RESUMEN
Biomolecular reagents that enable the specific molecular recognition of proteins play a crucial role in basic research as well as medicine. Up to now, antibodies (immunoglobulins) have been widely used for this purpose. Their predominant feature is the vast repertoire of antigen-binding sites that arise from a set of 6 hypervariable loops. However, antibodies suffer from practical disadvantages because of their complicated architecture, large size, and multiple functions. The lipocalins, on the other hand, have evolved as a protein family that primarily serves for the binding of small molecules. Here, we show that an engineered lipocalin, derived from human Lcn2, can specifically bind the T cell coreceptor CTLA-4 as a prescribed protein target with subnanomolar affinity. Crystallographic analysis reveals that its reshaped cup-like binding site, which is formed by 4 variable loops, provides perfect structural complementarity with this "antigen." Furthermore, comparison with the crystal structure of the uncomplexed engineered lipocalin indicates a pronounced induced-fit mechanism, a phenomenon so far considered typical for antibodies. By recognizing the same epitope on CTLA-4 that interacts with the counterreceptors B7.1/B7.2 on antigen-presenting cells the engineered Lcn2 exhibits strong, cross-species antagonistic activity, as evidenced by biological effects comparable with a CTLA-4-specific antibody. With its proven stimulatory activity on T cells in vivo, the CTLA-4 blocking lipocalin offers potential for immunotherapy of cancer and infectious disease. Beyond that, lipocalins with engineered antigen-binding sites, so-called Anticalins, provide a class of small ( approximately 180 residues), structurally simple, and robust binding proteins with applications in the life sciences in general.
Asunto(s)
Antígenos CD/metabolismo , Epítopos , Lipocalinas/metabolismo , Ingeniería de Proteínas , Proteínas de Fase Aguda/genética , Anticuerpos/química , Antígenos CD/química , Sitios de Unión , Antígeno CTLA-4 , Cristalografía por Rayos X , Humanos , Indicadores y Reactivos/síntesis química , Indicadores y Reactivos/química , Lipocalina 2 , Lipocalinas/química , Lipocalinas/genética , Unión Proteica , Conformación Proteica , Proteínas Proto-Oncogénicas/genéticaRESUMEN
Interleukin 4 (IL-4) is a pleiotropic cytokine which induces T-cell differentiation and class switching of B cells. It therefore plays a central role in the development of allergies and asthma. An IL-4 variant in which Glu9 was mutated to alanine shows an 800-fold drop in binding affinity towards its high-affinity receptor chain. As shown by surface plasmon resonance measurements, this mostly arises from a decreased association rate. Here, the crystal structure of this mutant is reported. It reveals that the protein has a virtually identical structure to the wild type, showing that the unusual behaviour of the mutated protein is not a consequence of misfolding. The possibility that polar interactions in the encounter complex have a steering effect is discussed.
Asunto(s)
Interleucina-4/química , Alanina/genética , Sustitución de Aminoácidos , Cristalización , Cristalografía por Rayos X , Ácido Glutámico/genética , Humanos , Interleucina-4/genética , Modelos Moleculares , Mutación , Conformación Proteica , Proteínas Recombinantes/químicaRESUMEN
The heterotrimeric complex of the human major histocompatibity complex (MHC) molecule HLA-A*0201, beta2-microglobulin and the decameric peptide GVYDGREHTV derived from the melanoma antigen (MAGE-A4 protein has been determined by X-ray crystallography at 1.4 A resolution. MAGE-A4 belongs to a family of genes that are specifically expressed in a variety of tumours. MAGE-A4-derived peptides are presented by MHC molecules at the cell surface to cytotoxic T-lymphocytes. As the HLA-A*0201:MAGE-A4 complex occurs only on tumour cells, it is considered to be an appropriate target for immunotherapy. The structure presented here reveals potential epitopes specific to the complex and indicates which peptide residues could be recognised by T-cell receptors. In addition, as the structure could be refined anisotropically, it was possible to describe the movements of the bound peptide in more detail.
Asunto(s)
Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Antígenos HLA-A/química , Antígenos HLA-A/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Secuencia de Aminoácidos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Sitios de Unión , Dicroismo Circular , Cristalografía por Rayos X , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Antígenos HLA-A/inmunología , Humanos , Inmunoterapia , Ligandos , Modelos Moleculares , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/metabolismo , Fragmentos de Péptidos/inmunología , Polietilenglicoles/química , Polietilenglicoles/metabolismo , Unión Proteica , Conformación Proteica , Desnaturalización Proteica , Receptores de Antígenos de Linfocitos T/inmunología , Temperatura , Termodinámica , Microglobulina beta-2/química , Microglobulina beta-2/metabolismoRESUMEN
Homodimeric bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor beta (TGF-beta) superfamily that induces bone formation and regeneration, and determines important steps during early stages of embryonic development in vertebrates and non-vertebrates. BMP-2 can interact with two types of receptor chains, as well as with proteins of the extracellular matrix and several regulatory proteins. We report here the crystal structure of human BMP-2 determined by molecular replacement and refined to an R-value of 24.2 % at 2.7 A resolution. A common scaffold of BMP-2, BMP-7 and the TGF-betas, i.e. the cystine-knot motif and two finger-like double-stranded beta-sheets, can be superimposed with r. m.s. deviations of around 1 A. In contrast to the TGF-betas, the structure of BMP-2 shows differences in the flexibility of the N terminus and the orientation of the central alpha-helix as well as two external loops at the fingertips with respect to the scaffold. This is also known from the BMP-7 model. Small secondary structure elements in the loop regions of BMP-2 and BMP-7 seem to be specific for the respective BMP-subgroup. Two identical helix-finger clefts and two distinct cavities located around the central 2-fold axis of the dimer show characteristic shapes, polarity and surface charges. The possible function of these specific features in the interaction of BMP-2 with its binding partners is discussed.
Asunto(s)
Proteínas Morfogenéticas Óseas/química , Secuencia de Aminoácidos , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/clasificación , Cristalografía por Rayos X , Dimerización , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Reproducibilidad de los Resultados , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/clasificaciónRESUMEN
cis-Biphenyl-2,3-dihydrodiol-2,3-dehydrogenase (BphB) is involved in the aerobic biodegradation of polychlorinated biphenyls (PCBs). The crystal structure of the NAD+-enzyme complex was determined by molecular replacement and refined to an R-value of 17.9% at 2.0 A. As a member of the short-chain alcohol dehydrogenase/reductase (SDR) family, the overall protein fold and positioning of the catalytic triad in BphB are very similar to those observed in other SDR enzymes, although small differences occur in the cofactor binding site. Modeling studies indicate that the substrate is bound in a deep hydrophobic cleft close to the nicotinamide moiety of the NAD+ cofactor. These studies further suggest that Asn143 is a key determinant of substrate specificity. A two-step reaction mechanism is proposed for cis-dihydrodiol dehydrogenases.