Roberto Melero

Quaternary structures of tumor suppressor p53 and a specific p53–DNA complex

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.

A clustering approach to multireference alignment of single-particle projections in electron microscopy.

Two-dimensional analysis of projections of single-particles acquired by an electron microscope is a useful tool to help identifying the different kinds of projections present in a dataset and their different projection directions. Such analysis is also useful to distinguish between different kinds of particles or different particle conformations. In this paper we introduce a new algorithm for performing two-dimensional multireference alignment and classification that is based on a Hierarchical clustering approach using correntropy (instead of the more traditional correlation) and a modified criterion for the definition of the clusters specially suited for cases in which the Signal-to-Noise Ratio of the differences between classes is low. We show that our algorithm offers an improved sensitivity over current methods in use for distinguishing between different projection orientations and different particle conformations. This algorithm is publicly available through the software package Xmipp.

Averaging of Electron Subtomograms and Random Conical Tilt Reconstructions through Likelihood Optimization

The reference-free averaging of three-dimensional electron microscopy (3D-EM) reconstructions with empty regions in Fourier space represents a pressing problem in electron tomography and single-particle analysis. We present a maximum likelihood algorithm for the simultaneous alignment and classification of subtomograms or random conical tilt (RCT) reconstructions, where the Fourier components in the missing data regions are treated as hidden variables. The behavior of this algorithm was explored using tests on simulated data, while application to experimental data was shown to yield unsupervised class averages for subtomograms of groEL/groES complexes and RCT reconstructions of p53. The latter application served to obtain a reliable de novo structure for p53 that may resolve uncertainties about its quaternary structure.

The cryo-EM structure of the UPF–EJC complex shows UPF1 poised toward the RNA 3′ end

Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that degrades aberrant mRNAs containing premature termination codons (PTCs). NMD is triggered upon the assembly of the UPF surveillance complex near a PTC. In humans, UPF assembly is prompted by the exon junction complex (EJC). We investigated the molecular architecture of the human UPF complex bound to the EJC by cryo-EM and using positional restraints from additional EM, MS and biochemical interaction data. The heptameric assembly is built around UPF2, a scaffold protein with a ring structure that closes around the CH domain of UPF1, keeping the helicase region in an accessible and unwinding-competent state. UPF2 also positions UPF3 to interact with the EJC. The geometry is such that this transient complex poises UPF1 to elicit helicase activity toward the 3′ end of the mRNP.

Electron microscopy studies on the quaternary structure of p53 reveal different binding modes for p53 tetramers in complex with DNA.

The multidomain homotetrameric tumor suppressor p53 has two modes of binding dsDNA that are thought to be responsible for scanning and recognizing specific response elements (REs). The C termini bind nonspecifically to dsDNA. The four DNA-binding domains (DBDs) bind REs that have two symmetric 10 base-pair sequences. p53 bound to a 20-bp RE has the DBDs enveloping the DNA, which is in the center of the molecule surrounded by linker sequences to the tetramerization domain (Tet). We investigated by electron microscopy structures of p53 bound to DNA sequences consisting of a 20-bp RE with either 12 or 20 bp nonspecific extensions on either end. We found a variety of structures that give clues to recognition and scanning mechanisms. The 44- and 60-bp sequences gave rise to three and four classes of structures, respectively. One was similar to the known 20-bp structure, but the DBDs in the other classes were loosely arranged and incompatible with specific DNA recognition. Some of the complexes had density consistent with the C termini extending from Tet to the DNA, adjacent to the DBDs. Single-molecule fluorescence resonance energy transfer experiments detected the approach of the C termini towards the DBDs on addition of DNA. The structural data are consistent with p53 sliding along DNA via its C termini and the DNA-binding domains hopping on and off during searches for REs. The loose structures and posttranslational modifications account for the affinity of nonspecific DNA for p53 and point to a mechanism of enhancement of specificity by its binding to effector proteins.

Design of coiled-coil protein-origami cages that self-assemble in vitro and in vivo

Polypeptides and polynucleotides are natural programmable biopolymers that can self-assemble into complex tertiary structures. We describe a system analogous to designed DNA nanostructures in which protein coiled-coil (CC) dimers serve as building blocks for modular de novo design of polyhedral protein cages that efficiently self-assemble in vitro and in vivo. We produced and characterized >20 single-chain protein cages in three shapes—tetrahedron, four-sided pyramid, and triangular prism—with the largest containing >700 amino-acid residues and measuring 11 nm in diameter. Their stability and folding kinetics were similar to those of natural proteins. Solution small-angle X-ray scattering (SAXS), electron microscopy (EM), and biophysical analysis confirmed agreement of the expressed structures with the designs. We also demonstrated self-assembly of a tetrahedral structure in bacteria, mammalian cells, and mice without evidence of inflammation. A semi-automated computational design platform and a toolbox of CC building modules are provided to enable the design of protein cages in any polyhedral shape.

Modulation of the Chaperone DnaK Allosterism by the Nucleotide Exchange Factor GrpE

Background: DnaK is a Hsp70 (heat shock protein) molecular chaperone whose function is controlled in part by the nucleotide exchange factor GrpE. Results: We describe the structures of several complexes formed between GrpE and different DnaK variants. Conclusion: The DnaK-GrpE complex has several conformations, an important factor in regulating DnaK function. Significance: The information obtained explains the nucleotide exchange role of GrpE in much more detail. Hsp70 chaperones comprise two domains, the nucleotide-binding domain (Hsp70NBD), responsible for structural and functional changes in the chaperone, and the substrate-binding domain (Hsp70SBD), involved in substrate interaction. Substrate binding and release in Hsp70 is controlled by the nucleotide state of DnaKNBD, with ATP inducing the open, substrate-receptive DnaKSBD conformation, whereas ADP forces its closure. DnaK cycles between the two conformations through interaction with two cofactors, the Hsp40 co-chaperones (DnaJ in Escherichia coli) induce the ADP state, and the nucleotide exchange factors (GrpE in E. coli) induce the ATP state. X-ray crystallography showed that the GrpE dimer is a nucleotide exchange factor that works by interaction of one of its monomers with DnaKNBD. DnaKSBD location in this complex is debated; there is evidence that it interacts with the GrpE N-terminal disordered region, far from DnaKNBD. Although we confirmed this interaction using biochemical and biophysical techniques, our EM-based three-dimensional reconstruction of the DnaK-GrpE complex located DnaKSBD near DnaKNBD. This apparent discrepancy between the functional and structural results is explained by our finding that the tail region of the GrpE dimer in the DnaK-GrpE complex bends and its tip contacts DnaKSBD, whereas the DnaKNBD-DnaKSBD linker contacts the GrpE helical region. We suggest that these interactions define a more complex role for GrpE in the control of DnaK function.

Human nonsense-mediated mRNA decay factor UPF2 interacts directly with eRF3 and the SURF complex

Nonsense-mediated mRNA decay (NMD) is an mRNA degradation pathway that regulates gene expression and mRNA quality. A complex network of macromolecular interactions regulates NMD initiation, which is only partially understood. According to prevailing models, NMD begins by the assembly of the SURF (SMG1–UPF1–eRF1–eRF3) complex at the ribosome, followed by UPF1 activation by additional factors such as UPF2 and UPF3. Elucidating the interactions between NMD factors is essential to comprehend NMD, and here we demonstrate biochemically and structurally the interaction between human UPF2 and eukaryotic release factor 3 (eRF3). In addition, we find that UPF2 associates with SURF and ribosomes in cells, in an UPF3-independent manner. Binding assays using a collection of UPF2 truncated variants reveal that eRF3 binds to the C-terminal part of UPF2. This region of UPF2 is partially coincident with the UPF3-binding site as revealed by electron microscopy of the UPF2–eRF3 complex. Accordingly, we find that the interaction of UPF2 with UPF3b interferes with the assembly of the UPF2–eRF3 complex, and that UPF2 binds UPF3b more strongly than eRF3. Together, our results highlight the role of UPF2 as a platform for the transient interactions of several NMD factors, including several components of SURF.

Single-particle electron microscopy structure of UDP-glucose:glycoprotein glucosyltransferase suggests a selectivity mechanism for misfolded proteins

The enzyme UDP-glucose:glycoprotein glucosyltransferase (UGGT) mediates quality control of glycoproteins in the endoplasmic reticulum by attaching glucose to N-linked glycan of misfolded proteins. As a sensor, UGGT ensures that misfolded proteins are recognized by the lectin chaperones and do not leave the secretory pathway. The structure of UGGT and the mechanism of its selectivity for misfolded proteins have been unknown for 25 years. Here, we used negative-stain electron microscopy and small-angle X-ray scattering to determine the structure of UGGT from Drosophila melanogaster at 18-Å resolution. Three-dimensional reconstructions revealed a cage-like structure with a large central cavity. Particle classification revealed flexibility that precluded determination of a high-resolution structure. Introduction of biotinylation sites into a fungal UGGT expressed in Escherichia coli allowed identification of the catalytic and first thioredoxin-like domains. We also used hydrogen-deuterium exchange mass spectrometry to map the binding site of an accessory protein, Sep15, to the first thioredoxin-like domain. The UGGT structural features identified suggest that the central cavity contains the catalytic site and is lined with hydrophobic surfaces. This enhances the binding of misfolded substrates with exposed hydrophobic residues and excludes folded proteins with hydrophilic surfaces. In conclusion, we have determined the UGGT structure, which enabled us to develop a plausible functional model of the mechanism for UGGTs selectivity for misfolded glycoproteins.

Denoising of high-resolution single-particle electron-microscopy density maps by their approximation using three-dimensional Gaussian functions.

Cryo-electron microscopy (cryo-EM) of frozen-hydrated preparations of isolated macromolecular complexes is the method of choice to obtain the structure of complexes that cannot be easily studied by other experimental methods due to their flexibility or large size. An increasing number of macromolecular structures are currently being obtained at subnanometer resolution but the interpretation of structural details in such EM-derived maps is often difficult because of noise at these high-frequency signal components that reduces their contrast. In this paper, we show that the method for EM density-map approximation using Gaussian functions can be used for denoising of single-particle EM maps of high (typically subnanometer) resolution. We show its denoising performance using simulated and experimental EM density maps of several complexes.