a bacterially expressed polypeptide that mimics a H5 HA stem by protein minimization to focus the antibody response towards the HA stem. The HA mini-stem folds as a trimer mimicking the HA prefusion conformation. It is resistant to thermal/chemical stress, and it binds to conformation-specific, HA stem-directed broadly neutralizing antibodies with high affinity. Mice vaccinated with the group 1 HA mini-stems are protected from morbidity and mortality against lethal challenge by both group 1 (H5 and H1) and group 2 (H3) influenza viruses, the first report of cross-group protection. Passive transfer of immune serum demonstrates the protection is mediated by stem-specific antibodies. Furthermore, antibodies induced by these HA stems have broad HA reactivity, yet they do not have antibody-dependent enhancement activity.

Cell surface assembly of HIV gp41 six-helix bundles for facile, quantitative measurements of hetero-oligomeric interactions.

Helix-helix interactions are fundamental to many biological signals and systems and are found in homo- or heteromultimerization of signaling molecules as well as in the process of virus entry into the host. In HIV, virus-host membrane fusion during infection is mediated by the formation of six-helix bundles (6HBs) from homotrimers of gp41, from which a number of synthetic peptides have been derived as antagonists of virus entry. Using a yeast surface two-hybrid (YS2H) system, a platform designed to detect protein-protein interactions occurring through a secretory pathway, we reconstituted 6HB complexes on the yeast surface, quantitatively measured the equilibrium and kinetic constants of soluble 6HB, and delineated the residues influencing homo-oligomeric and hetero-oligomeric coiled-coil interactions. Hence, we present YS2H as a platform for the facile characterization and design of antagonistic peptides for inhibition of HIV and many other enveloped viruses relying on membrane fusion for infection, as well as cellular signaling events triggered by hetero-oligomeric coiled coils.

Conformational changes of the chaperone SecB upon binding to a model substrate--bovine pancreatic trypsin inhibitor (BPTI).

SecB is a homotetrameric cytosolic chaperone that forms part of the protein translocation machinery in E. coli. Due to SecB, nascent polypeptides are maintained in an unfolded translocation-competent state devoid of tertiary structure and thus are guided to the translocon. In vitro SecB rapidly binds to a variety of ligands in a non-native state. We have previously investigated the bound state conformation of the model substrate bovine pancreatic trypsin inhibitor (BPTI) as well as the conformation of SecB itself by using proximity relationships based on site-directed spin labeling and pyrene fluorescence methods. It was shown that SecB undergoes a conformational change during the process of substrate binding. Here, we generated SecB mutants containing but a single cysteine per subunit or an exposed highly reactive new cysteine after removal of the nearby intrinsic cysteines. Quantitative spin labeling was achieved with the methanethiosulfonate spin label (MTS) at positions C97 or E90C, respectively. Highfield (W-band) electron paramagnetic resonance (EPR) measurements revealed that with BPTI present the spin labels are exposed to a more polar/hydrophilic environment. Nanoscale distance measurements with double electron-electron resonance (DEER) were in excellent agreement with distances obtained by molecular modeling. Binding of BPTI also led to a slight change in distances between labels at C97 but not at E90C. While the shorter distance in the tetramer increased, the larger diagonal distance decreased. These findings can be explained by a widening of the tetrameric structure upon substrate binding much like the opening of two pairs of scissors.

Ligand-modulated parallel mechanical unfolding pathways of maltose-binding proteins.

Protein folding and unfolding are complex phenomena, and it is accepted that multidomain proteins generally follow multiple pathways. Maltose-binding protein (MBP) is a large (a two-domain, 370-amino acid residue) bacterial periplasmic protein involved in maltose uptake. Despite the large size, it has been shown to exhibit an apparent two-state equilibrium unfolding in bulk experiments. Single-molecule studies can uncover rare events that are masked by averaging in bulk studies. Here, we use single-molecule force spectroscopy to study the mechanical unfolding pathways of MBP and its precursor protein (preMBP) in the presence and absence of ligands. Our results show that MBP exhibits kinetic partitioning on mechanical stretching and unfolds via two parallel pathways: one of them involves a mechanically stable intermediate (path I) whereas the other is devoid of it (path II). The apoMBP unfolds via path I in 62% of the mechanical unfolding events, and the remaining 38% follow path II. In the case of maltose-bound MBP, the protein unfolds via the intermediate in 79% of the cases, the remaining 21% via path II. Similarly, on binding to maltotriose, a ligand whose binding strength with the polyprotein is similar to that of maltose, the occurrence of the intermediate is comparable (82% via path I) with that of maltose. The precursor protein preMBP also shows a similar behavior upon mechanical unfolding. The percentages of molecules unfolding via path I are 53% in the apo form and 68% and 72% upon binding to maltose and maltotriose, respectively, for preMBP. These observations demonstrate that ligand binding can modulate the mechanical unfolding pathways of proteins by a kinetic partitioning mechanism. This could be a general mechanism in the unfolding of other large two-domain ligand-binding proteins of the bacterial periplasmic space.

Conformational analysis and design of cross-strand disulfides in antiparallel ß-sheets.

Cross-strand disulfides bridge two cysteines in a registered pair of antiparallel ß-strands. A nonredundant data set comprising 5025 polypeptides containing 2311 disulfides was used to study cross-strand disulfides. Seventy-six cross-strand disulfides were found of which 75 and 1 occurred at non-hydrogen-bonded (NHB) and hydrogen-bonded (HB) registered pairs, respectively. Conformational analysis and modeling studies demonstrated that disulfide formation at HB pairs necessarily requires an extremely rare and positive χ¹ value for at least one of the cysteine residues. Disulfides at HB positions also have more unfavorable steric repulsion with the main chain. Thirteen pairs of disulfides were introduced in NHB and HB pairs in four model proteins: leucine binding protein (LBP), leucine, isoleucine, valine binding protein (LIVBP), maltose binding protein (MBP), and Top7. All mutants LIVBP T247C V331C showed disulfide formation either on purification, or on treatment with oxidants. Protein stability in both oxidized and reduced states of all mutants was measured. Relative to wild type, LBP and MBP mutants were destabilized with respect to chemical denaturation, although the sole exposed NHB LBP mutant showed an increase of 3.1°C in T(m). All Top7 mutants were characterized for stability through guanidinium thiocyanate chemical denaturation. Both exposed and two of the three buried NHB mutants were appreciably stabilized. All four HB Top7 mutants were destabilized (ΔΔG° = -3.3 to -6.7 kcal/mol). The data demonstrate that introduction of cross-strand disulfides at exposed NHB pairs is a robust method of improving protein stability. All four exposed Top7 disulfide mutants showed mild redox activity.

Disulfide conformation and design at helix N-termini.

To understand structural and thermodynamic features of disulfides within an alpha-helix, a non-redundant dataset comprising of 5025 polypeptide chains containing 2311 disulfides was examined. Thirty-five examples were found of intrahelical disulfides involving a CXXC motif between the N-Cap and third helical positions. GLY and PRO were the most common amino acids at positions 1 and 2, respectively. The N-Cap residue for disulfide bonded CXXC motifs had average (phi,psi) values of (-112 +/- 25.2 degrees , 106 +/- 25.4 degrees ). To further explore conformational requirements for intrahelical disulfides, CYS pairs were introduced at positions N-Cap-3; 1,4; 7,10 in two helices of an Escherichia coli thioredoxin mutant lacking its active site disulfide (nSS Trx). In both helices, disulfides formed spontaneously during purification only at positions N-Cap-3. Mutant stabilities were characterized by chemical denaturation studies (in both oxidized and reduced states) and differential scanning calorimetry (oxidized state only). All oxidized as well as reduced mutants were destabilized relative to nSS Trx. All mutants were redox active, but showed decreased activity relative to wild-type thioredoxin. Such engineered disulfides can be used to probe helix start sites in proteins of unknown structure and to introduce redox activity into proteins. Conversely, a protein with CYS residues at positions N-Cap and 3 of an alpha-helix is likely to have redox activity.

Stereochemical criteria for prediction of the effects of proline mutations on protein stability.

When incorporated into a polypeptide chain, proline (Pro) differs from all other naturally occurring amino acid residues in two important respects. The phi dihedral angle of Pro is constrained to values close to -65 degrees and Pro lacks an amide hydrogen. Consequently, mutations which result in introduction of Pro can significantly affect protein stability. In the present work, we describe a procedure to accurately predict the effect of Pro introduction on protein thermodynamic stability. Seventy-seven of the 97 non-Pro amino acid residues in the model protein, CcdB, were individually mutated to Pro, and the in vivo activity of each mutant was characterized. A decision tree to classify the mutation as perturbing or nonperturbing was created by correlating stereochemical properties of mutants to activity data. The stereochemical properties including main chain dihedral angle phi and main chain amide H-bonds (hydrogen bonds) were determined from 3D models of the mutant proteins built using MODELLER. We assessed the performance of the decision tree on a large dataset of 163 single-site Pro mutations of T4 lysozyme, 74 nsSNPs, and 52 other Pro substitutions from the literature. The overall accuracy of this algorithm was found to be 81% in the case of CcdB, 77% in the case of lysozyme, 76% in the case of nsSNPs, and 71% in the case of other Pro substitution data. The accuracy of Pro scanning mutagenesis for secondary structure assignment was also assessed and found to be at best 69%. Our prediction procedure will be useful in annotating uncharacterized nsSNPs of disease-associated proteins and for protein engineering and design.

Design and utility of temperature-sensitive Gal4 mutants for conditional gene expression in Drosophila.

Temperature-sensitive (ts) mutants are valuable tools to study the function of essential genes in vivo. Despite their widespread use, little is known about mechanisms responsible for the temperature-sensitive (ts) phenotype, or of the transferability of ts mutants of a specific gene between organisms. Since ts mutants are typically generated by random mutagenesis it is difficult to isolate such mutants without efficient screening procedures. We have recently shown that it is possible to obtain ts mutants at high frequency by targeted mutations at either predicted, buried residues important for protein stability or at functional, ligand binding residues. The former class of residues can be identified solely from amino acid sequence and the latter from Ala scanning mutagenesis or from a structure of the protein:ligand complex. Several ts mutants of Gal4 in yeast were generated by mutating both categories of residues. Two of these ts mutants were also shown to result in tight and rapid ts reporter gene-expression in Drosophila when driven by either the elav or GMR promoters. We suggest possible mechanisms that might be responsible for such transferable ts phenotypes and also discuss some of the limitations and difficulties involved in rational design of ts mutants.

Protein stabilization by introduction of cross-strand disulfides.

Disulfides cross-link residues in a protein that are separated in primary sequence and stabilize the protein through entropic destabilization of the unfolded state. While the removal of naturally occurring disulfides leads to protein destabilization, introduction of engineered disulfides does not always lead to significant stabilization of a protein. We have analyzed naturally occurring disulfides that span adjacent antiparallel strands of beta sheets (cross-strand disulfides). Cross-strand disulfides have recently been implicated as redox-based conformational switches in proteins such as gp120 and CD4. The propensity of these disulfides to act as conformational switches was postulated on the basis of the hypothesis that this class of disulfide is conformationally strained. In the present analysis, there was no evidence to suggest that cross-strand disulfides are more strained compared to other disulfides as assessed by their torsional energy. It was also observed that these disulfides occur solely at non-hydrogen-bonded (NHB) registered pairs of adjacent antiparallel strands and not at hydrogen-bonded (HB) positions as suggested previously. One of the half-cystines involved in cross-strand disulfide formation often occurs at an edge strand. Experimental confirmation of the stabilizing effects of such disulfides was carried out in Escherichia coli thioredoxin. Four pairs of cross-strand cysteines were introduced, two at HB and two at NHB pairs. Disulfides were formed in all four cases. However, as predicted from our analysis, disulfides at NHB positions resulted in an increase in melting temperature of 7-10 degrees C, while at HB positions there was a corresponding decrease of -7 degrees C. The reduced state of all proteins had similar stability.

NMR structural analysis of a peptide mimic of the bridging sheet of HIV-1 gp120 in methanol and water.

gp120 is a subunit of the Env (viral envelope protein) of HIV-1. The protein consists of inner and outer domains linked by a bridging sheet. Several gp120 residues that bind the neutralizing antibody 17b as well as the cellular co-receptor CCR5 (CC chemokine receptor 5), are located in the bridging sheet. Peptides that mimic the 17b-binding regions of gp120 would be useful potential immunogens for the generation of neutralizing antibodies against HIV-1. Towards this end, a 26-residue, four-stranded beta-sheet peptide was designed on the basis of the structure of the bridging sheet, and its structure was characterized in methanol by NMR. In methanol, amide and alpha-proton resonances were well resolved and dispersed. A number of interstrand NOEs (nuclear Overhauser effects) were observed, providing good evidence for multiple turn beta-hairpin structure. NOEs also provided good evidence for all Xxx-D-Pro bonds in the trans configuration and all three turns formed by a two residue D-Pro-Gly segment to be of type II' turn. The structure conforms well to the designed four-stranded beta-sheet structure. Approx. 20% of the peptide was estimated to adopt a folded conformation in water, as evidenced by CD spectroscopy. This was consistent with smaller, but still significant, downfield shifts of C(alpha)H protons relative to random-coil values. A second peptide was designed with two disulphide bonds to further constrain the peptide backbone. While structured in methanol, this peptide, like the previous one, also exhibits only partial structure formation in water, as evidenced by CD spectroscopy.