Surface-exposed Loops Research Articles

Mamastrovirus spike protein: Sequence and structural characterization as a basis for understanding cross-species transmission

Astroviruses (AstVs) are notable for their propensity for cross-species transmission; however, the molecular determinants underlying this phenomenon remain poorly understood. The spike protein, which is responsible for host cell entry and is a major antigenic determinant, is hypothesized to play a pivotal role. In this study, we observed high sequence variability in the spike region of AstV. Structural analyses have revealed variability, arising from diverse evolutionary relationships, among AstVs of the same host origin. AstV spike proteins can be categorized into six groups, each of which encompasses AstVs from diverse hosts that exhibit high degrees of structural similarity. These six groups correspond to branches observed in the phylogenetic tree. Notably, the spike surface-exposed loops emerged as focal hotspots for B-cell epitopes across groups, with sequence variability that may contribute to immune evasion upon host switching. Differences in the spike structures of AstVs infecting the same host raise the possibility of distinct tissue tropisms and corresponding clinical manifestations. Collectively, our findings provide insights into the roles of spike protein similarities and immune epitope diversity in driving AstV cross-species transmission. Understanding these molecular mechanisms is crucial to predicting and mitigating the emergence of novel AstV strains.

Investigating the importance of selected surface-exposed loops in HpuB for hemoglobin binding and utilization by Neisseria gonorrhoeae.

Neisseria gonorrhoeae is the etiological agent of the sexually transmitted infection gonorrhea. The pathogen is a global health challenge since no protective immunity results from infection, and far fewer treatment options are available with increasing antimicrobial resistance. With no efficacious vaccines, researchers are exploring new targets for vaccine development and innovative therapeutics. The outer membrane TonB-dependent transporters (TdTs) produced by N. gonorrhoeae are considered promising vaccine antigens as they are highly conserved and play crucial roles in overcoming nutritional immunity. One of these TdTs is part of the hemoglobin transport system comprised of HpuA and HpuB. This system allows N. gonorrhoeae to acquire iron from hemoglobin (hHb). In the current study, mutations in the hpuB gene were generated to better understand the structure-function relationships in HpuB. This study is one of the first to demonstrate that N. gonorrhoeae can bind to and utilize hemoglobin produced by animals other than humans. This study also determined that when HpuA is absent, mutations targeting extracellular loop 7 of HpuB led to defective hHb binding and utilization. However, when the lipoprotein HpuA is present, these loop 7 mutants recovered their ability to bind hHb, although the growth phenotype remained significantly impaired. Interestingly, loop 7 contains putative heme-binding motifs and a hypothetical α-helical region, both of which may be important for the use of hHb. Taken together, these results highlight the importance of loop 7 in the functionality of HpuB in binding hHb and extracting and internalizing iron.

Engineered protein cages with enhanced extracellular drug release for elevated antitumor efficacy

Human heavy chain ferritin (HFn) protein cage has been explored as a nanocarrier for targeted anticancer drug delivery. Here, we introduced a matrix metalloproteinases (MMPs)-cleavable sequence into the DE loop of HFn, creating an MMP-responsive variant, MR-HFn, for localized and extracellular drug release. The crystal structure of MR-HFn revealed that the addition of the MMPs recognition sequence did not affect the self-assembly of HFn but presented a surface-exposed loop susceptible to MMPs cleavage. Biochemical analysis indicated that this engineered protein cage is responsive to MMPs, enabling the targeted release of encapsulated drugs. To evaluate the therapeutic potential of this engineered protein cage, monosubstituted β-carboxy phthalocyanine zinc (CPZ), a type of photosensitizer, was loaded inside this protein cage. The prepared CPZ@MR-HFn showed higher uptake and stronger phototoxicity in MMPs overexpressed tumor cells, as well as enhanced penetration into multicellular tumor spheroids compared with its counterpart CPZ@HFn in vitro. In vivo, CPZ@MR-HFn displayed a higher tumor inhibitory rate than CPZ@HFn under illumination. These results indicated that MR-HFn is a promising nanocarrier for anticancer drug delivery and the MMP-responsive strategy here can also be adapted for other stimuli.

Chimeric virus-like particles of human norovirus constructed by structure-guided epitope grafting elicit cross-reactive immunity against both GI.1 and GII.4 genotypes.

Human norovirus (HuNoV) is highly infectious and can result in severe illnesses in the elderly and children. So far, there is no effective antiviral drug to treat HuNoV infection, and thus, the development of HuNoV vaccines is urgent. However, NoV evolves rapidly, and currently, at least 10 genogroups with numerous genotypes have been found. The genetic diversity of NoV and the lack of cross-protection between different genotypes pose challenges to the development of broadly protective vaccines. In this study, guided by structural alignment between GI.1 and GII.4 HuNoV VP1 proteins, several chimeric-type virus-like particles (VLPs) were designed through surface-exposed loop grafting. Mouse immunization studies show that two of the designed chimeric VLPs induced cross-immunity against both GI.1 and GII.4 HuNoVs. To our knowledge, this is the first designed chimeric VLPs that can induce cross-immune activities across different genogroups of HuNoV, which provides valuable strategies for the development of cross-reactive HuNoV vaccines.

Molecular Dynamics Mappings of the CCT/TRiC Complex-Mediated Protein Folding Cycle Using Diffracted X-ray Tracking.

The CCT/TRiC complex is a type II chaperonin that undergoes ATP-driven conformational changes during its functional cycle. Structural studies have provided valuable insights into the mechanism of this process, but real-time dynamics analyses of mammalian type II chaperonins are still scarce. We used diffracted X-ray tracking (DXT) to investigate the intramolecular dynamics of the CCT complex. We focused on three surface-exposed loop regions of the CCT1 subunit: the loop regions of the equatorial domain (E domain), the E and intermediate domain (I domain) juncture near the ATP-binding region, and the apical domain (A domain). Our results showed that the CCT1 subunit predominantly displayed rotational motion, with larger mean square displacement (MSD) values for twist (χ) angles compared with tilt (θ) angles. Nucleotide binding had a significant impact on the dynamics. In the absence of nucleotides, the region between the E and I domain juncture could act as a pivotal axis, allowing for greater motion of the E domain and A domain. In the presence of nucleotides, the nucleotides could wedge into the ATP-binding region, weakening the role of the region between the E and I domain juncture as the rotational axis and causing the CCT complex to adopt a more compact structure. This led to less expanded MSD curves for the E domain and A domain compared with nucleotide-absent conditions. This change may help to stabilize the functional conformation during substrate binding. This study is the first to use DXT to probe the real-time molecular dynamics of mammalian type II chaperonins at the millisecond level. Our findings provide new insights into the complex dynamics of chaperonins and their role in the functional folding cycle.

Serodominant SARS-CoV-2 Nucleocapsid Peptides Map to Unstructured Protein Regions.

The SARS-CoV-2 nucleocapsid (N) protein is highly immunogenic, and anti-N antibodies are commonly used as markers for prior infection. While several studies have examined or predicted the antigenic regions of N, these have lacked consensus and structural context. Using COVID-19 patient sera to probe an overlapping peptide array, we identified six public and four private epitope regions across N, some of which are unique to this study. We further report the first deposited X-ray structure of the stable dimerization domain at 2.05 Å as similar to all other reported structures. Structural mapping revealed that most epitopes are derived from surface-exposed loops on the stable domains or from the unstructured linker regions. An antibody response to an epitope in the stable RNA binding domain was found more frequently in sera from patients requiring intensive care. Since emerging amino acid variations in N map to immunogenic peptides, N protein variation could impact detection of seroconversion for variants of concern. IMPORTANCE As SARS-CoV-2 continues to evolve, a structural and genetic understanding of key viral epitopes will be essential to the development of next-generation diagnostics and vaccines. This study uses structural biology and epitope mapping to define the antigenic regions of the viral nucleocapsid protein in sera from a cohort of COVID-19 patients with diverse clinical outcomes. These results are interpreted in the context of prior structural and epitope mapping studies as well as in the context of emergent viral variants. This report serves as a resource for synthesizing the current state of the field toward improving strategies for future diagnostic and therapeutic design.

Structural and functional analysis of the cerato-platanin-like protein Cpl1 suggests diverging functions in smut fungi.

Plant-pathogenic fungi are causative agents of the majority of plant diseases and can lead to severe crop loss in infected populations. Fungal colonization is achieved by combining different strategies, such as avoiding and counteracting the plant immune system and manipulating the host metabolome. Of major importance are virulence factors secreted by fungi, which fulfil diverse functions to support the infection process. Most of these proteins are highly specialized, with structural and biochemical information often absent. Here, we present the atomic structures of the cerato-platanin-like protein Cpl1 from Ustilago maydis and its homologue Uvi2 from Ustilago hordei. Both proteins adopt a double-Ψβ-barrel architecture reminiscent of cerato-platanin proteins, a class so far not described in smut fungi. Our structure-function analysis shows that Cpl1 binds to soluble chitin fragments via two extended grooves at the dimer interface of the two monomer molecules. This carbohydrate-binding mode has not been observed previously and expands the repertoire of chitin-binding proteins. Cpl1 localizes to the cell wall of U. maydis and might synergize with cell wall-degrading and decorating proteins during maize infection. The architecture of Cpl1 harbouring four surface-exposed loop regions supports the idea that it might play a role in the spatial coordination of these proteins. While deletion of cpl1 has only mild effects on the virulence of U. maydis, a recent study showed that deletion of uvi2 strongly impairs U. hordei virulence. Our structural comparison between Cpl1 and Uvi2 reveals sequence variations in the loop regions that might explain a diverging function.

DNA accelerates the protease inhibition of a bacterial serpin chloropin.

Serine protease inhibitors (Serpins) are the most widely distributed protease inhibitors in nature and have been identified from all kingdoms of life. Eukaryotic serpins are most abundant with their activities often subject to modulation by cofactors; however, little is known about the regulation of prokaryotic serpins. To address this, here we prepared a recombinant bacteria serpin, termed chloropin, derived from green sulfur bacteria Chlorobium limicola and solved its crystal structure at 2.2Å resolution. This showed a canonical inhibitory serpin conformation of native chloropin with a surface-exposed reactive loop and a large central beta-sheet. Enzyme activity analysis showed that chloropin could inhibit multiple proteases, such as thrombin and KLK7 with second order inhibition rate constants at 2.5×104M-1s-1 and 4.5×104M-1s-1 respectively, consistent with its P1 arginine residue. Heparin could accelerate the thrombin inhibition by ∼17-fold with a bell-shaped dose-dependent curve as seen with heparin-mediated thrombin inhibition by antithrombin. Interestingly, supercoiled DNA could accelerate the inhibition of thrombin by chloropin by 74-fold, while linear DNA accelerated the reaction by 142-fold through a heparin-like template mechanism. In contrast, DNA did not affect the inhibition of thrombin by antithrombin. These results indicate that DNA is likely a natural modulator of chloropin protecting the cell from endogenous or exogenous environmental proteases, and prokaryotic serpins have diverged during evolution to use different surface subsites for activity modulation.

Characterization of surface-exposed structural loops as insertion sites for foreign antigen delivery in calicivirus-derived VLP platform.

Chimeric virus-like particles (cVLPs) show great potential in improving public health as they are safe and effective vaccine candidates. The capsid protein of caliciviruses has been described previously as a self-assembling, highly immunogenic delivery platform. The ability to significantly induce cellular and humoral immunity can be used to boost the immune response to low immunogenic foreign antigens displayed on the surface of VLPs. Capsid proteins of caliciviruses despite sequence differences share similar architecture with structural loops that can be genetically modified to present foreign epitopes on the surface of cVLPs. Here, based on the VP1 protein of norovirus (NoV), we investigated the impact of the localization of the epitope in different structural loops of the P domain on the immunogenicity of the presented epitope. In this study, three distinct loops of NoV VP1 protein were genetically modified to present a multivalent influenza virus epitope consisting of a tandem repeat of M2/NP epitopes. cVLPs presenting influenza virus-conserved epitopes in different localizations were produced in the insect cells and used to immunize BALB/c mice. Specific reaction to influenza epitopes was compared in sera from vaccinated mice to determine whether the localization of the foreign epitope has an impact on the immunogenicity.

Reduction-cleavable desferrioxamine B pulldown system enriches Ni(ii)-superoxide dismutase from a Streptomyces proteome.

Two resins with the hydroxamic acid siderophore desferrioxamine B (DFOB) immobilised as a free ligand or its Fe(iii) complex were prepared to screen the Streptomyces pilosus proteome for proteins involved in siderophore-mediated Fe(iii) uptake. The resin design included a disulfide bond to enable the release of bound proteins under mild reducing conditions. Proteomics analysis of the bound fractions did not identify proteins associated with siderophore-mediated Fe(iii) uptake, but identified nickel superoxide dismutase (NiSOD), which was enriched on the apo-DFOB-resin but not the Fe(iii)-DFOB-resin or the control resin. While DFOB is unable to sequester Fe(iii) from sites deeply buried in metalloproteins, the coordinatively unsaturated Ni(ii) ion in NiSOD is present in a surface-exposed loop region at the N-terminus, which might enable partial chelation. The results were consistent with the notion that the apo-DFOB-resin formed a ternary complex with NiSOD, which was not possible for either the coordinatively saturated Fe(iii)-DFOB-resin or the non-coordinating control resin systems. In support, ESI-TOF-MS measurements from a solution of a model Ni(ii)-SOD peptide and DFOB showed signals that correlated with a ternary Ni(ii)-SOD peptide-DFOB complex. Although any biological implications of a DFOB-NiSOD complex are unclear, the work shows that the metal coordination properties of siderophores might influence an array of metal-dependent biological processes beyond those established in iron uptake.

Allelic Variants of P66 Gene in Borrelia bavariensis Isolates from Patients with Ixodid Tick-Borne Borreliosis.

Protein P66 is one of the crucial virulence factors of Borrelia, inducing the production of specific antibodies in patients with ixodid tick-borne borreliosis (ITBB). Various species of Borrelia are characterized by genetic variability of the surface-exposed loop of P66. However, little is known about this variability in Borrelia bavariensis. Here we describe the variability of the nucleotide sequences of P66 gene locus in isolates of B. bavariensis. Analysis of nucleotide sequences of P66 in 27 isolates of B. bavariensis from ITBB patients revealed three allelic variants of this gene. The alignment score of amino acid sequences in the isolates showed amino acid replacements in various positions confirming the presence of three allelic variants. Two of them are characteristic only for some isolates of B. bavariensis of the Eurasian gene pool from various parts of the geographic ranges of B. bavariensis from various samples. At least three allelic variants of P66 B. bavariensis have been identified, which have different amino acid expression, occur with different frequency in ITBB patients and, presumably, can have different effects on the course of the infection.

Cry4Aa and Cry4Ba Mosquito-Active Toxins Utilize Different Domains in Binding to a Particular Culex ALP Isoform: A Functional Toxin Receptor Implicating Differential Actions on Target Larvae.

The three-domain Cry4Aa toxin produced from Bacillus thuringiensis subsp. israelensis was previously shown to be much more toxic to Culex mosquito larvae than its closely related toxin—Cry4Ba. The interaction of these two individual toxins with target receptors on susceptible larval midgut cells is likely to be the critical determinant in their differential toxicity. Here, two full-length membrane-bound alkaline phosphatase (mALP) isoforms from Culex quinquefasciatus larvae, Cq-mALP1263and Cq-mALP1264, predicted to be GPI-linked was cloned and functionally expressed in Spodoptera frugiperda (Sf9) cells as 57- and 61-kDa membrane-bound proteins, respectively. Bioinformatics analysis disclosed that both Cq-mALP isoforms share significant sequence similarity to Aedes aegypti-mALP—a Cry4Ba toxin receptor. In cytotoxicity assays, Sf9 cells expressing Cq-mALP1264, but not Cq-mALP1263, showed remarkably greater susceptibility to Cry4Aa than Cry4Ba, while immunolocalization studies revealed that both toxins were capable of binding to each Cq-mALP expressed on the cell membrane surface. Molecular docking of the Cq-mALP1264-modeled structure with individual Cry4 toxins revealed that Cry4Aa could bind to Cq-mALP1264 primarily through particular residues on three surface-exposed loops in the receptor-binding domain—DII, including Thr512, Tyr513 and Lys514 in the β10-β11loop. Dissimilarly, Cry4Ba appeared to utilize only certain residues in its C-terminal domain—DIII to interact with such a Culex counterpart receptor. Ala-substitutions of selected β10-β11loop residues (T512A, Y513A and K514A) revealed that only the K514A mutant displayed a drastic decrease in biotoxicity against C. quinquefasciatus larvae. Further substitution of Lys514 with Asp (K514D) revealed a further decrease in larval toxicity. Furthermore, in silico calculation of the binding affinity change (ΔΔGbind) in Cry4Aa-Cq-mALP1264 interactions upon these single-substitutions revealed that the K514D mutation displayed the largest ΔΔGbind value as compared to three other mutations, signifying an adverse impact of a negative charge at this critical receptor-binding position. Altogether, our present study has disclosed that these two related-Cry4 mosquito-active toxins conceivably exploited different domains in functional binding to the same Culex membrane-bound ALP isoform—Cq-mALP1264 for mediating differential toxicity against Culex target larvae.

Modular capsid decoration boosts adenovirus vaccine-induced humoral immunity against SARS-CoV-2

Adenovirus vector vaccines have been widely and successfully deployed in response to coronavirus disease 2019 (COVID-19). However, despite inducing potent T cell immunity, improvement of vaccine-specific antibody responses upon homologous boosting is modest compared with other technologies. Here, we describe a system enabling modular decoration of adenovirus capsid surfaces with antigens and demonstrate potent induction of humoral immunity against these displayed antigens. Ligand attachment via a covalent bond was achieved using a protein superglue, DogTag/DogCatcher (similar to SpyTag/SpyCatcher), in a rapid and spontaneous reaction requiring only co-incubation of ligand and vector components. DogTag was inserted into surface-exposed loops in the adenovirus hexon protein to allow attachment of DogCatcher-fused ligands on virus particles. Efficient coverage of the capsid surface was achieved using various ligands, with vector infectivity retained in each case. Capsid decoration shielded particles from vector neutralizing antibodies. In prime-boost regimens, adenovirus vectors decorated with the receptor-binding domain of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike induced >10-fold higher SARS-CoV-2 neutralization titers compared with an undecorated vector encoding spike. Importantly, decorated vectors achieved equivalent or superior T cell immunogenicity against encoded antigens compared with undecorated vectors. We propose capsid decoration using protein superglues as a novel strategy to improve efficacy and boostability of adenovirus-based vaccines and therapeutics.

Epitope-Based Vaccines against the Chlamydia trachomatis Major Outer Membrane Protein Variable Domain 4 Elicit Protection in Mice

Chlamydia trachomatis (Ct) is the most common bacterial sexual transmitted pathogen, yet a vaccine is not currently available. Here, we used the immunogenic bacteriophage MS2 virus-like particle (VLP) technology to engineer vaccines against the Ct major outer membrane protein variable domain 4 (MOMP-VD4), which contains a conserved neutralizing epitope (TTLNPTIAG). A previously described monoclonal antibody to the MOMP-VD4 (E4 mAb) is capable of neutralizing all urogenital Ct serovars and binds this core epitope, as well as several non-contiguous amino acids. This suggests that this core epitope may require conformational context in order to elicit neutralizing antibodies to Ct. In order to identify immunogens that could elicit neutralizing antibodies to the TTLNPTIAG epitope, we used two approaches. First, we used affinity selection with a bacteriophage MS2-VLP library displaying random peptides in a constrained, surface-exposed loop to identify potential E4 mAb mimotopes. After four rounds of affinity selection, we identified a VLP-displayed peptide (HMVGSTKWTN) that could bind to the E4 mAb and elicited serum IgG that bound weakly to Ct elementary bodies by ELISA. Second, two versions of the core conserved TTLNPTIAG epitope (TTLNPTIAG and TTLNPTIAGA) were recombinantly expressed on the coat protein of the MS2 VLP in a constrained, surface-exposed loop. Mouse immune sera IgG bound to Ct elementary bodies by ELISA. Immunization with these MS2 VLPs provided protection from vaginal Chlamydia infection in a murine challenge model. These data suggest that short peptide epitopes targeting the MOMP-VD4 could be appropriate for Ct vaccine design when displayed on an immunogenic bacteriophage VLP vaccine platform.

Cypin binds to fully polymerized microtubules and tubulin heterodimers via distinct domains

Proper branching of neuronal dendrites is crucial for healthy brain function. We previously reported that cypin, a guanine deaminase, binds to tubulin heterodimers via its collapsin response mediator protein (CRMP) homology domain (amino acids 350‐403), promoting microtubule assembly in a cell‐free system. This increased microtubule assembly results in increased dendrite number and branching. Here, we ask how cypin alters microtubules in neurons. We found that overexpression of cypin increases the number of and decreases the spacing between microtubules. We also observed that overexpression of cypin increases microtubule polymerization as evidenced by increased movement of end‐binding protein 3 (EB3) comets. To determine whether cypin binds polymerized microtubules in addition to tubulin heterodimers, we performed a series of biochemical and computational experiments. We found that cypin binds to fully formed microtubules but does not prefer microtubule ends or shafts. In addition, preliminary protein‐protein docking strategies suggest that cypin binds to microtubules through several unreported residues near the N‐terminal end of cypin, which form a surface‐exposed loop with several bulky residues. We used structure‐based approaches through docking analyses of “straight” polymerized microtubules and of “curved” soluble tubulin heterodimers and found that cypin binds free heterodimers though its CRMP homology domain along with a small amino acid region near the N‐terminus (residues 43‐63). Molecular dynamics data also suggest that the region of residues 43‐63 is highly flexible and may bind protein partners. Our results suggest that cypin regulates the microtubule cytoskeleton by promoting assembly and stabilizing microtubules.

Contribution of Surface-Exposed Loops on the HPV16 Capsid to Antigenic Domains Recognized by Vaccine or Natural Infection Induced Neutralizing Antibodies.

ABSTRACTHuman papillomavirus (HPV) is the causative agent of cervical and other cancers and represents a significant global health burden. HPV vaccines demonstrate excellent efficacy in clinical trials and effectiveness in national immunization programmes against the most prevalent genotype, HPV16. It is unclear whether the greater protection conferred by vaccine-induced antibodies, compared to natural infection antibodies, is due to differences in antibody magnitude and/or specificity. We explore the contribution of the surface-exposed loops of the major capsid protein to antigenic domains recognized by vaccine and natural infection neutralizing antibodies. Chimeric pseudoviruses incorporating individual (BC, DE, EF, FG, HI) or combined (All: BC/DE/EF/FG/HI) loop swaps between the target (HPV16) and control (HPV35) genotypes were generated, purified by ultracentrifugation and characterized by SDS-PAGE and electron microscopy. Neutralizing antibody data were subjected to hierarchical clustering and outcomes modeled on the HPV16 capsomer crystal model. Vaccine antibodies exhibited an FG loop preference followed by the EF and HI loops while natural infection antibodies displayed a more diverse pattern, most frequently against the EF loop followed by BC and FG. Both vaccine and natural infection antibodies demonstrated a clear requirement for multiple loops. Crystal modeling of these neutralizing antibody patterns suggested natural infection antibodies typically target the outer rim of the capsomer while vaccine antibodies target the central ring around the capsomer lumen. Chimeric pseudoviruses are useful tools for probing vaccine and natural infection antibody specificity. These data add to the evidence base for the effectiveness of an important public health intervention.IMPORTANCE The human papillomavirus type 16 (HPV16) major virus coat (capsid) protein is a target for antibodies induced by both natural infection and vaccination. Vaccine-induced immunity is highly protective against HPV16-related infection and disease while natural infection associated immunity significantly less so. For this study, we created chimeric functional pseudoviruses based upon an antigenically distant HPV genotype (HPV35) resistant to HPV16-specific antibodies with inserted capsid surface fragments (external loops) from HPV16. By using these chimeric pseudoviruses in functional neutralization assays we were able to highlight specific and distinct areas on the capsid surface recognized by both natural infection and vaccine induced antibodies. These data improve our understanding of the difference between natural infection and vaccine induced HPV16-specific immunity.

Multiomics analysis of rheumatoid arthritis yields sequence variants that have large effects on risk of the seropositive subset

ObjectivesTo find causal genes for rheumatoid arthritis (RA) and its seropositive (RF and/or ACPA positive) and seronegative subsets.MethodsWe performed a genome-wide association study (GWAS) of 31 313 RA cases (68%...

B-Cell Epitope Mapping of TprC and TprD Variants of Treponema pallidum Subspecies Informs Vaccine Development for Human Treponematoses.

Several recent studies have focused on the identification, functional analysis, and structural characterization of outer membrane proteins (OMPs) of Treponema pallidum (Tp). The Tp species encompasses the highly related pallidum, pertenue, and endemicum subspecies of this pathogen, known to be the causative agents of syphilis, yaws, and bejel, respectively. These studies highlighted the importance of identifying surface-exposed OMP regions and the identification of B-cell epitopes that could be protective and used in vaccine development efforts. We previously reported that the TprC and TprD OMPs of Tp are predicted to contain external loops scattered throughout the entire length of the proteins, several of which show a low degree of sequence variability among strains and subspecies. In this study, these models were corroborated using AlphaFold2, a state-of-the-art protein structure modeling software. Here, we identified B-cell epitopes across the full-length TprC and TprD variants using the Geysan pepscan mapping approach with antisera from rabbits infected with syphilis, yaws, and bejel strains and from animals immunized with refolded recombinant TprC proteins from three syphilis strains. Our results show that the humoral response is primarily directed to sequences predicted to be on surface-exposed loops of TprC and TprD proteins, and that the magnitude of the humoral response to individual epitopes differs among animals infected with various syphilis strains and Tp subspecies. Rather than exhibiting strain-specificity, antisera showed various degrees of cross-reactivity with variant sequences from other strains. The data support the further exploration of TprC and TprD as vaccine candidates.

A systematic approach for evaluating the role of surface-exposed loops in trypsin-like serine proteases applied to the 170 loop in coagulation factor VIIa

Proteases play a major role in many vital physiological processes. Trypsin-like serine proteases (TLPs), in particular, are paramount in proteolytic cascade systems such as blood coagulation and complement activation. The structural topology of TLPs is highly conserved, with the trypsin fold comprising two β-barrels connected by a number of variable surface-exposed loops that provide a surprising capacity for functional diversity and substrate specificity. To expand our understanding of the roles these loops play in substrate and co-factor interactions, we employ a systematic methodology akin to the natural truncations and insertions observed through evolution of TLPs. The approach explores a larger deletion space than classical random or directed mutagenesis. Using FVIIa as a model system, deletions of 1–7 amino acids through the surface exposed 170 loop, a vital allosteric regulator, was introduced. All variants were extensively evaluated by established functional assays and computational loop modelling with Rosetta. The approach revealed detailed structural and functional insights recapitulation and expanding on the main findings in relation to 170 loop functions elucidated over several decades using more cumbersome crystallization and single deletion/mutation methodologies. The larger deletion space was key in capturing the most active variant, which unexpectedly had a six-amino acid truncation. This variant would have remained undiscovered if only 2–3 deletions were considered, supporting the usefulness of the methodology in general protease engineering approaches. Our findings shed further light on the complex role that surface-exposed loops play in TLP function and supports the important role of loop length in the regulation and fine-tunning of enzymatic function throughout evolution.

Conserved Molecular Signatures in the Spike, Nucleocapsid, and Polymerase Proteins Specific for the Genus Betacoronavirus and Its Different Subgenera.

The genus Betacoronavirus, consisting of four main subgenera (Embecovirus, Merbecovirus, Nobecovirus, and Sarbecovirus), encompasses all clinically significant coronaviruses (CoVs), including SARS, MERS, and the SARS-CoV-2 virus responsible for current COVID-19 pandemic. Very few molecular characteristics are known that are specific for the genus Betacoronavirus or its different subgenera. In this study, our analyses of the sequences of four essential proteins of CoVs, viz., spike, nucleocapsid, envelope, and RNA-dependent RNA polymerase (RdRp), identified ten novel molecular signatures consisting of conserved signature indels (CSIs) in these proteins which are specific for the genus Betacoronavirus or its subgenera. Of these CSIs, two 14-aa-conserved deletions found within the heptad repeat motifs 1 and 2 of the spike protein are specific for all betacoronaviruses, except for their shared presence in the highly infectious avian coronavirus. Six additional CSIs present in the nucleocapsid protein and one CSI in the RdRp protein are distinctive characteristics of either the Merbecovirus, Nobecovirus, or Sarbecovirus subgenera. In addition, a 4-aa insert is present in the spike protein, which is uniquely shared by all viruses from the subgenera Merbecovirus, Nobecovirus, and Sarbecovirus, but absent in Embecovirus and all other genera of CoVs. This molecular signature provides evidence that viruses from the three subgenera sharing this CSI are more closely related to each other, and they evolved after the divergence of embecoviruses and other CoVs. As all CSIs specific for different groups of CoVs are flanked by conserved regions, their sequences provide novel means for identifying the above groups of CoVs and for developing novel diagnostic tests. Furthermore, our analyses of the structures of the spike and nucleocapsid proteins show that all identified CSIs are localized in the surface-exposed loops of these protein. It is postulated that these surface loops, through their interactions with other cellular proteins/ligands, play important roles in the biology/pathology of these viruses.