The homotetrameric tumor suppressor p53 consists of folded core and tetramerization domains, linked and flanked by intrinsically disordered segments that impede structure analysis by x-ray crystallography and NMR. Here, we solved the quaternary structure of human p53 in solution by a combination of small-angle x-ray scattering, which defined its shape, and NMR, which identified the core domain interfaces and showed that the folded domains had the same structure in the intact protein as in fragments. We combined the solution data with electron microscopy on immobilized samples that provided medium resolution 3D maps. Ab initio and rigid body modeling of scattering data revealed an elongated cross-shaped structure with a pair of loosely coupled core domain dimers at the ends, which are accessible for binding to DNA and partner proteins. The core domains in that open conformation closed around a specific DNA response element to form a compact complex whose structure was independently determined by electron microscopy. The structure of the DNA complex is consistent with that of the complex of four separate core domains and response element fragments solved by x-ray crystallography and contacts identified by NMR. Electron microscopy on the conformationally mobile, unbound p53 selected a minor compact conformation, which resembled the closed conformation, from the ensemble of predominantly open conformations. A multipronged structural approach could be generally useful for the structural characterization of the rapidly growing number of multidomain proteins with intrinsically disordered regions.DNA binding ͉ intrinsically unfolded ͉ modular ͉ natively disordered ͉ protein T he tumor suppressor p53 is a tetrameric, multidomain transcription factor that plays a central role in the cell cycle and maintaining genomic integrity (1, 2). It binds to specific DNA response elements, is integrated in various signaling networks by a multitude of protein-protein interactions, and is controlled by extensive posttranslational modifications (1, 3, 4). p53 protein is a homotetramer of 4 ϫ 393 residues. Each chain consists of two folded domains [the core, and the tetramerization domain (323-360)] that are linked by an intrinsically disordered sequence. The transactivation domain (1-67) (5), proline-rich region (67-94), nuclear localization signal (NLS)-containing region (303-323) (6), and C-terminal negative regulatory domain (360-393) are also intrinsically disordered (7-9) (see refs. 10 and 11 for reviews). The DNAbinding core domain (residues 94-294) binds to sequencespecific response elements associated with p53 target gene promoters (12)(13)(14). The structures of the core domain complexes with DNA have been solved by crystallography (15)(16)(17), and in solution in the absence of DNA by NMR (18). The structure of the tetramerization domain has been solved by both NMR and x-ray crystallography (19-21).Structural studies on full-length p53 have been impeded both by its intrinsic instability and the presence of disordered regions (...
determined to be 50 and 2000 , respectively, using a calibrated oscillating quartz crystal thickness monitor. After deposition, the devices were encapsulated with epoxy (Loctite quick-set epoxy) under an argon atmosphere in order to minimize exposure to oxygen and moisture. All device measurements were made at room temperature.Photoluminescence and electroluminescence spectra were measured on a SPEX Fluorolog-2 equipped with a liquid N 2 cooled InGaAs detector (800± 1600 nm), or on a spectrometer consisting of an ISA-SPEX Triax 180 spectrograph equipped with a liquid N 2 cooled CCD detector (Hamamatsu CCD, 1024 64 pixel, 400±1100 nm). Emission quantum yields were measured by relative actinometry with H 2 TPP (u = 0.11) or ZnTPP (u = 0.033). Near-IR quantum yields were determined for Yb(TPP)L(OEt) on the CCD fluorescence system using the visible H 2 TPP and ZnTPP actinometers. Then the emission quantum yields for the Nd and Er complexes were determined relative to the Yb(TPP)L(OEt) using the SPEX Fluorolog-2 with the InGaAs detector.Power for electroluminescence (EL) measurements was supplied using a Keithley 228 voltage/current source. A 100 W primary standard quartz halogen lamp was used to calibrate the Triax 180 spectrograph/CCD detector system in irradiance units (lW cm 2 nm ±1 ). Measurements were made normal to the surface of the devices, and in the computation of the EL quantum efficiencies it was assumed that the spatial distribution of the emission was Lambertian [23]. External device quantum efficiencies were calculated as described in the literature [27].
NADPH-cytochrome P450 reductase (CPR), a diflavin reductase, plays a key role in the mammalian P450 mono-oxygenase system. In its crystal structure, the two flavins are close together, positioned for interflavin electron transfer but not for electron transfer to cytochrome P450. A number of lines of evidence suggest that domain motion is important in the action of the enzyme. We report NMR and small-angle x-ray scattering experiments addressing directly the question of domain organization in human CPR. Comparison of the 1H-15N heteronuclear single quantum correlation spectrum of CPR with that of the isolated FMN domain permitted identification of residues in the FMN domain whose environment differs in the two situations. These include several residues that are solvent-exposed in the CPR crystal structure, indicating the existence of a second conformation in which the FMN domain is involved in a different interdomain interface. Small-angle x-ray scattering experiments showed that oxidized and NADPH-reduced CPRs have different overall shapes. The scattering curve of the reduced enzyme can be adequately explained by the crystal structure, whereas analysis of the data for the oxidized enzyme indicates that it exists as a mixture of approximately equal amounts of two conformations, one consistent with the crystal structure and one a more extended structure consistent with that inferred from the NMR data. The correlation between the effects of adenosine 2′,5′-bisphosphate and NADPH on the scattering curve and their effects on the rate of interflavin electron transfer suggests that this conformational equilibrium is physiologically relevant.
We demonstrate here the applicability of X-ray scattering for studying molecular conformation of multimeric proteins in solution by using synchrotron radiation to extend the range of data collection to include medium angles (ca. 3-4 degrees). We have been able to define the solution structure of the dissimilatory nitrite reductase of Achromobacter xylosoxidans (AxNiR), an enzyme for which there are conflicting reports as to the nature of its multimeric structure. Quantitative interpretation of the X-ray scattering profile, based on a modeling study using the high-resolution crystal structure data for the nitrite reductase from the related organism Achromobacter cycloclastes (AcNiR), provides a detailed model for the trimeric structure of AxNiR in solution. Sedimentation equilibrium centrifugation gave an M(r) of 103,000, consistent with such a trimeric structure.
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