RESUMO
Asparaginyl endopeptidases (AEPs) are a class of enzymes commonly associated with proteolysis in the maturation of seed storage proteins. However, a subset of AEPs work preferentially as peptide ligases, coupling release of a leaving group to formation of a new peptide bond. These "ligase-type" AEPs require only short recognition motifs to ligate a range of targets, making them useful tools in peptide and protein engineering for cyclisation of peptides or ligation of separate peptides into larger products. Here we report the recombinant expression, ligase activity and cyclisation kinetics of three new AEPs from the cyclotide producing plant Oldenlandia affinis with superior kinetics to the prototypical recombinant AEP ligase OaAEP1b. These AEPs work preferentially as ligases at both acidic and neutral pH and we term them "canonical AEP ligases" to distinguish them from other AEPs where activity preferences shift according to pH. We show that these ligases intrinsically favour ligation over hydrolysis, are highly efficient at cyclising two unrelated peptides and are compatible with organic co-solvents. Finally, we demonstrate the broad scope of recombinant AEPs in biotechnology by the backbone cyclisation of an intrinsically disordered protein, the 25 kDa malarial vaccine candidate Plasmodium falciparum merozoite surface protein 2 (MSP2).
Assuntos
Cisteína Endopeptidases/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Ligases/metabolismo , Proteínas de Plantas/metabolismo , Antígenos de Protozoários/metabolismo , Ciclização , Modelos Moleculares , Engenharia de Proteínas , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
The interaction between apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2) plays a key role in the invasion of red blood cells by Plasmodium parasites. Disruption of this critical protein-protein interaction represents a promising avenue for antimalarial drug discovery. In this work, we exploited a 13-residue ß-hairpin based on the C-terminal loop of RON2 to probe a conserved binding site on Plasmodium falciparum AMA1. A series of mutations was synthetically engineered into ß-hairpin peptides to establish structure-activity relationships. The best mutations improved the binding affinity of the ß-hairpin peptide by ~7-fold for 3D7 AMA1 and ~14-fold for FVO AMA1. We determined the crystal structures of several ß-hairpin peptides in complex with AMA1 in order to define the structural features and specific interactions that contribute to improved binding affinity. The same mutations in the longer RON2sp2 peptide (residues 2027-2055 of RON2) increased the binding affinity by >30-fold for 3D7 and FVO AMA1, producing KD values of 2.1nM and 0.4nM, respectively. To our knowledge, this is the most potent strain-transcending peptide reported to date and represents a valuable tool to characterize the AMA1-RON2 interaction.
Assuntos
Antígenos de Protozoários/metabolismo , Antimaláricos/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Cristalografia por Raios X , Proteínas de Membrana/genética , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Proteínas de Protozoários/genética , Relação Estrutura-AtividadeRESUMO
Malaria remains a significant global health burden. The development of an effective malaria vaccine remains as a major challenge with the potential to significantly reduce morbidity and mortality. While Plasmodium spp. have been shown to contain a large number of intrinsically disordered proteins (IDPs) or disordered protein regions, the relationship of protein structure to subcellular localisation and adaptive immune responses remains unclear. In this study, we employed several computational prediction algorithms to identify IDPs at the proteome level of six Plasmodium spp. and to investigate the potential impact of protein disorder on adaptive immunity against P. falciparum parasites. IDPs were shown to be particularly enriched within nuclear proteins, apical proteins, exported proteins and proteins localised to the parasitophorous vacuole. Furthermore, several leading vaccine candidates, and proteins with known roles in host-cell invasion, have extensive regions of disorder. Presentation of peptides by MHC molecules plays an important role in adaptive immune responses, and we show that IDP regions are predicted to contain relatively few MHC class I and II binding peptides owing to inherent differences in amino acid composition compared to structured domains. In contrast, linear B-cell epitopes were predicted to be enriched in IDPs. Tandem repeat regions and non-synonymous single nucleotide polymorphisms were found to be strongly associated with regions of disorder. In summary, immune responses against IDPs appear to have characteristics distinct from those against structured protein domains, with increased antibody recognition of linear epitopes but some constraints for MHC presentation and issues of polymorphisms. These findings have major implications for vaccine design, and understanding immunity to malaria.
Assuntos
Proteínas Intrinsicamente Desordenadas/imunologia , Plasmodium/imunologia , Proteoma , Proteômica , Proteínas de Protozoários/imunologia , Sequência de Aminoácidos , Aminoácidos , Biologia Computacional/métodos , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Peptídeos/química , Peptídeos/imunologia , Plasmodium falciparum/imunologia , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Sequências de Repetição em TandemRESUMO
Apical membrane antigen 1 (AMA1) of the human malaria parasite Plasmodium falciparum has been implicated in invasion of the host erythrocyte. It interacts with malarial rhoptry neck (RON) proteins in the moving junction that forms between the host cell and the invading parasite. Agents that block this interaction inhibit invasion and may serve as promising leads for anti-malarial drug development. The invasion-inhibitory peptide R1 binds to a hydrophobic cleft on AMA1, which is an attractive target site for small molecules that block parasite invasion. In this work, truncation and mutational analyses show that Phe5-Phe9, Phe12 and Arg15 in R1 are the most important residues for high affinity binding to AMA1. These residues interact with two well-defined binding hot spots on AMA1. Computational solvent mapping reveals that one of these hot spots is suitable for small molecule targeting. We also confirm that R1 in solution binds to AMA1 with 1:1 stoichiometry and adopts a secondary structure consistent with the major form of R1 observed in the crystal structure of the complex. Our results provide a basis for designing high affinity inhibitors of the AMA1-RON2 interaction.
Assuntos
Antígenos de Protozoários/química , Malária Falciparum/parasitologia , Proteínas de Membrana/química , Peptídeos/química , Plasmodium falciparum/química , Proteínas de Protozoários/química , Antígenos de Protozoários/genética , Cristalografia por Raios X , Interações Hospedeiro-Parasita , Humanos , Malária Falciparum/genética , Proteínas de Membrana/genética , Ligação Proteica , Proteínas de Protozoários/genética , Espalhamento a Baixo Ângulo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismoRESUMO
Apical membrane antigen 1 (AMA1) is essential for malaria parasite invasion of erythrocytes and is therefore an attractive target for drug development. Peptides that bind AMA1 have been identified from random peptide libraries expressed on the surface of phage. Of these, R1, which binds to a hydrophobic ligand binding site on AMA1, was a particularly potent inhibitor of parasite invasion of erythrocytes in vitro. The solution structure of R1 contains a turn-like conformation between residues 5-10. Here the importance of residues in this turn-like structure for binding to AMA1 was examined by site-directed mutagenesis and NMR spectroscopy. The peptide was expressed as a fusion protein following replacement of Met16 by Leu in order to accommodate cyanogen bromide cleavage. This modified peptide (R2) displayed the same affinity for AMA1 as R1, showing that the identity of the side chain at position 16 was not critical for binding. Substitution of Phe5, Pro7, Leu8, and Phe9 with alanine led to significant (7.5- to >350-fold) decreases in affinity for AMA1. Comparison of backbone amide and C(α) H chemical shifts for these R2 analogues with corresponding values for R2 showed no significant changes, with the exception of R2(P7A), where slightly larger differences were observed, particularly for residues flanking position 7. The absence of significant changes in the secondary chemical shifts suggests that these mutations had little effect on the solution conformation of R2. The identification of a nonpolar region of these peptides containing residues essential for AMA1 binding establishes a basis for the design of anti-malarial drugs based on R1 mimetics.
Assuntos
Antimaláricos/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Peptídeos/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Antimaláricos/química , Sítios de Ligação/genética , Desenho de Fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Humanos , Técnicas In Vitro , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/genética , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidade , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Virulência/efeitos dos fármacosRESUMO
Merozoite surface protein 2 (MSP2), one of the most abundant proteins on the surface of the merozoite stage of Plasmodium falciparum, is a potential component of a malaria vaccine, having shown some efficacy in a clinical trial in Papua New Guinea. MSP2 is a GPI-anchored protein consisting of conserved N- and C-terminal domains and a variable central region. Previous studies have shown that it is an intrinsically unstructured protein with a high propensity for fibril formation, in which the conserved N-terminal domain has a key role. Secondary structure predictions suggest that MSP2 contains long stretches of random coil with very little alpha-helix or beta-strand. Circular dichroism spectroscopy confirms this prediction under physiological conditions (pH 7.4) and in more acidic solutions (pH 6.2 and 3.4). Pulsed field gradient NMR diffusion measurements showed that MSP2 under physiological conditions has a large effective hydrodynamic radius consistent with an intrinsic pre-molten globule state, as defined by Uversky. This was supported by sedimentation velocity studies in the analytical ultracentrifuge. NMR resonance assignments have been obtained for FC27 MSP2, allowing the residual secondary structure and backbone dynamics to be defined. There is some motional restriction in the conserved C-terminal region in the vicinity of an intramolecular disulfide bond. Two other regions show motional restrictions, both of which display helical structure propensities. One of these helical regions is within the conserved N-terminal domain, which adopts essentially the same conformation in full-length MSP2 as in corresponding peptide fragments. We see no evidence of long-range interactions in the full-length protein. MSP2 associates with lipid micelles, but predominantly through the N-terminal region rather than the C terminus, which is GPI-anchored to the membrane in the parasite.
Assuntos
Antígenos de Protozoários/química , Lipídeos/química , Plasmodium falciparum , Proteínas de Protozoários/química , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/genética , Dicroísmo Circular , Dissulfetos/química , Micelas , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Peptídeos/química , Peptídeos/genética , Fosforilcolina/análogos & derivados , Estrutura Secundária de Proteína , Proteínas de Protozoários/genética , Análise de Sequência de Proteína , Soluções , UltracentrifugaçãoRESUMO
Apical membrane antigen 1 (AMA1) is a leading malaria vaccine candidate that possesses polymorphisms that may pose a problem for a vaccine based on this antigen. Knowledge of the distribution of the polymorphic sites on the surface of AMA1 is necessary to obtain a detailed understanding of their significance for vaccine development. For this reason we have sought to determine the three-dimensional structure of AMA1 using x-ray crystallography. The central two-thirds of AMA1 is relatively conserved among Plasmodium species as well as more distantly related apicomplexan parasites, and contains two clusters of disulfide-bonded cysteines termed domains I and II. The crystal structure of this fragment of AMA1 reported here reveals that domains I+II consists of two intimately associated PAN domains. PAN domain I contains many long loops that extend from the domain core and form a scaffold for numerous polymorphic residues. This extreme adaptation of a PAN domain reveals how malaria parasites have introduced significant flexibility and variation into AMA1 to evade protective human antibody responses. The polymorphisms on the AMA1 surface are exclusively located on one side of the molecule, presumably because this region of AMA1 is most accessible to antibodies reacting with the parasite surface. Moreover, the most highly polymorphic residues surround a conserved hydrophobic trough that is ringed by domain I and domain II loops. Precedents set by viral receptor proteins would suggest that this is likely to be the AMA1 receptor binding pocket.
Assuntos
Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Plasmodium falciparum/química , Plasmodium falciparum/genética , Polimorfismo Genético/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas de Protozoários/metabolismo , Alinhamento de SequênciaRESUMO
Combination B is a malaria vaccine that comprises recombinant Plasmodium falciparum (P. falciparum) blood-stage proteins MSP1, MSP2 and RESA, formulated with the adjuvant Montanide ISA 720. A phase I-IIb double-blind randomised placebo-controlled trial was undertaken in 120 children aged 5-9 years. Subjects were randomised in four groups: (i) No sulphadoxine-pyrimethamine (SP)+vaccine, (ii) No SP+placebo, (iii) SP+vaccine, (iv) SP+placebo. 15 microg of each protein were given in the thigh, at both first and second injection (4 weeks apart). The placebo was adjuvant emulsified with saline. No serious or severe AEs occurred. Moderate AEs were seen in 3% of the vaccine and 3% of the placebo recipients after first injection and in 12 and 10% after second injection. The vaccine induced significant antibody responses to all three antigens but triggered an IFN-gamma response to MSP1 only. At Week 12, the IFN-gamma response to MSP1 was substantially higher in the vaccine group where No SP had been given. Combination B proved to be safe and immunogenic in children aged 5-9 years. Vaccine immunogenicity was neither impaired by circulating parasites nor increased after pre-treatment with SP and pre-treatment is not advisable in future trials of malaria vaccines, at least for those including blood-stage antigens.
Assuntos
Vacinas Antimaláricas/imunologia , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Animais , Anticorpos Antiprotozoários/análise , Anticorpos Antiprotozoários/biossíntese , Divisão Celular , Criança , Pré-Escolar , Estudos de Coortes , Citocinas/análise , Citocinas/biossíntese , Método Duplo-Cego , Ensaio de Imunoadsorção Enzimática , Humanos , Imunização , Imunoglobulina G/análise , Imunoglobulina G/biossíntese , Interferon gama/biossíntese , Interleucina-10/análise , Interleucina-10/biossíntese , Interleucina-4/análise , Interleucina-4/biossíntese , Vacinas Antimaláricas/efeitos adversos , Malária Falciparum/epidemiologia , Monócitos/imunologia , Papua Nova Guiné/epidemiologia , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Apical membrane antigen-1 (AMA1) is a prime vaccine candidate for inclusion in a vaccine against malaria. It is known that the disulphide bond stabilised conformation of this antigen is important for eliciting a protective antibody response, however little is known about the epitopes within this molecule that are targeted by the immune response. We have used a peptide approach for the identification and characterisation of such regions. In this study, the in vitro refolded, recombinant ectodomain of AMA1 from the D strain of Plasmodium chabaudi adami, was digested with trypsin and individual peptide fragments examined for antigenic activity. We found that a tryptic fragment, which was derived from a loop-like structure within the putative domain I of the intact AMA1 molecule, was highly reactive with antibodies from the sera of hyperimmune mice. Two different synthetic peptide constructs incorporating this antigenically active fragment were assembled. The first consisted of two separate peptide chains which were linked through a disulphide bond formed using chemo-selective chemistry. A larger 45-mer loop peptide, generated by the oxidation of two cysteine residues close to the N- and C-termini of the 45-mer, represented the complete loop structure and incorporated the tryptic fragment. Each peptide construct was also able to elicit production of high titres of antibodies in mice and furthermore, the 45-residue loop peptide elicited antibodies capable of binding to AMA1 with titres comparable to those present in a mouse which had recovered from multiple exposures to P. chabaudi adami parasites. Passive immunisation with anti-loop antibodies did not suppress the development of parasitaemia in mice challenged with P. chabaudi adami suggesting that although highly immunogenic, the peptides represented inadequate or inappropriate epitopes for vaccination purposes.
Assuntos
Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/imunologia , Proteínas de Membrana/imunologia , Fragmentos de Peptídeos/imunologia , Plasmodium chabaudi/imunologia , Proteínas de Protozoários/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/biossíntese , Dimerização , Feminino , Proteínas de Membrana/química , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Proteínas de Protozoários/química , Tripsina/farmacologiaRESUMO
Phage display has become a widely used tool for the identification of proteins or peptides with affinity for a variety of biomolecules. The versatility, simplicity and cost effectiveness of this application has pervaded a wide variety of research areas. Although not without its limitations, phage display has provided a convenient methodology for obtaining ligands to study the function, structure and diagnostic or therapeutic potential of various macromolecules. This review highlights some recent research employing this technology that serves to illustrate its utility in various research and clinical applications.