RESUMEN
The RIPK1-RIPK3 necrosome is an amyloid signaling complex that initiates TNF-induced necroptosis, serving in human immune defense, cancer, and neurodegenerative diseases. RIPK1 and RIPK3 associate through their RIP homotypic interaction motifs with consensus sequences IQIG (RIPK1) and VQVG (RIPK3). Using solid-state nuclear magnetic resonance, we determined the high-resolution structure of the RIPK1-RIPK3 core. RIPK1 and RIPK3 alternately stack (RIPK1, RIPK3, RIPK1, RIPK3, etc.) to form heterotypic ß sheets. Two such ß sheets bind together along a compact hydrophobic interface featuring an unusual ladder of alternating Ser (from RIPK1) and Cys (from RIPK3). The crystal structure of a four-residue RIPK3 consensus sequence is consistent with the architecture determined by NMR. The RIPK1-RIPK3 core is the first detailed structure of a hetero-amyloid and provides a potential explanation for the specificity of hetero- over homo-amyloid formation and a structural basis for understanding the mechanisms of signal transduction.
Asunto(s)
Amiloide/química , Proteína Serina-Treonina Quinasas de Interacción con Receptores/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Humanos , Resonancia Magnética Nuclear Biomolecular , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Alineación de SecuenciaRESUMEN
The interaction of lipids with subunit c from F1F0 ATP synthase is studied by biophysical methods. Subunit c from both Escherichia coli and Streptococcus pneumoniae interacts and copurifies with cardiolipin. Solid state NMR data on oligomeric rings of F0 show that the cardiolipin interacts with the c subunit in membrane bilayers. These studies offer strong support for the hypothesis that F0 has specific interactions with cardiolipin.
Asunto(s)
Cardiolipinas/química , Espectroscopía de Resonancia Magnética/métodos , ATPasas de Translocación de Protón Mitocondriales/química , Subunidades de ProteínaRESUMEN
A transverse-dephasing optimized S(3)E (spin-state selective excitation) method is implemented in solid-state NMR experiments of uniformly labeled protein samples, and it is shown to provide a simultaneous significant gain in both resolution (up to a factor of 2.2) and sensitivity (up to a factor of 1.4). This is illustrated with high-resolution NCO and NCA correlations of a microcrystalline sample of the oxidized form of the 153 residue human Cu(II)Zn(II) superoxide dismutase (SOD), a dimeric paramagnetic enzyme of 32 kDa. This method allows the resolution of 145 signals in the highly crowded carbonyl region in the NCO correlation spectrum.
Asunto(s)
Isótopos de Carbono/química , Espectroscopía de Resonancia Magnética/métodos , Proteínas/química , Cristalización , Espectroscopía de Resonancia Magnética/instrumentación , Resonancia Magnética Nuclear Biomolecular/métodos , Superóxido Dismutasa/químicaRESUMEN
The advantages offered by ultra-fast (>60 kHz) magic angle spinning (MAS) rotation for the study of biological samples, notably containing paramagnetic centers are explored. It is shown that optimal conditions for performing solid-state (13)C NMR under 60 kHz MAS are obtained with low-power CW (1)H decoupling, as well as after a low-power (1)H,(13)C cross-polarization step at a double-quantum matching condition. Acquisition with low-power decoupling highlights the existence of rotational decoupling sidebands. The sideband intensities and the existence of first and second rotary conditions are explained in the framework of the Floquet-van Vleck theory. As a result, optimal (13)C spectra of the oxidized, paramagnetic form of human copper zinc superoxide dismutase (SOD) can be obtained employing rf-fields which do not exceed 40 kHz during the whole experiment. This enables the removal of unwanted heating which can lead to deterioration of the sample. Furthermore, combined with the short (1)H T(1)s, this allows the repetition rate of the experiments to be shortened from 3 s to 500 ms, thus compensating for the sensitivity loss due to the smaller sample volume in a 1.3 mm rotor. The result is that 2D (13)C-(13)C correlation could be acquired in about 24 h on less than 1 mg of SOD sample.
Asunto(s)
Isótopos de Carbono/química , Hidrógeno/química , Espectroscopía de Resonancia Magnética/métodos , Superóxido Dismutasa/química , Estabilidad de Enzimas , Humanos , Cinética , Protones , Teoría Cuántica , Rotación , TermodinámicaRESUMEN
A magic angle spinning (MAS) NMR technique to transfer polarization from protons to a specific set of the (13)C spins is introduced for the study of biomolecular samples in the solid-state. Ultrafast (>60 kHz) MAS and low irradiation rf fields are used to achieve band-selective Hartmann-Hahn cross-polarization (CP) between the whole proton bath and carbons whose resonances are close to the (13)C-transmitter offset. When compared to conventional, broadband (1)H-(13)C CP, the band-selective experiment can be established without any loss of sensitivity when polarizing the aliphatic signals of a protein sample, and with a significant gain when polarizing carbonyls. This scheme can be used as a building block in 2D (13)C-(13)C homonuclear correlation experiments to obtain a faster and more sensitive characterization of biological solids.
Asunto(s)
Isótopos de Carbono/química , Espectroscopía de Resonancia Magnética/métodos , Protones , Carbono/química , Modelos Moleculares , Conformación Molecular , Estructura Molecular , TemperaturaRESUMEN
A two-dimensional proton-mediated carbon-carbon correlation experiment that relies on through-bond heteronuclear magnetization transfers is demonstrated in the context of solid-state NMR of proteins. This new experiment, dubbed J-CHHC by analogy to the previously developed dipolar CHHC techniques, is shown to provide selective and sensitive correlations in the methyl region of 2D spectra of crystalline organic compounds. The method is then demonstrated on a microcrystalline sample of the dimeric protein Crh (2 x 10.4 kDa). A total of 34 new proton-proton contacts involving side-chain methyl groups were observed in the J-CHHC spectrum, which had not been observed with the conventional experiment. The contacts were then used as additional distance restraints for the 3D structure determination of this microcrystalline protein. Upon addition of these new distance restraints, which are in large part located in the hydrophobic core of the protein, the root-mean-square deviation with respect to the X-ray structure of the backbone atom coordinates of the 10 best conformers of the new ensemble of structures is reduced from 1.8 to 1.1 A.