In-Ja L. Byeon

Polyglutamine disruption of the huntingtin exon 1 N terminus triggers a complex aggregation mechanism

Simple polyglutamine (polyQ) peptides aggregate in vitro via a nucleated growth pathway directly yielding amyloid-like aggregates. We show here that the 17-amino-acid flanking sequence (HTTNT) N-terminal to the polyQ in the toxic huntingtin exon 1 fragment imparts onto this peptide a complex alternative aggregation mechanism. In isolation, the HTTNT peptide is a compact coil that resists aggregation. When polyQ is fused to this sequence, it induces in HTTNT, in a repeat-length dependent fashion, a more extended conformation that greatly enhances its aggregation into globular oligomers with HTTNT cores and exposed polyQ. In a second step, a new, amyloid-like aggregate is formed with a core composed of both HTTNT and polyQ. The results indicate unprecedented complexity in how primary sequence controls aggregation within a substantially disordered peptide and have implications for the molecular mechanism of Huntingtons disease.

Structural convergence between Cryo-EM and NMR reveals intersubunit interactions critical for HIV-1 capsid function.

Mature HIV-1 particles contain conical-shaped capsids that enclose the viral RNA genome and perform essential functions in the virus life cycle. Previous structural analysis of two- and three-dimensional arrays of the capsid protein (CA) hexamer revealed three interfaces. Here, we present a cryoEM study of a tubular assembly of CA and a high-resolution NMR structure of the CA C-terminal domain (CTD) dimer. In the solution dimer structure, the monomers exhibit different relative orientations compared to previous X-ray structures. The solution structure fits well into the EM density map, suggesting that the dimer interface is retained in the assembled CA. We also identified a CTD-CTD interface at the local three-fold axis in the cryoEM map and confirmed its functional importance by mutagenesis. In the tubular assembly, CA intermolecular interfaces vary slightly, accommodating the asymmetry present in tubes. This provides the necessary plasticity to allow for controlled virus capsid dis/assembly.

Tumor suppressor p16INK4A: determination of solution structure and analyses of its interaction with cyclin-dependent kinase 4.

The solution structure of the tumor suppressor p16INK4A has been determined by NMR, and important recognition regions of both cdk4 and p16INK4A have been identified. The tertiary structure of p16INK4A contains four helix-turn-helix motifs linked by three loops. Twelve tumorigenic mutants of p16INK4A have been constructed and analyzed for their structure and activity, and new mutants have been designed rationally. A fragment of 58 residues at the N terminus of cdk4 important for p16INK4A binding has been identified. The importance of this region was further verified by mutational analysis of cdk4. These results and docking experiments have been used to assess possible modes of binding between p16INK4A and cdk4.

NMR structure of human restriction factor APOBEC3A reveals substrate binding and enzyme specificity

Human APOBEC3A (A3A) is a single-stranded DNA (ssDNA) cytidine deaminase that restricts viral pathogens and endogenous retrotransposons and plays a role in the innate immune response. Furthermore, its potential to act as a genomic DNA mutator has implications for a role in carcinogenesis. A deeper understanding of A3A’s deaminase and nucleic acid binding properties, which is central to its biological activities, has been limited by the lack of structural information. Here, we report the NMR solution structure of A3A and show that the critical interface for interaction with ssDNA substrates includes residues extending beyond the catalytic center. Importantly, by monitoring deaminase activity in real time, we find that A3A displays similar catalytic activity on A3A-specific TTCA- or A3G-specific CCCA-containing substrates, involving key determinants immediately 5′ of the reactive C. Our results afford novel mechanistic insights into A3A-mediated deamination and provide the structural basis for further molecular studies.

The C terminus of p53 binds the N-terminal domain of MDM2

The p53 tumor suppressor interacts with its negative regulator Mdm2 via the formers N-terminal region and core domain, yet the extreme p53 C-terminal region contains lysine residues ubiquitinated by Mdm2 and can bear post-translational modifications that inhibit Mdm2-p53 association. We show that the Mdm2-p53 interaction is decreased upon deletion, mutation or acetylation of the p53 C terminus. Mdm2 decreases the association of full-length but not C-terminally deleted p53 with a DNA target sequence in vitro and in cells. Further, using multiple approaches, we show that a peptide from the p53 C terminus directly binds the Mdm2 N terminus in vitro. We also show that p300-acetylated p53 inefficiently binds Mdm2 in vitro, and Nutlin-3 treatment induces C-terminal modification(s) of p53 in cells, explaining the low efficiency of Nutlin-3 in dissociating p53-MDM2 in vitro.

Solid-State NMR Studies of HIV-1 Capsid Protein Assemblies

In mature HIV-1 virions, the 26.6 kDa CA protein is assembled into a characteristic cone-shaped core (capsid) that encloses the RNA viral genome. The assembled capsid structure is best described by a fullerene cone model that is made up from a hexameric lattice containing a variable number of CA pentamers, thus allowing for closure of tubular or conical structures. In this paper, we present a solid-state NMR analysis of the wild-type HIV-1 CA protein, prepared as conical and spherical assemblies that are stable and are not affected by magic angle spinning of the samples at frequencies between 10 and 25 kHz. Multidimensional homo- and heteronuclear correlation spectra of CA assemblies of uniformly (13)C,(15)N-labeled CA exhibit narrow lines, indicative of the conformational homogeneity of the protein in these assemblies. For the conical assemblies, partial residue-specific resonance assignments were obtained. Analysis of the NMR spectra recorded for the conical and spherical assemblies indicates that the CA protein structure is not significantly different in the different morphologies. The present results demonstrate that the assemblies of CA protein are amenable to detailed structural analysis by solid-state NMR spectroscopy.

A protein contortionist: core mutations of GB1 that induce dimerization and domain swapping.

Immunoglobulin-binding domain B1 of streptococcal protein G (GB1), a small (56 residues), stable, single-domain protein, is one of the most extensively used model systems in the area of protein folding and design. Recently, NMR and X-ray structures of a quintuple GB1 core mutant (L5V/A26F/F30V/Y33F/A34F) that showed an unexpected, intertwined tetrameric architecture were determined. Here, we report the NMR structure of another mutant, derived from the tetramer by reverting the single amino acid position F26 back to the wild-type sequence A26. The structure reveals a domain-swapped dimer that involves exchange of the second beta-hairpin. The resulting overall structure comprises an eight-stranded beta-sheet whose concave side is covered by two alpha helices. The dimer dissociates into a partially folded, monomeric species with a dissociation constant of 93(+/-10)microM.

Tumor Suppressor p16INK4A: Structural Characterization of Wild-Type and Mutant Proteins by NMR and Circular Dichroism†

The tumor suppressor p16INK4A with eight N-terminal amino acids deleted (p16/delta 1-8) was expressed in Escherichia coli without any fusion artifacts and purified. The integrity of p16/delta 1-8 was confirmed by mass spectrometry, and its activity was demonstrated by in vitro cdk4 inhibition assay. Various physical methods were used to characterize the molecular and structural properties of p16/delta 1-8. The protein was found to oligomerize in vitro, as demonstrated by gel electrophoresis, mass spectrometry, and NMR. Various approaches, including changes of concentration and pH, additions of salts, detergents, and various organic solvents, and construction of a C-terminal deletion mutant and a cysteine mutant were used to try to reduce the extent of oligomerization. Only decreasing the protein concentration was found to reduce oligomerization. The affinity between p16 molecules in vivo was demonstrated by the yeast two-hybrid system. The protein was found to be very unstable on the basis of urea- and guanidinium chloride-induced denaturation studies monitored by NMR and CD, respectively. Despite these unfavorable properties, total NMR assignments were accomplished with uniform 13C and 15N isotope labeling. All multidimensional NMR experiments were performed at a very low concentration of 0.2 mM. The secondary structure was then determined from the NMR data. The results of NMR and CD studies indicate that the protein is highly alpha-helical, and the ankyrin repeat sequences show helix-turn-helix structures. This is the first structural information obtained for the important motif of ankyrin repeats. Overall, p16/delta 1-8 appears to be conformationally flexible. In order to understand the structural basis of the functional changes for some mutants existing in tumor cells, several missense mutants of p16/delta 1-8 were constructed. Four of them were expressed at high levels and purified. The molecular and structural properties of these mutants were analyzed by CD and NMR and compared with the corresponding properties of wild-type p16/delta 1-8. The results suggest that the functional changes in P114L and G101W are likely to be related to global conformational changes. In addition, we have demonstrated that the tendency of aggregation increases significantly by a single D84H mutation.

Sequential phosphorylation and multisite interactions characterize specific target recognition by the FHA domain of Ki67

The forkhead-associated (FHA) domain of human Ki67 interacts with the human nucleolar protein hNIFK, recognizing a 44-residue fragment, hNIFK226–269, phosphorylated at Thr234. Here we show that high-affinity binding requires sequential phosphorylation by two kinases, CDK1 and GSK3, yielding pThr238, pThr234 and pSer230. We have determined the solution structure of Ki67FHA in complex with the triply phosphorylated peptide hNIFK226–2693P, revealing not only local recognition of pThr234 but also the extension of the β-sheet of the FHA domain by the addition of a β-strand of hNIFK. The structure of an FHA domain in complex with a biologically relevant binding partner provides insights into ligand specificity and potentially links the cancer marker protein Ki67 to a signaling pathway associated with cell fate specification.

Motions on the millisecond time scale and multiple conformations of HIV-1 capsid protein: implications for structural polymorphism of CA assemblies.

The capsid protein (CA) of human immunodeficiency virus 1 (HIV-1) assembles into a cone-like structure that encloses the viral RNA genome. Interestingly, significant heterogeneity in shape and organization of capsids can be observed in mature HIV-1 virions. In vitro, CA also exhibits structural polymorphism and can assemble into various morphologies, such as cones, tubes, and spheres. Many intermolecular contacts that are critical for CA assembly are formed by its C-terminal domain (CTD), a dimerization domain, which was found to adopt different orientations in several X-ray and NMR structures of the CTD dimer and full-length CA proteins. Tyr145 (Y145), residue two in our CTD construct used for NMR structure determination, but not present in the crystallographic constructs, was found to be crucial for infectivity and engaged in numerous interactions at the CTD dimer interface. Here we investigate the origin of CA structural plasticity using solid-state NMR and solution NMR spectroscopy. In the solid state, the hinge region connecting the NTD and CTD is flexible on the millisecond time scale, as evidenced by the backbone motions of Y145 in CA conical assemblies and in two CTD constructs (137-231 and 142-231), allowing the protein to access multiple conformations essential for pleimorphic capsid assemblies. In solution, the CTD dimer exists as two major conformers, whose relative populations differ for the different CTD constructs. In the longer CTD (144-231) construct that contains the hinge region between the NTD and CTD, the populations of the two conformers are likely determined by the protonation state of the E175 side chain that is located at the dimer interface and within hydrogen-bonding distance of the W184 side chain on the other monomer. At pH 6.5, the major conformer exhibits the same dimer interface as full-length CA. In the short CTD (150-231) construct, no pH-dependent conformational shift is observed. These findings suggest that the presence of structural plasticity at the CTD dimer interface permits pleiotropic HIV-1 capsid assembly, resulting in varied capsid morphologies.