RESUMO
Overexpression of the epidermal growth factor (EGF) receptor (EGFR) in cancer cells correlates with tumor malignancy and poor prognosis for cancer patients. For this reason, the EGFR has become one of the main targets of anticancer therapies. Structural data obtained in the last few years have revealed the molecular mechanism for ligand-induced EGFR dimerization and subsequent signal transduction, and also how this signal is blocked by either monoclonal antibodies or small molecules. Nimotuzumab (also known as h-R3) is a humanized antibody that targets the EGFR and has been successful in the clinics. In this work, we report the crystal structure of the Fab fragment of Nimotuzumab, revealing some unique structural features in the heavy variable domain. Furthermore, competition assays show that Nimotuzumab binds to domain III of the extracellular region of the EGFR, within an area that overlaps with both the surface patch recognized by Cetuximab (another anti-EGFR antibody) and the binding site for EGF. A computer model of the Nimotuzumab-EGFR complex, constructed by docking and molecular dynamics simulations and supported by mutagenesis studies, unveils a novel mechanism of action, with Nimotuzumab blocking EGF binding while still allowing the receptor to adopt its active conformation, hence warranting a basal level of signaling.
Assuntos
Anticorpos Monoclonais/química , Antineoplásicos/química , Receptores ErbB/química , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Sítios de Ligação , Ligação Competitiva , Cristalografia por Raios X , Receptores ErbB/genética , Receptores ErbB/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Ligantes , Camundongos , Modelos Moleculares , Conformação Molecular , Mutação , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Multiple-sequence alignment of glycoside hydrolase (GH) families 32, 43, 62, and 68 revealed three conserved blocks, each containing an acidic residue at an equivalent position in all the enzymes. A detailed analysis of the site-directed mutations so far performed on invertases (GH32), arabinanases (GH43), and bacterial fructosyltransferases (GH68) indicated a direct implication of the conserved residues Asp/Glu (block I), Asp (block II), and Glu (block III) in substrate binding and hydrolysis. These residues are close in space in the 5-bladed beta-propeller fold determined for Cellvibrio japonicus alpha-L-arabinanase Arb43A [Nurizzo et al., Nat Struct Biol 2002;9:665-668] and Bacillus subtilis endo-1,5-alpha-L-arabinanase. A sequence-structure compatibility search using 3D-PSSM, mGenTHREADER, INBGU, and SAM-T02 programs predicted indistinctly the 5-bladed beta-propeller fold of Arb43A and the 6-bladed beta-propeller fold of sialidase/neuraminidase (GH33, GH34, and GH83) as the most reliable topologies for GH families 32, 62, and 68. We conclude that the identified acidic residues are located at the active site of a beta-propeller architecture in GH32, GH43, GH62, and GH68, operating with a canonical reaction mechanism of either inversion (GH43 and likely GH62) or retention (GH32 and GH68) of the anomeric configuration. Also, we propose that the beta-propeller architecture accommodates distinct binding sites for the acceptor saccharide in glycosyl transfer reaction.
Assuntos
Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Biologia Computacional , Sequência Conservada , Glicosídeo Hidrolases/classificação , Glicosídeo Hidrolases/genética , Hidrólise , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Dobramento de Proteína , Alinhamento de Sequência , SoftwareRESUMO
The endophytic bacterium Gluconacetobacter diazotrophicus SRT4 secretes a constitutively expressed levansucrase (LsdA; EC 2.4.1.10), which converts sucrose to fructo-oligosaccharides and levan. Fully active LsdA was purified to high homogeneity by non-denaturing reversed-phase HPLC and was crystallized at room temperature by the hanging-drop vapour-diffusion method using ammonium sulfate and ethanol as precipitants. The crystals are extremely sensitive, but native data have been collected to 2.5 A under cryogenic conditions using synchrotron radiation. LsdA crystals belong to the orthorhombic space group P22(1)2(1) or P2(1)2(1)2, with unit-cell parameters a = 53.80, b = 119.39, c = 215.10 A.