Determinants Of Specificity Research Articles

Distinct substrate specificities of the three catalytic subunits of the Trichomonas vaginalis proteasome.

The protozoan parasite Trichomonas vaginalis (Tv) causes trichomoniasis, the most common non-viral sexually transmitted infection in the world. Although Tv has been linked to significant health complications, only two closely related 5-nitroimidazole drugs are approved for its treatment. The emergence of resistance to these drugs and lack of alternative treatment options poses an increasing threat to public health, making development of novel anti-Trichomonas compounds an urgent need. The proteasome, a critical enzyme complex found in all eukaryotes has three catalytic subunits, β1, β2, and β5 and has been validated as a drug target to treat trichomoniasis. With the goal of developing tools to study the Tv proteasome, we isolated the enzyme complex and identified inhibitors that preferentially inactivate either one or two of the three catalytic subunits. Using a mass spectrometry-based peptide digestion assay, these inhibitors were used to define the substrate preferences of the β1, β2 and β5 subunits. Subsequently, three model fluorogenic substrates were designed, each specific for one of the catalytic subunits. This novel substrate profiling methodology will allow for individual subunit characterization of other proteasomes of interest. Using the new substrates, we screened a library of 284 peptide epoxyketone inhibitors against Tv and determined the subunits targeted by the most active compounds. The data show that inhibition of the Tv β5 subunit alone is toxic to the parasite. Taken together, the optimized proteasome subunit substrates will be instrumental for understanding the molecular determinants of proteasome specificity and for accelerating drug development against trichomoniasis.

Unraveling the Molecular Determinants of Catalytic Efficiency and Substrate Specificity in l-Amino Acid Decarboxylases.

l-Tryptophan decarboxylase (TDC) and l-3,4-dihydroxyphenylalanine decarboxylase (DDC) catalyze the decarboxylation of l-tryptophan, 5-hydroxytryptophan, and l-3,4-dihydroxyphenylalanine. In this study, we analyzed the amino acid compositions of the substrate-binding pockets of TDC from Catharanthus roseus (CrTDC) and DDC from Sus scrofa (SsDDC), explored the specificity of key amino acids within these pockets, and elucidated mechanisms influencing substrate selectivity and catalytic activity in both enzymes, using whole-cell catalysis to screen mutants and determine enzymatic kinetic parameters. The results demonstrated that residues Ala-103 and Val-122 in CrTDC, along with their corresponding sites Thr-82 and Ile-101 in SsDDC, significantly influence substrate selectivity and catalytic efficiency. Molecular dynamics simulations revealed that substrate selectivity and catalytic efficiency depends on the nucleophilic attack distance between the substrate's amino group and the C4' of pyridoxal 5'-phosphate. This study elucidates the catalytic mechanisms and structural bases of TDC and DDC, guiding enhancements in the related aromatic monoamine biosynthesis.

Toxic small alarmone synthetase FaRel2 inhibits translation by pyrophosphorylating tRNAGly and tRNAThr.

Translation-targeting toxic small alarmone synthetases (toxSAS) are effectors of bacterial toxin-antitoxin systems that pyrophosphorylate the 3'-CCA end of transfer RNA (tRNA) to prevent aminoacylation. toxSAS are implicated in antiphage immunity: Phage detection triggers the toxSAS activity to shut down viral production. We show that the toxSAS FaRel2 inspects the tRNA acceptor stem to specifically select tRNAGly and tRNAThr. The first, second, fourth, and fifth base pairs of the stem act as the specificity determinants. We show that the toxSASs PhRel2 and CapRelSJ46 differ in tRNA specificity from FaRel2 and rationalize this through structural modeling: While the universal 3'-CCA end slots into a highly conserved CCA recognition groove, the acceptor stem recognition region is variable across toxSAS diversity. As phages use tRNA isoacceptors to overcome tRNA-targeting defenses, we hypothesize that highly evolvable modular tRNA recognition allows for the escape of viral countermeasures through tRNA substrate specificity switching.

The Trajectory of Damaged-Base Eversion into the Active Site of Apurinic/Apyrimidinic Endonuclease APE1 Regulates This Enzyme's Substrate Specificity.

Apurinic/apyrimidinic endonuclease 1 (APE1) is responsible for the hydrolysis of the phosphodiester bond on the 5' side of an apurinic/apyrimidinic site during base excision repair. Moreover, in DNA, this enzyme can recognize nucleotides containing such damaged bases as 5,6-dihydro-2'-deoxyuridine (DHU), 2'-deoxyuridine (dU), alpha-2'-deoxyadenosine (αA), and 1,N6-ethenoadenosine (εA). Previously, by pulsed electron-electron double resonance spectroscopy and pre-steady-state kinetic analysis, we have revealed multistep DNA rearrangements during the formation of the catalytic complex. In the present study, the modeling of the eversion trajectory of nucleotides with various damaged bases was performed by directed molecular dynamics simulations. It was found that each damaged base at the beginning of the eversion interacts with protein loop Val196-Arg201, which should be moved to enable further nucleotide eversion. This movement involves a shift in loop Val196-Arg201 away from loop Asn253-Thr257 and requires the disruption of contacts between these loops. The Glu260Ala substitution facilitates the separation of the two loops. Moreover, conformational changes in the Asn253-Thr257 loop should occur in the second half of the lesion eversion trajectory. All these perturbations within the protein globule tend to reduce steric interactions of each damaged base with the protein during the eversion of the nucleotide from DNA and movement to the active site. These perturbations are important determinants of substrate specificity of endonuclease APE1.

Functional and structural analyses of IMP-27 metallo-β-lactamase: evolution of IMP-type enzymes to overcome Zn(II) deprivation.

IMP-type metallo-β-lactamases are di-Zn(II) enzymes that can inactivate a wide range of bicyclic β-lactam agents used in clinical practice. IMP-27 shares 82% amino acid sequence identity with IMP-1, the first IMP-type enzyme identified. Herein, we conducted structural determination, kinetic, and chelating agent resistance analyses of IMP-27. Once determined, IMP-27 was then compared to its mutant, namely, G262S, and IMP-1. Crystallographic structural analysis of IMP-27 showed an overall structure comparable to that of IMP-1 and other IMP-type enzymes; the positions of the zinc (Zn) ions varied across enzymes. Kinetic analysis showed that IMP-27 had lower catalytic efficiency against penicillins, ceftazidime, cephalexin, and imipenem than IMP-1; however, it had higher affinity and catalytic efficiency against meropenem, especially in the presence of Zn(II). This suggests that the catalytic site of IMP-27 is optimized to hydrolyze meropenem during molecular evolution at the expense of catalytic efficiency against penicillins. However, Zn(II) content analysis after EDTA treatment revealed no significant difference between enzymes. Moreover, analysis of IMP-27 mutants indicated that the differences in kinetic properties and chelator resistance between IMP-1 and IMP-27 were mainly due to an amino acid substitution at position 262.IMPORTANCEThe residue at position 262 has been reported as a key determinant of substrate specificity in IMP-type enzymes. Among more than 80 IMP-type metallo-β-lactamase (MBL) variants, IMP-27 was the first reported IMP-type MBL isolated from Proteus mirabilis. This enzyme has a glycine residue at position 262, which is occupied by serine in IMP-1. Compared with IMP-1, IMP-27 had a significantly higher affinity and catalytic efficiency against meropenem and improved metal-binding capacity, maintaining its activity under Zn(II)-limited conditions better than IMP-1. The analysis of the IMP-27 mutants indicated that differences between IMP-27 and IMP-1 were mainly due to an amino acid substitution at position 262. In the case of IMP-27, the G262S mutation optimized the catalytic site of IMP-27 for meropenem hydrolysis, at the expense of catalytic efficiency against penicillins.

Crystal Structure and Mutagenesis of an XYP Subfamily Cyclodipeptide Synthase Reveal Key Determinants of Enzyme Activity and Substrate Specificity.

Cyclodipeptide synthases (CDPSs) catalyze the synthesis of diverse cyclodipeptides (CDPs) by utilizing two aminoacyl-tRNA (aa-tRNA) substrates in a sequential ping-pong reaction mechanism. Numerous CDPSs have been characterized to provide precursors for diketopiperazines (DKPs) with diverse structural characteristics and biological activities. BcmA, belonging to the XYP subfamily, is a cyclo(l-Ile-l-Leu)-synthesizing CDPS involved in the biosynthesis of the antibiotic bicyclomycin. The structural basis and determinants influencing BcmA enzyme activity and substrate selectivity are not well understood. Here, we report the crystal structure of SsBcmA from Streptomyces sapporonensis. Through structural comparison and systematic site-directed mutagenesis, we highlight the significance of key residues located in the aminoacyl-binding pocket for enzyme activity and substrate specificity. In particular, the nonconserved residues D161 and K165 in pocket P2 are essential for the activity of SsBcmA without significant alteration of the substrate specificity, while the conserved residues F158 as well as F210 and S211 in P2 are responsible for determining substrate selectivity. These findings facilitate the understanding of how CDPSs selectively accept hydrophobic substrates and provide additional clues for the engineering of these enzymes for synthetic biology applications.

Isolation, characterization, and receptor-binding protein specificity of phages PAS7, PAS59 and PAS61 infecting Shiga toxin-producing Escherichia coli O103 and O146

Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen with 6,534 annual reported cases in the EU in 2021. This pathotype generally contains strains with smooth LPS with O-antigen serogroup O157 being the predominant serogroup in the US. However, non-O157 STEC serogroups are becoming increasingly prevalent. Here we announce the complete genomes of three newly isolated phages that infect STEC serogroups O103 and O146, namely Escherichia phages vB_EcoP_PAS7, vB_EcoP_PAS59 and vB_EcoP_PAS61. The genome sequences revealed that they belong to three distinct genera, namely the newly proposed genus Cepavirus within the Slopekvirinae subfamily, the genus Suseptimavirus and the genus Uetakevirus, respectively. We identified the tailspikes of phages PAS7 and PAS61 as a primary specificity determinant for the O-antigens O103 and O146, respectively, and predicted their active site in silico.

Specific anti-toxic immunotherapy: use in medical practice and perspectives

The results of use of approaches and means of antitoxic immunotherapy in medical practice are summarized: passive transfer of various variants of antibodies specific to toxic compounds, vaccination – specific active immunization with vaccines carrying determinants of the immunochemical specificity of target toxic compounds. The practical effectiveness of the known approaches of passive transfer and vaccination is associated with the ability of specific antibodies, by binding target bioactive compounds with pronounced toxicity, to change the availability of corresponding structures in the body-targets and, in the presence of a sufficient number of specific antibodies with high binding ability to the target compounds, neutralize their toxicity. In medical practice, polyspecific heterologous antisera or the gamma globulin fraction of antisera (extremely rare monoclonal antibodies of narrow specificity) are widely used for passive transfer as antidotes in the treatment of victims in order to prevent deaths and extensive necrosis of soft tissues at the site of the bite of poisonous snakes and insects. Active immunization – vaccination with appropriate antigenic drugs should create in immunized individuals a state of humoral immunity with the corresponding characteristics of antibody formation specific to the target compound. When a target toxic compound enters an immunized organism, it is also possible to neutralize its toxicity. The most successful experience in using the principles of active immunization as a technology for specific antitoxic therapy is associated with the practice of using toxoids for toxinemic infections. Specific practical techniques used to achieve the effectiveness of possible approaches to specific antitoxic immunotherapy in the form of passive transfer of specific antibodies or their fragments are considered: to combat lethal infections in the pathogenesis of which the toxic effects of bacterial exotoxins are significant; when treating victims of snake and insect bites, exposure to poisons of marine organisms, algae and plant toxins; in the treatment of severe intoxication with certain low molecular weight toxic substances: digoxin, colchicine, tricyclic antidepressants. The most successful experiences of using the principles of active immunization as a technology for specific antitoxic therapy, based on the use of toxoids with specificity for diphtheria, tetanus, botulism, cholera, typhoid fever, dysentery, gas gangrene and other toxinemic infections, are also considered. The fairly high immunogenicity of toxoids with the possibility of activating both constitutional and adaptive immunity has become the basis for their use as macromolecular carriers of hapten analogues of narcotic substances – a promising direction in drug addiction, in the implementation of which a number of experimental molecular and combined vaccines of opiates, methamphetamine, cocaine, and nicotine. This variant of practical efforts in medicine can be regarded as a new direction of specific antitoxic immunotherapy – an option to combat drug addiction by vaccinating drug addicts.

The crystal structure of Nictaba reveals its carbohydrate-binding properties and a new lectin dimerization mode.

Nictaba is a (GlcNAc)n-binding, stress-inducible lectin from Nicotiana tabacum that serves as a representative for the Nictaba-related lectins, a group of proteins that play pivotal roles in plant defense mechanisms and stress response pathways. Despite extensive research into biological activities and physiological role(s) of the lectin, the three-dimensional structure of Nictaba remained largely unknown. Here, we report crystal structures for Nictaba in the apo form and bound to chitotriose. The structures reveal that the Nictaba protomer has a jelly-roll fold, similar to the cucumber lectin Cus17, but exhibit a unique and previously unseen mode of dimerization. The chitotriose binding mode, similar to Cus17, centers around the central GlcNAc residue, providing insights into the determinants of specificity of Nictaba towards carbohydrate structures. By integrating these structural insights with inputs from glycan arrays, molecular docking, and molecular dynamics simulations, we propose that Nictaba employs a single carbohydrate-recognition domain within each of the two subunits in the dimer to display pronounced specificity towards GlcNAc-containing carbohydrates. Furthermore, we identified amino acid residues involved in the extended binding site capable of accommodating structurally diverse high-mannose and complex N-glycans. Glycan array and in silico analyses revealed interactions centered around the conserved Man3GlcNAc2 core, explaining the broad recognition of N-glycan structures. Collectively, the structural and biochemical insights presented here fill a void into the atlas of lectin structure-function relationships and pave the way for future developments in plant stress biology and lectin-based applications.

Specificity determinants of pathogens in forest

Abstract Host‐specific pathogens have long been suggested to act as a major driver of species diversity in tropical forests. However, determining the degree of host specificity of potential pathogens coupled to key cellular characteristics of infection is difficult and time‐consuming. These challenges have delayed progress in relating the functional specificity of pathogenic fungi to ecological consequences. We tested the pathogenicity of 27 (of 215) fungi that were isolated from surface sterilized roots of seedlings from four common tree species in a diverse subtropical forest. Inoculation experiments showed that six fungi exhibited strong pathogenicity on the host seedlings. Five of these only infected their specific hosts (i.e. host‐specific pathogen species). Green fluorescent protein labelling revealed that in three host‐specific pathogens fungal hyphae were able to grow into the vascular tissues only in their specific host plant, in contrast to other fungal‐host combinations that exhibit non‐pathogenic interactions. This fluorescent labelling technique allows direct tracking of the progress of fungal infection in different host tissues. Synthesis. By coupling the green fluorescent protein technique with standard host inoculation experiments, we determined the developmental differences between infections by pathogenic and non‐pathogenic fungi in a relatively simple and straightforward way. Our work provides a useful tool for rapidly screening and categorizing host–fungal interactions, reflecting the functional basis of host specificity of pathogenic fungi. More broadly, this technique can contribute to understanding the roles of pathogens in species coexistence and biodiversity maintenance in forest communities.

Enzyme-Sialylation-Controlled Chemical Sulfation of Glycan Epitopes for Decoding the Binding of Siglec Ligands.

Widely distributed in nature, sulfated glycan epitopes play important roles in diverse pathophysiological processes. However, due to their structural complexity, the preparation of glycan epitopes with structurally defined sulfation patterns is challenging, which significantly hampers the detailed elucidation of their biological functions at the molecular level. Here, we introduce a strategy for site-specific chemical sulfation of glycan epitopes, leveraging enzymatic sialylation and desialylation processes to precisely control the regio-specificity of sulfation of disaccharide or trisaccharide glycan backbones. Using this method, a sulfated glycan library covering the most common sialylated glycan epitopes was prepared in high yield and efficiency. By screening a microarray prepared with this glycan library, we systematically probed their binding specificity with human Siglecs (sialic acid-binding immunoglobulin-type lectins), many of which function as glyco-immune checkpoints to suppress immune system activation. Our investigation revealed that sulfation and sialylation patterns serve as important determinants of Siglec binding affinity and specificity. Thus, these findings offer new insights for the development of research tools and potential therapeutic agents targeting glyco-immune checkpoints by modulating the Siglec signaling pathway.

Antibody sequence determinants of viral antigen specificity.

Throughout life, humans experience repeated exposure to viral antigens through infection and vaccination, resulting in the generation of diverse, antigen-specific antibody repertoires. A paramount feature of antibodies that enables their critical contributions in counteracting recurrent and novel pathogens, and consequently fostering their utility as valuable targets for therapeutic and vaccine development, is the exquisite specificity displayed against their target antigens. Yet, there is still limited understanding of the determinants of antibody-antigen specificity, particularly as a function of antibody sequence. In recent years, experimental characterization of antibody repertoires has led to novel insights into fundamental properties of antibody sequences but has been largely decoupled from at-scale antigen specificity analysis. Here, using the LIBRA-seq technology, we generated a large data set mapping antibody sequence to antigen specificity for thousands of B cells, by screening the repertoires of a set of healthy individuals against 20 viral antigens representing diverse pathogens of biomedical significance. Analysis uncovered virus-specific patterns in variable gene usage, gene pairing, somatic hypermutation, as well as the presence of convergent antiviral signatures across multiple individuals, including the presence of public antibody clonotypes. Notably, our results showed that, for B-cell receptors originating from different individuals but leveraging an identical combination of heavy and light chain variable genes, there is a specific CDRH3 identity threshold above which B cells appear to exclusively share the same antigen specificity. This finding provides a quantifiable measure of the relationship between antibody sequence and antigen specificity and further defines experimentally grounded criteria for defining public antibody clonality.IMPORTANCEThe B-cell compartment of the humoral immune system plays a critical role in the generation of antibodies upon new and repeated pathogen exposure. This study provides an unprecedented level of detail on the molecular characteristics of antibody repertoires that are specific to each of the different target pathogens studied here and provides empirical evidence in support of a 70% CDRH3 amino acid identity threshold in pairs of B cells encoded by identical IGHV:IGL(K)V genes, as a means of defining public clonality and therefore predicting B-cell antigen specificity in different individuals. This is of exceptional importance when leveraging public clonality as a method to annotate B-cell receptor data otherwise lacking antigen specificity information. Understanding the fundamental rules of antibody-antigen interactions can lead to transformative new approaches for the development of antibody therapeutics and vaccines against current and emerging viruses.

Heterozygous and generalist MxA super-restrictors overcome breadth-specificity tradeoffs in antiviral restriction.

Antiviral restriction factors such as MxA (myxovirus resistance protein A) inhibit a broad range of viruses. However, they face the challenge of maintaining this breadth as viruses evolve to escape their defense. Viral escape drives restriction factors to evolve rapidly, selecting for amino acid changes at their virus-binding interfaces to regain defense. How do restriction factors balance the breadth of antiviral functions against the need to evolve specificity against individual escaping viruses? We explored this question in human MxA, which uses its rapidly evolving loop L4 as the specificity determinant for orthomyxoviruses such as THOV and IAV. Previous combinatorial mutagenesis of rapidly evolving residues in human MxA loop L4 revealed variants with a ten-fold increase in potency against THOV. However, this strategy did not yield improved IAV restriction, suggesting a strong tradeoff between antiviral specificity and breadth. Here, using a modified combinatorial mutagenesis strategy, we find 'super-restrictor' MxA variants with over ten-fold enhanced restriction of the avian IAV strain H5N1 but reduced THOV restriction. Analysis of super-restrictor MxA variants reveals that the identity of residue 561 explains most of MxA's breadth-specificity tradeoff in H5N1 versus THOV restriction. However, rare 'generalist' super-restrictors with enhanced restriction of both viruses allow MxA to overcome the breadth-specificity tradeoff. Finally, we show that a heterozygous combination of two 'specialist' super-restrictors, one against THOV and the other against IAV, enhances restriction against both viruses. Thus, two strategies enable restriction factors such as MxA to increase their restriction of diverse viruses to overcome breadth-specificity tradeoffs that may be pervasive in host-virus conflicts.

Characterization of a cytochrome P450 that catalyzes the O-demethylation of lignin-derived benzoates

Cytochromes P450 (P450s) are a superfamily of heme-containing enzymes possessing a broad range of monooxygenase activities. One such activity is O-demethylation, an essential and rate-determining step in emerging strategies to valorize lignin that employ carbon-carbon bond cleavage. We recently identified PbdA, a P450 from Rhodococcus jostii RHA1, and PbdB, its cognate reductase, which catalyze the O-demethylation of para-methoxylated benzoates (p-MBAs) to initiate growth of RHA1 on these compounds. PbdA had the highest affinity (Kd = 3.8 ± 0.6 μM) and apparent specificity (kcat/KM = 20 000 ± 3 000 M-1 s-1) for p-MBA. The enzyme also O-demethylated two related lignin-derived aromatic compounds with remarkable efficiency: veratrate and isovanillate. PbdA also catalyzed the hydroxylation and dehydrogenation of p-EB even though RHA1 did not grow on this compound. Atomic-resolution structures of PbdA in complex with p-MBA, p-EB and veratrate revealed a cluster of three residues that form hydrogen bonds with the substrates’ carboxylate: Ser87, Ser237 and Arg84. Substitution of these residues resulted in lower affinity and O-demethylation activity on p-MBA as well as increased affinity for the acetyl analogue, p-methoxyacetophenone. The S87A and S237A variants of PbdA also catalyzed the O-demethylation of an aldehyde analogue of p-MBA, p-methoxy-benzaldehyde, while the R84M variant did not, despite binding this compound with high affinity. These results suggest that Ser87, Ser237 and Arg84 are not only important determinants of specificity but also help to orientate that substrate correctly in the active site. This study facilitates the design of biocatalysts for lignin valorization.

Cryo-EM structures of the Spo11 core complex bound to DNA.

DNA double-strand breaks that initiate meiotic recombination are formed by the topoisomerase-relative enzyme Spo11, supported by conserved auxiliary factors. Because high-resolution structural data have not been available, many questions remain about the architecture of Spo11 and its partners and how they engage with DNA. We report cryo-electron microscopy structures at up to 3.3-Å resolution of DNA-bound core complexes of Saccharomyces cerevisiae Spo11 with Rec102, Rec104 and Ski8. In these structures, monomeric core complexes make extensive contacts with the DNA backbone and with the recessed 3'-OH and first 5' overhanging nucleotide, establishing the molecular determinants of DNA end-binding specificity and providing insight into DNA cleavage preferences in vivo. The structures of individual subunits and their interfaces, supported by functional data in yeast, provide insight into the role of metal ions in DNA binding and uncover unexpected structural variation in homologs of the Top6BL component of the core complex.

Acceptor Subsite Mutants of Limosilactobacillus fermentum NCC 2970 GtfB 4,3-α-Glucanotransferase Regulate the Ratio of (α1 → 3)/(α1 → 6) Linkages in Biosynthesized α-Glucans.

Limosilactobacillus fermentum NCC 2970 GtfB (Lf2970 GtfB) is the only characterized 4,3-α-glucanotransferase (4,3-α-GTase) in the glycoside hydrolase (GH) 70 family belonging to the GtfB subfamily. However, the mechanism for its (α1 → 3) linkage formation remains unclear, and the structural determinants of its linkage specificity remain to be explored. Here, sequence alignment and structural comparison were conducted to identify key amino acids that may be critical for linkage specificity. Five residues of Lf2970 GtfB (D991, G1028, A1398, T1400, and E1405), located at donor and acceptor subsites, were selected for mutation. Product structure analysis revealed that D991 and G1028, located near the acceptor binding subsites, played crucial roles in linkage formation. Besides native (α1 → 4) and (α1 → 3) linkages, mutants G1028R and D991N showed 8 and 10% (α1 → 6) linkage increases compared to 1% for wild-type in products. Additionally, molecular docking studies demonstrated that the orientation of acceptor binding in G1028R and D991N mutants was favorable for (α1 → 6) linkage synthesis. However, the mutation at positions A1398, T1400, and E1405 indicated that the donor subsites contribute less to the linkage specificity. These results shed light on the structural determinants of linkage specificity of 4,3-α-GTase Lf2970 GtfB and provided insights into the structure-function relationship of family GH70.

Multi-method analysis revealed the mechanism of substrate selectivity in NHase: A gatekeeper residue at the activity center

Recognizing the critical need to elucidate the molecular determinants of this selectivity offers a pathway to engineer enzymes with broader and more versatile catalytic capabilities. Through integrated methods including phylogenetic analysis, molecular docking, and structural analysis, we identified a pivotal amino acid residue, αTrp116, linking the substrate binding pocket and the active site of a NHase from Pseudonocardia thermophila JCM 3095 (PtNHase). This residue acts as a crucial determinant of substrate specificity within the NHase enzyme. The mutant αW116R modified the substrate specificity of PtNHase, significantly enhancing its catalytic efficiency towards aromatic substrates. The catalytic activity for aromatic compounds such as 3-Cyanopyridine was 14-fold that of the wild-type, whereas its activity for aliphatic substrates diminished to one-sixth. MD simulations revealed that replacing αTrp116 with Arg allowed aromatic nitrile substrates to achieve more favorable conformations within the active site. Based on the mutant αW116R, we further constructed a combinatorial variant Pt-4, tailored for aromatic substrates, which exhibited an enzyme activity 50 times that of the wild-type. These results highlight the critical influence of amino acid residues in the enzyme's active site on substrate specificity and offer fresh perspectives and approaches for the evolution of enzymes.

Natural diversifying evolution of nonribosomal peptide synthetases in a defensive symbiont reveals nonmodular functional constraints.

The modular architecture of nonribosomal peptide synthetases (NRPSs) has inspired efforts to study their evolution and engineering. In this study, we analyze in detail a unique family of NRPSs from the defensive intracellular bacterial symbiont, Candidatus Endobryopsis kahalalidifaciens (Ca. E. kahalalidifaciens). We show that intensive and indiscriminate recombination events erase trivial sequence covariations induced by phylogenetic relatedness, revealing nonmodular functional constraints and clear recombination units. Moreover, we reveal unique substrate specificity determinants for multiple enzymatic domains, allowing us to accurately predict and experimentally discover the products of an orphan NRPS in Ca. E. kahalalidifaciens directly from environmental samples of its algal host. Finally, we expanded our analysis to 1,531 diverse NRPS pathways and revealed similar functional constraints to those observed in Ca. E. kahalalidifaciens' NRPSs. Our findings reveal the sequence bases of genetic exchange, functional constraints, and substrate specificity in Ca. E. kahalalidifaciens' NRPSs, and highlight them as a uniquely primed system for diversifying evolution.

A structural basis of T cell cross-reactivity to native and spliced self-antigens presented by HLA-DQ8

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that has a strong HLA association, where a number of self-epitopes have been implicated in disease pathogenesis. Human pancreatic islet-infiltrating CD4+ T cell clones not only respond to proinsulin C-peptide (PI40-54; GQVELGGGPGAGSLQ) but also cross-react with a hybrid insulin peptide (HIP; PI40-47-IAPP74-80; GQVELGGG-NAVEVLK) presented by HLA-DQ8. How T cell receptors recognise self-peptide and cross-react to HIPs is unclear. We investigated the cross-reactivity of the CD4+ T cell clones reactive to native PI40-54 epitope and multiple HIPs fused at the same N-terminus (PI40-54) to the degradation products of two highly expressed pancreatic islet proteins, Neuropeptide Y (NPY68-74) and amyloid polypeptide (IAPP23-29 and IAPP74-80). We observed that five out of the selected SKW3 T cell lines expressing TCRs isolated from CD4+ T cells of people with T1D responded to multiple HIPs. Despite shared TRAV26-1-TRBV5-1 gene usage in some T cells, these clones cross-reacted to varying degrees with the PI40-54 and HIP epitopes. Crystal structures of two TRAV26-1+-TRBV5-1+ T cell receptors (TCRs) in complex with PI40-54 and HIPs bound to HLA-DQ8 revealed that the two TCRs had distinct mechanisms responsible for their differential recognition of the PI40-54 and HIP epitopes. Alanine scanning mutagenesis of the PI40-54 and HIPs determined that the P2, P7 and P8 residues in these epitopes were key determinants of TCR specificity. Accordingly, we provide a molecular basis for cross-reactivity towards native insulin and HIP epitopes presented by HLA-DQ8.

Dissecting the neuroprotective interaction between the BH4 domain of BCL-w and the IP3 receptor

BCL-w is a BCL-2 family protein that promotes cell survival in tissue- and disease-specific contexts. The canonical anti-apoptotic functionality of BCL-w is mediated by a surface groove that traps the BCL-2 homology 3 (BH3) α-helices of pro-apoptotic members, blocking cell death. A distinct N-terminal portion of BCL-w, termed the BCL-2 homology 4 (BH4) domain, selectively protects axons from paclitaxel-induced degeneration by modulating IP3 receptors, a noncanonical BCL-2 family target. Given the potential of BCL-w BH4 mimetics to prevent or mitigate chemotherapy-induced peripheral neuropathy, we sought to characterize the interaction between BCL-w BH4 and the IP3 receptor, combining "staple" and alanine scanning approaches with molecular dynamics simulations. We generated and identified stapled BCL-w BH4 peptides with optimized IP3 receptor binding and neuroprotective activities. Point mutagenesis further revealed the sequence determinants for BCL-w BH4 specificity, providing a blueprint for therapeutic targeting of IP3 receptors to achieve neuroprotection.