RESUMEN
Anomalous diffraction signals from typical native macromolecules are very weak, frustrating their use in de novo structure determination. Here, native SAD procedures are described to enhance signal to noise in anomalous diffraction by using multiple crystals in combination with synchrotron X-rays at 6â keV. Increased anomalous signals were obtained at 6â keV compared with 7â keV X-ray energy, which was used for previous native SAD analyses. A feasibility test of multi-crystal-based native SAD phasing was performed at 3.2â Å resolution for a known tyrosine protein kinase domain, and real-life applications were made to two novel membrane proteins at about 3.0â Å resolution. The three applications collectively serve to validate the robust feasibility of native SAD phasing at lower energy.
Asunto(s)
Proteínas Bacterianas/química , Cristalografía por Rayos X/métodos , Receptores ErbB/química , Conformación Proteica , Bacillus subtilis/química , Humanos , Listeria monocytogenes/química , Proteínas de Transporte de Membrana/química , Modelos Moleculares , Estructura Terciaria de Proteína , Reproducibilidad de los Resultados , SincrotronesRESUMEN
Vitamin A homeostasis is critical to normal cellular function. Retinol-binding protein (RBP) is the sole specific carrier in the bloodstream for hydrophobic retinol, the main form in which vitamin A is transported. The integral membrane receptor STRA6 mediates cellular uptake of vitamin A by recognizing RBP-retinol to trigger release and internalization of retinol. We present the structure of zebrafish STRA6 determined to 3.9-angstrom resolution by single-particle cryo-electron microscopy. STRA6 has one intramembrane and nine transmembrane helices in an intricate dimeric assembly. Unexpectedly, calmodulin is bound tightly to STRA6 in a noncanonical arrangement. Residues involved with RBP binding map to an archlike structure that covers a deep lipophilic cleft. This cleft is open to the membrane, suggesting a possible mode for internalization of retinol through direct diffusion into the lipid bilayer.
Asunto(s)
Proteínas de la Membrana/química , Proteínas de Transporte de Membrana/química , Proteínas de Unión al Retinol/química , Vitamina A/metabolismo , Proteínas de Pez Cebra/química , Animales , Transporte Biológico , Calcio/química , Calmodulina/química , Microscopía por Crioelectrón , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genética , Unión Proteica , Conformación Proteica en Hélice alfa , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas de Unión al Retinol/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Calcium homeostasis balances passive calcium leak and active calcium uptake. Human Bax inhibitor-1 (hBI-1) is an antiapoptotic protein that mediates a calcium leak and is representative of a highly conserved and widely distributed family, the transmembrane Bax inhibitor motif (TMBIM) proteins. Here, we present crystal structures of a bacterial homolog and characterize its calcium leak activity. The structure has a seven-transmembrane-helix fold that features two triple-helix sandwiches wrapped around a central C-terminal helix. Structures obtained in closed and open conformations are reversibly interconvertible by change of pH. A hydrogen-bonded, pKa (where Ka is the acid dissociation constant)-perturbed pair of conserved aspartate residues explains the pH dependence of this transition, and biochemical studies show that pH regulates calcium influx in proteoliposomes. Homology models for hBI-1 provide insights into TMBIM-mediated calcium leak and cytoprotective activity.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Calcio/metabolismo , Membrana Celular/metabolismo , Proteínas de la Membrana/química , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Humanos , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Estructura Secundaria de ProteínaRESUMEN
Structural and functional studies of many mammalian systems are critically dependent on abundant supplies of recombinant multiprotein complexes. Mammalian cells are often the most ideal, if not the only suitable host for such experiments. This is due to their intrinsic capability to generate functional mammalian proteins. This advantage is frequently countered by problems with yields in expression, time required to generate overexpressing lines, and elevated costs. Coexpression of multiple proteins adds another level of complexity to these experiments, as cells need to be screened and selected for expression of suitable levels of each component. Here, we present an efficient fluorescence marking procedure for establishing stable cell lines that overexpress two proteins in coordination, and we validate the method in the production of recombinant monoclonal antibody Fab fragments. This procedure may readily be expanded to systems of greater complexity, comprising more than two components.
Asunto(s)
Citometría de Flujo/métodos , Ingeniería Genética/métodos , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/aislamiento & purificación , Técnicas de Cultivo de Célula , Clonación Molecular , Vectores Genéticos/genética , Células HEK293 , Humanos , Fragmentos Fab de Inmunoglobulinas/biosíntesis , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , TransfecciónRESUMEN
Crystal structure analyses for biological macromolecules without known structural relatives entail solving the crystallographic phase problem. Typical de novo phase evaluations depend on incorporating heavier atoms than those found natively; most commonly, multi- or single-wavelength anomalous diffraction (MAD or SAD) experiments exploit selenomethionyl proteins. Here, we realize routine structure determination using intrinsic anomalous scattering from native macromolecules. We devised robust procedures for enhancing the signal-to-noise ratio in the slight anomalous scattering from generic native structures by combining data measured from multiple crystals at lower-than-usual x-ray energy. Using this multicrystal SAD method (5 to 13 equivalent crystals), we determined structures at modest resolution (2.8 to 2.3 angstroms) for native proteins varying in size (127 to 1148 unique residues) and number of sulfur sites (3 to 28). With no requirement for heavy-atom incorporation, such experiments provide an attractive alternative to selenomethionyl SAD experiments.
Asunto(s)
Cristalografía por Rayos X/métodos , Conformación Proteica , Proteínas/química , Proteínas Bacterianas/química , Interpretación Estadística de Datos , Proteínas Ligadas a GPI/química , Histidina Quinasa , Modelos Moleculares , Proteínas del Tejido Nervioso/química , Netrinas , Proteínas Quinasas/química , Estructura Terciaria de Proteína , Selenometionina/química , Relación Señal-Ruido , Azufre/química , Difracción de Rayos XRESUMEN
BACKGROUND: The GXGD-type diaspartyl intramembrane protease, presenilin, constitutes the catalytic core of the γ-secretase multi-protein complex responsible for activating critical signaling cascades during development and for the production of ß-amyloid peptides (Aß) implicated in Alzheimer's disease. The only other known GXGD-type diaspartyl intramembrane proteases are the eukaryotic signal peptide peptidases (SPPs). The presence of presenilin-like enzymes outside eukaryots has not been demonstrated. Here we report the existence of presenilin-like GXGD-type diaspartyl intramembrane proteases in archaea. METHODOLOGY AND PRINCIPAL FINDINGS: We have employed in vitro activity assays to show that MCMJR1, a polytopic membrane protein from the archaeon Methanoculleus marisnigri JR1, is an intramembrane protease bearing the signature YD and GXGD catalytic motifs of presenilin-like enzymes. Mass spectrometry analysis showed MCMJR1 could cleave model intramembrane protease substrates at several sites within their transmembrane region. Remarkably, MCMJR1 could also cleave substrates derived from the ß-amyloid precursor protein (APP) without the need of protein co-factors, as required by presenilin. Two distinct cleavage sites within the transmembrane domain of APP could be identified, one of which coincided with Aß40, the predominant site processed by γ-secretase. Finally, an established presenilin and SPP transition-state analog inhibitor could inhibit MCMJR1. CONCLUSIONS AND SIGNIFICANCE: Our findings suggest that a primitive GXGD-type diaspartyl intramembrane protease from archaea can recapitulate key biochemical properties of eukaryotic presenilins and SPPs. MCMJR1 promises to be a more tractable, simpler system for in depth structural and mechanistic studies of GXGD-type diaspartyl intramembrane proteases.
Asunto(s)
Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Membrana Celular/enzimología , Methanomicrobiaceae/enzimología , Péptido Hidrolasas/metabolismo , Presenilinas/metabolismo , Secuencia de Aminoácidos , Proteínas Arqueales/química , Membrana Celular/química , Membrana Celular/genética , Humanos , Methanomicrobiaceae/química , Methanomicrobiaceae/genética , Datos de Secuencia Molecular , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Presenilinas/química , Presenilinas/genética , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Alineación de SecuenciaRESUMEN
G-protein-coupled receptors (GPCRs) respond to external stimuli by activating heterotrimeric G proteins inside the cell. There is increasing evidence that many GPCRs exist as dimers or higher oligomers, but the biochemical nature of such dimers and what roles they have, if any, in signal transduction remains unclear. We conducted a comprehensive study of dimerization of the 5HT2c serotonin receptor using disulphide-trapping experiments. We found a dimer interface between transmembrane (TM) helices IV and V that is markedly sensitive to the state of receptor activation. This dimer seems to be quasisymmetrical in interfacial geometry and asymmetrical in its association with its cognate G alpha protein. We also found a second interface at TM I helices, which is insensitive to the state of activation.
Asunto(s)
Modelos Moleculares , Estructura Terciaria de Proteína , Receptor de Serotonina 5-HT2C/química , Receptor de Serotonina 5-HT2C/metabolismo , Línea Celular , Cisteína/genética , Dimerización , Humanos , Immunoblotting , Inmunoprecipitación , Ligandos , MutagénesisRESUMEN
Structural and functional analyses for many mammalian systems depend on having abundant supplies of recombinant multi-protein complexes that can be produced best, or only, in mammalian cells. We present an efficient fluorescence marking procedure for establishing stable cell lines that overexpress two proteins in co-ordination, and we validate the method in the production of monoclonal antibody Fab fragments. The procedure has worked without fail on all seven of seven trials on Fabs, which are being used in the crystallization of G-protein coupled receptors. This manner of efficient selection may readily be adapted for the co-production of other complexes of two or more proteins.
Asunto(s)
Color , Proteínas/metabolismo , Anticuerpos Monoclonales/biosíntesis , Línea Celular , Separación Celular , Fluorescencia , Humanos , Microscopía ConfocalRESUMEN
mAbs that are sensitive to protein conformation can be helpful in studies of protein structure and function; in particular, mAb fragments are useful reagents in membrane protein crystallization. We immunized mice with the rat 5HT2c serotonin receptor and derived clonal hybridoma cells, which we tested for specific antigen reactivity by using the complementarity of purified protein from bacteria and receptor-embedded mammalian cell membranes. Nine mAbs met our criteria for specificity, affinity, and sensitivity to conformational features. Epitopes were mapped in various additional tests. Five of the nine mAbs have cytoplasmic epitopes, and two of these are sensitive to the ligand state of the receptor. These properties should be useful both for structural analysis and in probes of function.