RESUMO
Protein engineering approaches are often a combination of rational design and directed evolution using display technologies. Here, we test "loop grafting," a rational design method, on three-finger fold proteins. These small reticulated proteins have exceptional affinity and specificity for their diverse molecular targets, display protease-resistance, and are highly stable and poorly immunogenic. The wealth of structural knowledge makes them good candidates for protein engineering of new functionality. Our goal is to enhance the efficacy of these mini-proteins by modifying their pharmacological properties in order to extend their use in imaging, diagnostics and therapeutic applications. Using the interaction of three-finger fold toxins with muscarinic and adrenergic receptors as a model, chimeric toxins have been engineered by substituting loops on toxin MT7 by those from toxin MT1. The pharmacological impact of these grafts was examined using binding experiments on muscarinic receptors M1 and M4 and on the α(1A)-adrenoceptor. Some of the designed chimeric proteins have impressive gain of function on certain receptor subtypes achieving an original selectivity profile with high affinity for muscarinic receptor M1 and α(1A)-adrenoceptor. Structure-function analysis supported by crystallographic data for MT1 and two chimeras permits a molecular based interpretation of these gains and details the merits of this protein engineering technique. The results obtained shed light on how loop permutation can be used to design new three-finger proteins with original pharmacological profiles.
Assuntos
Receptores Adrenérgicos/efeitos dos fármacos , Receptores Muscarínicos/efeitos dos fármacos , Toxinas Biológicas/toxicidade , Sequência de Aminoácidos , Cristalização , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Engenharia de Proteínas , Homologia de Sequência de AminoácidosRESUMO
In the present study, we show that histidines 310 and 435 at the CH2-CH3 interface of the Fc portion of human IgG1 can coordinate a Zn2+ and participate in the control of the CH2-CH2 interdomain opening. Structures obtained in the absence of Zn2+ have a reduced interdomain gap that likely hamper FcγR binding. This closed conformation of the Fc is stabilized by inter-CH2 domain sugar contacts. Zinc appears to counteract the sugar mediated constriction, suggesting that zinc could be an important control factor in IgG1/FcγR interactions. The results of binding studies performed in the presence of EDTA on FcγR expressing cells supports this hypothesis. When a mutated Fc fragment, in which histidines 310 and 435 have been substituted by lysines (Fc H/K), was compared with the wild-type Fc in crystallographic studies, we found that the mutations leave the interface unaltered but have a long-range effect on the CH2 interdomain separation. Moreover, these substitutions have a differential effect on the binding of IgG1 to Fcγ receptors and their functions. Interaction with the inhibitory FcγRIIB is strongly perturbed by the mutations and mutant IgG1 H/K only weakly engages this receptor. By contrast, higher affinity FcγR are mostly unaffected.
Assuntos
Imunoglobulina G/imunologia , Domínios e Motivos de Interação entre Proteínas , Receptores de IgG/imunologia , Zinco/metabolismo , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Células Jurkat , Modelos Moleculares , Mutação , Receptores de IgG/química , Receptores de IgG/genética , Receptores de IgG/metabolismo , Zinco/químicaRESUMO
A novel heterodimeric three-finger neurotoxin, irditoxin, was isolated from venom of the brown treesnake Boiga irregularis (Colubridae). Irditoxin subunit amino acid sequences were determined by Edman degradation and cDNA sequencing. The crystal structure revealed two subunits with a three-finger protein fold, typical for "nonconventional" toxins such as denmotoxin, bucandin, and candoxin. This is the first colubrid three-finger toxin dimer, covalently connected via an interchain disulfide bond. Irditoxin showed taxon-specific lethality toward birds and lizards and was nontoxic toward mice. It produced a potent neuromuscular blockade at the avian neuromuscular junction (IC(50)=10 nM), comparable to alpha-bungarotoxin, but was three orders of magnitude less effective at the mammalian neuromuscular junction. Covalently linked heterodimeric three-finger toxins found in colubrid venoms constitute a new class of venom peptides, which may be a useful source of new neurobiology probes and therapeutic leads.
Assuntos
Neurotoxinas/química , Neurotoxinas/metabolismo , Venenos de Serpentes/química , Venenos de Serpentes/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Ligação Competitiva , Colubridae/metabolismo , Sequência Conservada , Cristalografia por Raios X , DNA Complementar/genética , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Neurotoxinas/isolamento & purificação , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Alinhamento de Sequência , Venenos de Serpentes/isolamento & purificação , Especificidade da Espécie , Homologia Estrutural de ProteínaRESUMO
Human prostate-specific antigen (PSA or KLK3) is an important marker for the diagnosis and management of prostate cancer. This is an androgen-regulated glycoprotein of the kallikrein-related protease family secreted by prostatic epithelial cells. Its physiological function is to cleave semenogelins in the seminal coagulum and its enzymatic activity is strongly modulated by zinc ions. Here we present the first crystal structure of human PSA in complex with monoclonal antibody (mAb) 8G8F5 that enhances its enzymatic activity. The mAb recognizes an epitope composed of five discontinuous segments including residues from the kallikrein loop and stabilizes PSA in an "open and active conformation" that accelerates catalysis. We also present the crystal structure of PSA in complex with both the mAb 8G8F5 and a fluorogenic substrate Mu-KGISSQY-AFC, derived from semenogelin I. By exploiting the inhibition of PSA by zinc ions, we were able to obtain a substrate acyl intermediate covalently linked to the catalytic serine, at pH 7.3 but not at pH 5.5. Moreover, the inhibition of PSA activity by zinc was found to be modulated by pH variations but not by the antibody binding. The correlation of the different data with the physiological conditions under which PSA can cleave semenogelins is discussed.
Assuntos
Anticorpos Monoclonais/química , Antígeno Prostático Específico/química , Proteínas Secretadas pela Vesícula Seminal/química , Sequência de Aminoácidos , Cristalografia por Raios X , Dimerização , Epitopos/química , Humanos , Ligação de Hidrogênio , Região Variável de Imunoglobulina/química , Calicreínas/química , Modelos Moleculares , Dados de Sequência Molecular , Antígeno Prostático Específico/antagonistas & inibidores , Estrutura Quaternária de Proteína , Alinhamento de Sequência , Especificidade por Substrato , Zinco/farmacologiaRESUMO
Boiga dendrophila (mangrove catsnake) is a colubrid snake that lives in Southeast Asian lowland rainforests and mangrove swamps and that preys primarily on birds. We have isolated, purified, and sequenced a novel toxin from its venom, which we named denmotoxin. It is a monomeric polypeptide of 77 amino acid residues with five disulfide bridges. In organ bath experiments, it displayed potent postsynaptic neuromuscular activity and irreversibly inhibited indirectly stimulated twitches in chick biventer cervicis nerve-muscle preparations. In contrast, it induced much smaller and readily reversible inhibition of electrically induced twitches in mouse hemidiaphragm nerve-muscle preparations. More precisely, the chick muscle alpha(1)betagammadelta-nicotinic acetylcholine receptor was 100-fold more susceptible compared with the mouse receptor. These data indicate that denmotoxin has a bird-specific postsynaptic activity. We chemically synthesized denmotoxin, crystallized it, and solved its crystal structure at 1.9 A by the molecular replacement method. The toxin structure adopts a non-conventional three-finger fold with an additional (fifth) disulfide bond in the first loop and seven additional residues at its N terminus, which is blocked by a pyroglutamic acid residue. This is the first crystal structure of a three-finger toxin from colubrid snake venom and the first fully characterized bird-specific toxin. Denmotoxin illustrates the relationship between toxin specificity and the primary prey type that constitutes the snake's diet.
Assuntos
Venenos de Serpentes/metabolismo , Sequência de Aminoácidos , Animais , Galinhas , Masculino , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Músculos/efeitos dos fármacos , Músculos/metabolismo , Neurotoxinas/química , Receptores Nicotínicos/metabolismo , Homologia de Sequência de Aminoácidos , Venenos de Serpentes/química , Venenos de Serpentes/toxicidade , Serpentes , Sinapses/efeitos dos fármacosRESUMO
The binding of IgG antibodies to receptors for the Fc region of IgG (FcgammaR) is a critical step for the initiation and/or the control of effector immune functions once immune complexes have been formed. Site-directed and random mutagenesis as well as domain-swapping, NMR and X-ray cristallography have made it possible to get detailed insights in the molecular mechanisms that govern IgG/FcgammaR interactions and to define some of the structural determinants that impact IgG binding to the various FcgammaR. It has demonstrated the role of particular stretches and individual residues located in the lower hinge region of the CH2 domain and in the CH2 and CH3 domains of the Fc region. The importance of the sugar components linked to asparagine 297 in the binding properties of IgG1, the human IgG isotype the most widely used in antibody-based therapies, has been also highlighted. These data have led to the engineering of a new generation of monoclonal antibodies for therapeutic use with optimized effector functions.
Assuntos
Anticorpos Monoclonais/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Receptores Fc/imunologia , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/uso terapêutico , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Imunoglobulina G/genética , Imunoglobulina G/uso terapêutico , Mutagênese Sítio-Dirigida/métodos , Ligação Proteica/genética , Ligação Proteica/imunologia , Receptores Fc/genéticaRESUMO
Proteins and peptides with variable degrees of disorder are a challenge for protein crystallization. These may be completely disordered or just contain regions with a high degree of mobility that may be represented by a multitude of discretely defined conformations. These difficulties are not insurmountable, but it may be unreasonable to expect a clean result from a structural point of view. The complex between a murine monoclonal antibody (19D9D6) and a synthetic peptide that encompasses the first 45 residues of the core protein of Hepatitis C virus that is poorly structured in solution has been crystallized. In order to make the crystallization possible, use was made of a single immunoglobulin-binding domain of protein L from Peptostreptococcus magnus (PpL), a bacterial protein that can bind the variable region (Fv) of a large population of antibodies through its light chain with no interference with antibody-antigen recognition. Crystals were obtained in different space groups where the size of the cavity that accommodates the peptide is different, although many of the crystal contacts and the overall lattice are preserved. The peptide can be considered to be semi-disordered and the larger cavity accommodates a better ordered peptide than the smaller one. The lattice is of interest for the design of a scaffold system for the crystallization of peptide-tagged proteins since a cavity that accommodates a disordered entity might be able to host ordered proteins of the same size and shape as the cavity. Here, the differences between the lattices formed by this trimolecular complex are described and it is discussed how such a system may be adapted to the crystallization of peptide-tagged proteins.
Assuntos
Anticorpos Monoclonais/química , Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Fragmentos Fab das Imunoglobulinas/química , Peptídeos/química , Animais , Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/imunologia , Cristalografia por Raios X , Proteínas de Ligação a DNA/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Camundongos , Peptídeos/imunologia , Estrutura Quaternária de ProteínaRESUMO
The first crystal structure of a complex between a hepatitis C virus (HCV) core protein-derived peptide (residues 13-40) and the Ab fragment of a murine mAb (19D9D6) has been solved, allowing determination of the recognized epitope and elucidation of its conformation. This Ab, raised against the first 120 residues of the core protein, recognizes core particles and strongly competes with anticore human Abs, suggesting that it is highly representative of the human anti-HCV core response. Its epitope lies within the first 45 aa of the protein, the major antigenic segment of core recognized both by murine and human Abs. Surprisingly, the recognized epitope (29-37: QIVGGVYLL) has an unusual preponderance of hydrophobic residues, some of which are buried in a small hydrophobic core in the nuclear magnetic resonance structure of the peptide (2-45) in solution, suggesting that the Ab may induce a structural rearrangement upon recognition. The flexibility may reside entirely within the Ag, since the Fab'-peptide complex structure at 2.34 A shows that the Ab binding site is hardly perturbed by complexation. Given that the recognized residues are unlikely to be solvent exposed, we are left with the interesting possibility that Ab-core interactions may take place in a nonaqueous environment.
Assuntos
Anticorpos Monoclonais/química , Hepacivirus/química , Hepacivirus/imunologia , Antígenos da Hepatite C/química , Epitopos Imunodominantes/química , Proteínas do Core Viral/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Complexo Antígeno-Anticorpo/química , Sítios de Ligação de Anticorpos , Ligação Competitiva/imunologia , Cristalografia por Raios X , Antígenos da Hepatite C/imunologia , Interações Hidrofóbicas e Hidrofílicas , Epitopos Imunodominantes/imunologia , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Conformação Proteica , Soluções , Proteínas do Core Viral/imunologiaRESUMO
Animal toxins are small proteins built on the basis of a few disulfide bonded frameworks. Because of their high variability in sequence and biologic function, these proteins are now used as templates for protein engineering. Here we report the extensive characterization of the structure and dynamics of two toxin folds, the "three-finger" fold and the short alpha/beta scorpion fold found in snake and scorpion venoms, respectively. These two folds have a very different architecture; the short alpha/beta scorpion fold is highly compact, whereas the "three-finger" fold is a beta structure presenting large flexible loops. First, the crystal structure of the snake toxin alpha was solved at 1.8-A resolution. Then, long molecular dynamics simulations (10 ns) in water boxes of the snake toxin alpha and the scorpion charybdotoxin were performed, starting either from the crystal or the solution structure. For both proteins, the crystal structure is stabilized by more hydrogen bonds than the solution structure, and the trajectory starting from the X-ray structure is more stable than the trajectory started from the NMR structure. The trajectories started from the X-ray structure are in agreement with the experimental NMR and X-ray data about the protein dynamics. Both proteins exhibit fast motions with an amplitude correlated to their secondary structure. In contrast, slower motions are essentially only observed in toxin alpha. The regions submitted to rare motions during the simulations are those that exhibit millisecond time-scale motions. Lastly, the structural variations within each fold family are described. The localization and the amplitude of these variations suggest that the regions presenting large-scale motions should be those tolerant to large insertions or deletions.