RESUMO
As a novel alternative to established surface display or combinatorial chemistry approaches for the discovery of therapeutic peptides, we present a method for the isolation of small, cysteine-rich domains from bovine antibody ultralong complementarity-determining regions (CDRs). We show for the first time that isolated bovine antibody knob domains can function as autonomous entities by binding antigen outside the confines of the antibody scaffold. This yields antibody fragments so small as to be considered peptides, each stabilised by an intricate, bespoke arrangement of disulphide bonds. For drug discovery, cow immunisations harness the immune system to generate knob domains with affinities in the picomolar to low nanomolar range, orders of magnitude higher than unoptimized peptides from naïve library screening. Using this approach, knob domain peptides that tightly bound Complement component C5 were obtained, at scale, using conventional antibody discovery and peptide purification techniques.
Assuntos
Anticorpos/química , Dissulfetos/isolamento & purificação , Domínios de Imunoglobulina , Fragmentos de Peptídeos/isolamento & purificação , Domínios e Motivos de Interação entre Proteínas , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Afinidade de Anticorpos , Formação de Anticorpos , Especificidade de Anticorpos , Antígenos/genética , Antígenos/imunologia , Linfócitos B/fisiologia , Bovinos , Complemento C5/química , Complemento C5/genética , Complemento C5/imunologia , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Dissulfetos/química , Dissulfetos/imunologia , Mapeamento de Epitopos/métodos , Humanos , Imunização , Domínios de Imunoglobulina/genética , Modelos Moleculares , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Domínios e Motivos de Interação entre Proteínas/genéticaRESUMO
Thrombin activation of C5 connects thrombosis to inflammation. Complement research in whole blood ex vivo necessitates anticoagulation, which potentially interferes with the inflammatory modulation by thrombin. We challenged the concept of thrombin as an activator of native C5 by analyzing complement activation and C5 cleavage in human whole blood anticoagulated with Gly-Pro-Arg-Pro (GPRP), a peptide targeting fibrin polymerization downstream of thrombin, allowing complete endogenous thrombin generation. GPRP dose-dependently inhibited coagulation but allowed for platelet activation in accordance with thrombin generation. Spontaneous and bacterial-induced complement activation by Escherichia coli and Staphylococcus aureus, analyzed at the level of C3 and C5, were similar in blood anticoagulated with GPRP and the thrombin inhibitor lepirudin. In the GPRP model, endogenous thrombin, even at supra-physiologic concentrations, did not cleave native C5, despite efficiently cleaving commercially sourced purified C5 protein, both in buffer and when added to C5-deficient serum. In normal serum, only exogenously added, commercially sourced C5 was cleaved, whereas the native plasma C5 remained intact. Crucially, affinity-purified C5, eluted under mild conditions using an MgCl2 solution, was not cleaved by thrombin. Acidification of plasma to pH ≤ 6.8 by hydrochloric or lactic acid induced a C5 antigenic change, nonreversible by pH neutralization, that permitted cleavage by thrombin. Circular dichroism on purified C5 confirmed the structural change during acidification. Thus, we propose that pH-induced conformational change allows thrombin-mediated cleavage of C5 and that, contrary to previous reports, thrombin does not cleave plasma C5 in its native form, suggesting that thrombin cleavage of C5 may be restricted to certain pathophysiological conditions.
Assuntos
Complemento C5 , Trombina , Coagulação Sanguínea , Ativação do Complemento , Fibrina , HumanosRESUMO
Among the major challenges in the development of biopharmaceuticals are structural heterogeneity and aggregation. The development of a successful therapeutic monoclonal antibody (mAb) requires both a highly active and also stable molecule. Whilst a range of experimental (biophysical) approaches exist to track changes in stability of proteins, routine prediction of stability remains challenging. The fluorescence red edge excitation shift (REES) phenomenon is sensitive to a range of changes in protein structure. Based on recent work, we have found that quantifying the REES effect is extremely sensitive to changes in protein conformational state and dynamics. Given the extreme sensitivity, potentially this tool could provide a 'fingerprint' of the structure and stability of a protein. Such a tool would be useful in the discovery and development of biopharamceuticals and so we have explored our hypothesis with a panel of therapeutic mAbs. We demonstrate that the quantified REES data show remarkable sensitivity, being able to discern between structurally identical antibodies and showing sensitivity to unfolding and aggregation. The approach works across a broad concentration range (µg-mg/ml) and is highly consistent. We show that the approach can be applied alongside traditional characterisation testing within the context of a forced degradation study (FDS). Most importantly, we demonstrate the approach is able to predict the stability of mAbs both in the short (hours), medium (days) and long-term (months). The quantified REES data will find immediate use in the biopharmaceutical industry in quality assurance, formulation and development. The approach benefits from low technical complexity, is rapid and uses instrumentation which exists in most biochemistry laboratories without modification.
Assuntos
Anticorpos Monoclonais/química , Conformação Proteica , Estabilidade Proteica , Espectrometria de FluorescênciaRESUMO
Complement component C5 is the target of the mAb eculizumab and is the focus of a sustained drug discovery effort to prevent complement-induced inflammation in a range of autoimmune diseases. The immune evasion protein OmCI binds to and potently inactivates C5; this tight-binding interaction can be exploited to affinity-purify C5 protein from serum, offering a vastly simplified protocol compared with existing methods. However, breaking the high-affinity interaction requires conditions that risk denaturing or activating C5. We performed structure-guided in silico mutagenesis to identify prospective OmCI residues that contribute significantly to the binding affinity. We tested our predictions in vitro, using site-directed mutagenesis, and characterized mutants using a range of biophysical techniques, as well as functional assays. Our biophysical analyses suggest that the C5-OmCI interaction is complex with potential for multiple binding modes. We present single mutations that lower the affinity of OmCI for C5 and combinations of mutations that significantly decrease or entirely abrogate formation of the complex. The affinity-attenuated forms of OmCI are suitable for affinity purification and allow elution under mild conditions that are nondenaturing or activating to C5. We present the rational design, biophysical characterization, and experimental validation of affinity-reduced forms of OmCI as tool reagents to enable the affinity purification of C5.
Assuntos
Complemento C5/isolamento & purificação , Descoberta de Drogas , Animais , Sítios de Ligação , Desenho de Fármacos , Humanos , Evasão da Resposta Imune , Mutagênese Sítio-Dirigida , Ligação Proteica , Purificação por Afinidade em TandemRESUMO
Microbial virulence is a complex and often multifactorial phenotype, intricately linked to a pathogen's evolutionary trajectory. Toxicity, the ability to destroy host cell membranes, and adhesion, the ability to adhere to human tissues, are the major virulence factors of many bacterial pathogens, including Staphylococcus aureus. Here, we assayed the toxicity and adhesiveness of 90 MRSA (methicillin resistant S. aureus) isolates and found that while there was remarkably little variation in adhesion, toxicity varied by over an order of magnitude between isolates, suggesting different evolutionary selection pressures acting on these two traits. We performed a genome-wide association study (GWAS) and identified a large number of loci, as well as a putative network of epistatically interacting loci, that significantly associated with toxicity. Despite this apparent complexity in toxicity regulation, a predictive model based on a set of significant single nucleotide polymorphisms (SNPs) and insertion and deletions events (indels) showed a high degree of accuracy in predicting an isolate's toxicity solely from the genetic signature at these sites. Our results thus highlight the potential of using sequence data to determine clinically relevant parameters and have further implications for understanding the microbial virulence of this opportunistic pathogen.
Assuntos
Genoma Bacteriano , Staphylococcus aureus Resistente à Meticilina/genética , Modelos Genéticos , Virulência/genética , Estudo de Associação Genômica Ampla , Mutação INDEL , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Albumin, the major circulating protein in blood plasma, can be subjected to an increased level of glycation in a diabetic context. Albumin exerts crucial pharmacological activities through its drug binding capacity, i.e., ketoprofen, and via its esterase-like activity, allowing the conversion of prodrugs into active drugs. In this study, the impact of the glucose-mediated glycation on the pharmacological and biochemical properties of human albumin was investigated. Aggregation product levels and the redox state were quantified to assess the impact of glycation-mediated changes on the structural properties of albumin. Glucose-mediated changes in ketoprofen binding properties and esterase-like activity were evaluated using fluorescence spectroscopy and p-nitrophenyl acetate hydrolysis assays, respectively. With the exception of oxidative parameters, significant dose-dependent alterations in biochemical and functional properties of in vitro glycated albumin were observed. We also found that the dose-dependent increase in levels of glycation and protein aggregation and average molecular mass changes correlated with a gradual decrease in the affinity of albumin for ketoprofen and its esterase-like property. In parallel, significant alterations in both pharmacological properties were also evidenced in albumin purified from diabetic patients. Partial least-squares regression analyses established a significant correlation between glycation-mediated changes in biochemical and pharmacological properties of albumin, highlighting the important role for glycation in the variability of the drug response in a diabetic situation.
Assuntos
Albuminas/química , Albuminas/metabolismo , Cromatografia de Afinidade , Produtos Finais de Glicação Avançada , Glicosilação , Humanos , Cetoprofeno/química , Cetoprofeno/metabolismo , Espectrometria de Massas , Ligação Proteica , Estrutura Secundária de Proteína , Albumina Sérica/química , Albumina Sérica/metabolismo , Espectrometria de Fluorescência , Albumina Sérica GlicadaRESUMO
The ubiquitous bacterial pathogen, Staphylococcus aureus, expresses a large arsenal of virulence factors essential for pathogenesis. The phenol-soluble modulins (PSMs) are a family of cytolytic peptide toxins which have multiple roles in staphylococcal virulence. To gain an insight into which specific factors are important in PSM-mediated cell membrane disruption, the lytic activity of individual PSM peptides against phospholipid vesicles and T cells was investigated. Vesicles were most susceptible to lysis by the PSMα subclass of peptides (α1-3 in particular), when containing between 10 and 30mol% cholesterol, which for these vesicles is the mixed solid ordered (so)-liquid ordered (lo) phase. Our results show that the PSMß class of peptides has little effect on vesicles at concentrations comparable to that of the PSMα class and exhibited no cytotoxicity. Furthermore, within the PSMα class, differences emerged with PSMα4 showing decreased vesicle and cytotoxic activity in comparison to its counterparts, in contrast to previous studies. In order to understand this, peptides were studied using helical wheel projections and circular dichroism measurements. The degree of amphipathicity, alpha-helicity and properties such as charge and hydrophobicity were calculated, allowing a structure-function relationship to be inferred. The degree of alpha-helicity of the peptides was the single most important property of the seven peptides studied in predicting their lytic activity. These results help to redefine this class of peptide toxins and also highlight certain membrane parameters required for efficient lysis.
RESUMO
The four-component polypeptides of the 2-oxoacid dehydrogenase complex from the thermophilic archaeon Thermoplasma acidophilum assemble to give an active multienzyme complex possessing activity with the branched-chain 2-oxoacids derived from leucine, isoleucine and valine, and with pyruvate. The dihydrolipoyl acyl-transferase (E2) core of the complex is composed of identical trimer-forming units that assemble into a novel 42-mer structure comprising octahedral and icosahedral geometric aspects. From our previously determined structure of this catalytic core, the inter-trimer interactions involve a tyrosine residue near the C-terminus secured in a hydrophobic pocket of an adjacent trimer like a ball-and-socket joint. In the present study, we have deleted the five C-terminal amino acids of the E2 polypeptide (IIYEI) and shown by equilibrium centrifugation that it now only assembles into a trimeric enzyme. This was confirmed by SAXS analysis, although this technique showed the presence of approximately 20% hexamers. The crystal structure of the trimeric truncated E2 core has been determined and shown to be virtually identical with the ones observed in the 42-mer, demonstrating that removal of the C-terminal anchor does not significantly affect the individual monomer or trimer structures. The truncated E2 is still able to bind both 2-oxoacid decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) components to give an active complex with catalytic activity similar to the native multienzyme complex. This is the first report of an active mini-complex for this enzyme, and raises the question of why all 2-oxoacid dehydrogenase complexes assemble into such large structures.
Assuntos
Proteínas Arqueais/química , Complexos Multienzimáticos/química , Oxirredutases/química , Thermoplasma/enzimologia , Proteínas Arqueais/genética , Cristalografia por Raios X , Di-Hidrolipoamida Desidrogenase/química , Estabilidade Enzimática , Temperatura Alta , Cinética , Complexos Multienzimáticos/genética , Oxirredutases/genética , Conformação Proteica , Espalhamento a Baixo ÂnguloRESUMO
Boronic acids can interact with Lewis bases to generate boronate anions, and they can also bind with diol units to form cyclic boronate esters. Boronic acid based receptor designs originated when Lorand and Edwards used the pH drop observed upon the addition of saccharides to boronic acids to determine their association constants. The inherent acidity of the boronic acid is enhanced when 1,2-, 1,3-, or 1,4-diols react with boronic acids to form cyclic boronic esters (5, 6, or 7 membered rings) in aqueous media, and these interactions form the cornerstone of diol-based receptors used in the construction of sensors and separation systems. In addition, the recognition of saccharides through boronic acid complex (or boronic ester) formation often relies on an interaction between a Lewis acidic boronic acid and a Lewis base (proximal tertiary amine or anion). These properties of boronic acids have led to them being exploited in sensing and separation systems for anions (Lewis bases) and saccharides (diols). The fast and stable bond formation between boronic acids and diols to form boronate esters can serve as the basis for forming reversible molecular assemblies. In spite of the stability of the boronate esters' covalent B-O bonds, their formation is reversible under certain conditions or under the action of certain external stimuli. The reversibility of boronate ester formation and Lewis acid-base interactions has also resulted in the development and use of boronic acids within multicomponent systems. The dynamic covalent functionality of boronic acids with structure-directing potential has led researchers to develop a variety of self-organizing systems including macrocycles, cages, capsules, and polymers. This Account gives an overview of research published about boronic acids over the last 5 years. We hope that this Account will inspire others to continue the work on boronic acids and reversible covalent chemistry.
Assuntos
Ácidos Borônicos/química , Ésteres/química , Corantes Fluorescentes/química , Ciclização , Dopamina/química , Eletroforese , Estrutura Molecular , OxirreduçãoRESUMO
Background: Serum albumin binding is an established mechanism to extend the serum half-life of antibody fragments and peptides. The cysteine rich knob domains, isolated from bovine antibody ultralong CDRH3, are the smallest single chain antibody fragments described to date and versatile tools for protein engineering. Methods: Here, we used phage display of bovine immune material to derive knob domains against human and rodent serum albumins. These were used to engineer bispecific Fab fragments, by using the framework III loop as a site for knob domain insertion. Results: By this route, neutralisation of the canonical antigen (TNFα) was retained but extended pharmacokinetics in-vivo were achieved through albumin binding. Structural characterisation revealed correct folding of the knob domain and identified broadly common but non-cross-reactive epitopes. Additionally, we show that these albumin binding knob domains can be chemically synthesised to achieve dual IL-17A neutralisation and albumin binding in a single chemical entity. Conclusions: This study enables antibody and chemical engineering from bovine immune material, via an accessible discovery platform.
Assuntos
Anticorpos Biespecíficos , Albumina Sérica , Animais , Bovinos , Humanos , Albumina Sérica/metabolismo , Fragmentos Fab das Imunoglobulinas , Epitopos , Técnicas de Visualização da Superfície CelularRESUMO
Synthetic receptors for diols that incorporate boronic acid motifs have been developed as new sensors and separation tools. Utilizing the reversible interactions of diols with boronic acids to form boronic esters under new binding regimes has provided new hydrogel constructs that have found use as dye-displacement sensors and electrophoretic separation tools; similarly, molecular boronic-acid-containing chemosensors were constructed that offer applications in the sensing of diols. This review provides a somewhat-personal perspective of developments in boronic-acid-mediated sensing and separation, placed in the context of the seminal works of others in the area, as well as offering a concise summary of the contributions of the co-authors in the area.
RESUMO
We recently characterized an interaction between the Staphylococcus aureus immune evasion molecule Staphylococcus aureus binder of Ig (Sbi) and complement C3, an interaction mediated primarily through the binding of C3d(g) to Sbi domain IV. Events related to these studies prompted us to investigate via mutagenesis the binding interface of C3d for Sbi domain IV (Sbi-IV), as well as to revisit the controversial issue of the complement receptor 2 (CR2) binding site of C3d. Specifically, we had shown that Sbi domains III and IV fragment binding to C3dg inhibited the latter's binding to CR2. Moreover, a published cocrystal structure of C3d bound to complement inhibitory C-terminal domain of extracellular fibrinogen-binding protein (Efb-C), a structural and functional homolog of Sbi-IV, showed Efb-C binding to a region on the concave face of C3d previously implicated in CR2 binding by our mutagenesis data but not confirmed in the CR2(short consensus repeat [SCR]1-2):C3d cocrystal structure. We have now analyzed by surface plasmon resonance the binding of a series of variant C3dg molecules to biosensor-bound Sbi-IV or CR2(SCR1-2). We found that mutations to the concave face acidic pocket of C3d significantly affected binding to both Sbi-IV and CR2, although there was divergence in which residues were most important in each case. By contrast, no binding defects were seen for mutations made to the sideface of C3d implicated from the cocrystal structure to be involved in binding CR2(SCR1-2). The results with Sbi-IV suggest a mode of binding highly similar to that visualized in the Efb-C:C3d complex. The results with CR2 confirm our earlier mapping studies and cast even further doubt on the physiologic relevance of the complex visualized in the C3d:CR2 cocrystal.
Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte/química , Complemento C3d/química , Evasão da Resposta Imune , Receptores de Complemento 3d/química , Staphylococcus aureus/imunologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Complemento C3b/antagonistas & inibidores , Complemento C3b/genética , Complemento C3b/metabolismo , Complemento C3d/genética , Complemento C3d/metabolismo , Cristalização , Cristalografia por Raios X , Análise Mutacional de DNA , Humanos , Evasão da Resposta Imune/genética , Camundongos , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Receptores de Complemento 3d/antagonistas & inibidores , Receptores de Complemento 3d/genética , Staphylococcus aureus/genéticaRESUMO
The primary host response to Staphylococcus aureus infection occurs via complement. Complement is an elegant evolutionarily conserved system, playing essential roles in early defences by working in concert with immune cells to survey, label and destroy microbial intruders and coordinate inflammation. Currently the exact mechanisms employed by S. aureus to manipulate and evade complement is not clear and is hindered by the lack of accurate molecular tools that can report on complement deposition on the bacterial surface. Current gold-standard detection methods employ labelled complement-specific antibodies and flow cytometry to determine complement deposited on bacteria. These methods are restricted by virtue of the expression of the S. aureus immunoglobulin binding proteins, Protein A and Sbi. In this study we describe the use of a novel antibody-independent C3 probe derived from the staphylococcal Sbi protein, specifically Sbi-IV domain. Here we show that biotin-labelled Sbi-IV interacts specifically with deposited C3 products on the staphylococcal surface and thus can be used to measure complement fixation on wild-type cells expressing a full repertoire of immune evasion proteins. Lastly, our data indicates that genetically diverse S. aureus strains restrict complement to different degrees suggesting that complement evasion is a variable virulence trait among S. aureus isolates.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Proteínas de Bactérias , Biotina/metabolismo , Complemento C3/metabolismo , Humanos , Ligação Proteica , Staphylococcus aureus/metabolismoRESUMO
Cysteine-rich knob domains can be isolated from the ultralong heavy-chain complementarity-determining region (CDR) 3, which are unique to a subset of bovine antibodies, to create antibody fragments of ~4 kDa. Advantageously, the N- and C- termini of these small binding domains are in close proximity, and we propose that this may offer a practical route to engineer extrinsic binding specificity into proteins. To test this, we transplanted knob domains into various loops of rat serum albumin, targeting sites that were distal to the interface with the neonatal Fc receptor. Using knob domains raised against the clinically validated drug target complement component C5, we produced potent inhibitors, which exhibit an extended plasma half-life in vivo via attenuated renal clearance and neonatal Fc receptor-mediated avoidance of lysosomal catabolism. The same approach was also used to modify a Camelid VHH, targeting a framework loop situated at the opposing end of the domain to the CDRs, to produce a small, single-chain bispecific antibody and a dual inhibitor of Complement C3 and C5. This study presents new protein inhibitors of the complement cascade and demonstrates a broadly applicable method to engineer target specificity within polypeptide chains, using bovine knob domains.
Assuntos
Anticorpos Biespecíficos , Regiões Determinantes de Complementaridade , Animais , Anticorpos Biespecíficos/química , Bovinos , Ativação do Complemento , Regiões Determinantes de Complementaridade/química , Domínios Proteicos , RatosRESUMO
Staphylococcus aureus is an opportunistic pathogen that is able to thwart an effective host immune response by producing a range of immune evasion molecules, including S. aureus binder of IgG (Sbi) which interacts directly with the central complement component C3, its fragments and associated regulators. Recently we reported the first structure of a disulfide-linked human C3d17C dimer and highlighted its potential role in modulating B-cell activation. Here we present an X-ray crystal structure of a disulfide-linked human C3d17C dimer, which undergoes a structurally stabilising N-terminal 3D domain swap when in complex with Sbi. These structural studies, in combination with circular dichroism and fluorescence spectroscopic analyses, reveal the mechanism underpinning this unique helix swap event and could explain the origins of a previously discovered N-terminally truncated C3dg dimer isolated from rat serum. Overall, our study unveils a novel staphylococcal complement evasion mechanism which enables the pathogen to harness the ability of dimeric C3d to modulate B-cell activation.
Assuntos
Proteínas de Bactérias , Staphylococcus aureus , Animais , Proteínas de Transporte/metabolismo , Dissulfetos/metabolismo , Ratos , Staphylococcus/metabolismoRESUMO
Cleavage of C3 to C3a and C3b plays a central role in the generation of complement-mediated defences. Although the thioester-mediated surface deposition of C3b has been well-studied, fluid phase dimers of C3 fragments remain largely unexplored. Here we show C3 cleavage results in the spontaneous formation of C3b dimers and present the first X-ray crystal structure of a disulphide-linked human C3d dimer. Binding studies reveal these dimers are capable of crosslinking complement receptor 2 and preliminary cell-based analyses suggest they could modulate B cell activation to influence tolerogenic pathways. Altogether, insights into the physiologically-relevant functions of C3d(g) dimers gained from our findings will pave the way to enhancing our understanding surrounding the importance of complement in the fluid phase and could inform the design of novel therapies for immune system disorders in the future.
Assuntos
Complemento C3d/química , Modelos Moleculares , Multimerização Proteica , Complemento C3/química , Complemento C3/imunologia , Complemento C3d/imunologia , Humanos , Ativação Linfocitária/imunologia , Linfócitos/imunologia , Linfócitos/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Conformação Proteica , Proteólise , Proteínas Recombinantes/química , Relação Estrutura-AtividadeRESUMO
Cysteine-rich knob domains found in the ultralong complementarity determining regions of a subset of bovine antibodies are capable of functioning autonomously as 3-6 kDa peptides. While they can be expressed recombinantly in cellular systems, in this paper we show that knob domains are also readily amenable to a chemical synthesis, with a co-crystal structure of a chemically synthesized knob domain in complex with an antigen showing structural equivalence to the biological product. For drug discovery, following the immunization of cattle, knob domain peptides can be synthesized directly from antibody sequence data, combining the power and diversity of the bovine immune repertoire with the ability to rapidly incorporate nonbiological modifications. We demonstrate that, through rational design with non-natural amino acids, a paratope diversity can be massively expanded, in this case improving the efficacy of an allosteric peptide. As a potential route to further improve stability, we also performed head-to-tail cyclizations, exploiting the proximity of the N and C termini to synthesize functional, fully cyclic antibody fragments. Lastly, we highlight the stability of knob domains in plasma and, through pharmacokinetic studies, use palmitoylation as a route to extend the plasma half-life of knob domains in vivo. This study presents an antibody-derived medicinal chemistry platform, with protocols for solid-phase synthesis of knob domains, together with the characterization of their molecular structures, in vitro pharmacology, and pharmacokinetics.
Assuntos
Regiões Determinantes de Complementaridade/química , Fragmentos de Imunoglobulinas/química , Peptídeos Cíclicos/síntese química , Sequência de Aminoácidos , Animais , Bovinos , Fragmentos de Imunoglobulinas/sangue , Fragmentos de Imunoglobulinas/farmacologia , Masculino , Modelos Moleculares , Peptídeos Cíclicos/sangue , Peptídeos Cíclicos/farmacocinética , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Ratos Sprague-Dawley , Técnicas de Síntese em Fase Sólida , Espectrometria de Massas em Tandem , TermodinâmicaRESUMO
Bovines have evolved a subset of antibodies with ultra-long heavy chain complementarity determining regions that harbour cysteine-rich knob domains. To produce high-affinity peptides, we previously isolated autonomous 3-6 kDa knob domains from bovine antibodies. Here, we show that binding of four knob domain peptides elicits a range of effects on the clinically validated drug target complement C5. Allosteric mechanisms predominated, with one peptide selectively inhibiting C5 cleavage by the alternative pathway C5 convertase, revealing a targetable mechanistic difference between the classical and alternative pathway C5 convertases. Taking a hybrid biophysical approach, we present C5-knob domain co-crystal structures and, by solution methods, observed allosteric effects propagating >50 Å from the binding sites. This study expands the therapeutic scope of C5, presents new inhibitors, and introduces knob domains as new, low molecular weight antibody fragments, with therapeutic potential.
Antibodies are proteins produced by the immune system that can selectively bind to other molecules and modify their behaviour. Cows are highly equipped at fighting-off disease-causing microbes due to the unique shape of some of their antibodies. Unlike other jawed vertebrates, cows' antibodies contain an ultra-long loop region that contains a 'knob domain' which sticks out from the rest of the antibody. Recent research has shown that when detached, the knob domain behaves like an antibody fragment, and can independently bind to a range of different proteins. Antibody fragments are commonly developed in the laboratory to target proteins associated with certain diseases, such as arthritis and cancer. But it was unclear whether the knob domains from cows' antibodies could also have therapeutic potential. To investigate this, Macpherson et al. studied how knob domains attach to complement C5, a protein in the inflammatory pathway which is a drug target for various diseases, including severe COVID-19. The experiments identified various knob domains that bind to complement C5 and inhibits its activity by altering its structure or movement. Further tests studying the structure of these interactions, led to the discovery of a common mechanism by which inhibitors can modify the behaviour of this inflammatory protein. Complement C5 is involved in numerous molecular pathways in the immune system, which means many of the drugs developed to inhibit its activity can also leave patients vulnerable to infection. However, one of the knob domains identified by Macpherson et al. was found to reduce the activity of complement C5 in some pathways, whilst leaving other pathways intact. This could potentially reduce the risk of bacterial infections which sometimes arise following treatment with these types of inhibitors. These findings highlight a new approach for developing drug inhibitors for complement C5. Furthermore, the ability of knob domains to bind to multiple sites of complement C5 suggests that this fragment could be used to target proteins associated with other diseases.
Assuntos
Regulação Alostérica/efeitos dos fármacos , Complemento C5/antagonistas & inibidores , Descoberta de Drogas , Peptídeos/química , Peptídeos/farmacologia , Animais , Bovinos , Complemento C5/química , Complemento C5/metabolismo , Simulação de Acoplamento Molecular , Conformação Proteica/efeitos dos fármacosRESUMO
The incorporation of the specialized carbohydrate affinity ligand methacrylamido phenylboronic acid in polyacrylamide gels for SDS-PAGE analysis has been successful for the separation of carbohydrates and has here been adapted for the analysis of post-translationally modified proteins. While conventional SDS-PAGE analysis cannot distinguish between glycated and unglycated proteins, methacrylamido phenylboronate acrylamide gel electrophoresis (mP-AGE) in low loading shows dramatic retention of delta-gluconolactone modified proteins, while the mobility of the unmodified proteins remains unchanged. With gels containing 1% methacrylamido phenylboronate, mP-AGE analysis of gluconoylated recombinant protein Sbi results in the retention of the modified protein at a position expected for a protein that has quadrupled its expected molecular size. Subsequently, mP-AGE was tested on HSA, a protein that is known to undergo glycation under physiological conditions. mP-AGE could distinguish between various carbohydrate-protein adducts, using in vitro glycated HSA, and discriminate early from late glycation states of the protein. Enzymatically glycosylated proteins show no altered retention in the phenylboronate-incorporated gels, rendering this method highly selective for glycated proteins. We reveal that a trident interaction between phenylboronate and the Amadori cis 1,2 diol and amine group provides the molecular basis of this specificity. These results epitomize mP-AGE as an important new proteomics tool for the detection, separation, visualization and identification of protein glycation. This method will aid the design of inhibitors of unwanted carbohydrate modifications in recombinant protein production, ageing, diabetes, cardiovascular diseases and Alzheimer's disease.
Assuntos
Ácidos Borônicos/química , Eletroforese em Gel de Poliacrilamida/métodos , Glicosilação , Proteômica/métodos , Albumina Sérica/análise , Eletroforese em Gel de Poliacrilamida/economia , Humanos , Estrutura Molecular , Fosforilação , Processamento de Proteína Pós-Traducional , Proteômica/economia , Albumina Sérica/metabolismoRESUMO
The Gram-positive bacterium Staphylococcus aureus, similar to other pathogens, binds human complement regulators Factor H and Factor H related protein 1 (FHR-1) from human serum. Here we identify the secreted protein Sbi (Staphylococcus aureus binder of IgG) as a ligand that interacts with Factor H by a-to our knowledge-new type of interaction. Factor H binds to Sbi in combination with C3b or C3d, and forms tripartite SbiratioC3ratioFactor H complexes. Apparently, the type of C3 influences the stability of the complex; surface plasmon resonance studies revealed a higher stability of C3d complexed to Sbi, as compared to C3b or C3. As part of this tripartite complex, Factor H is functionally active and displays complement regulatory activity. Sbi, by recruiting Factor H and C3b, acts as a potent complement inhibitor, and inhibits alternative pathway-mediated lyses of rabbit erythrocytes by human serum and sera of other species. Thus, Sbi is a multifunctional bacterial protein, which binds host complement components Factor H and C3 as well as IgG and beta(2)-glycoprotein I and interferes with innate immune recognition.