A two-site flexible clamp mechanism for RET-GDNF-GFRα1 assembly reveals both conformational adaptation and strict geometric spacing.

RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFRα co-receptors recognize and activate RET stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here, we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2 and CLD3. Comparison with a cryo-electron microscopy structure of a ligand-engaged zebrafish RETECD-GDNF-GFRα1a complex indicates conformational changes within a clade-specific CLD3 loop adjacent to the co-receptor. Our observations indicate that RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFRα co-receptors, while its rigid arm recognizes a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualize linear arrays of RETECD-GDNF-GFRα1a suggesting that a conserved contact stabilizes higher-order species. Our study reveals that ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands.

Three-Dimensional Architecture of the Human BRCA1-A Histone Deubiquitinase Core Complex.

BRCA1 is a tumor suppressor found to be mutated in hereditary breast and ovarian cancer and plays key roles in the maintenance of genomic stability by homologous recombination repair. It is recruited to damaged chromatin as a component of the BRCA1-A deubiquitinase, which cleaves K63-linked ubiquitin chains attached to histone H2A and H2AX. BRCA1-A contributes to checkpoint regulation, repair pathway choice, and HR repair efficiency through molecular mechanisms that remain largely obscure. The structure of an active core complex comprising two Abraxas/BRCC36/BRCC45/MERIT40 tetramers determined by negative-stain electron microscopy (EM) reveals a distorted V-shape architecture in which a dimer of Abraxas/BRCC36 heterodimers sits at the base, with BRCC45/Merit40 pairs occupying each arm. The location and ubiquitin-binding activity of BRCC45 suggest that it may provide accessory interactions with nucleosome-linked ubiquitin chains that contribute to their efficient processing. Our data also suggest how ataxia telangiectasia mutated (ATM)-dependent BRCA1 dimerization may stabilize self-association of the entire BRCA1-A complex.

Role of calpain in the formation of human papillomavirus type 16 E1

The human papillomavirus (HPV) type 16 E1^E4 (16E1^E4) protein is expressed in the middle to upper layers of infected epithelium and has several roles within the virus life cycle. It is apparent that within the epithelium there are multiple species of 16E1^E4 that differ in length and/or degree of phosphorylation and that some or all of these can associate with the cellular keratin networks, leading to network disruption. We show here that the cellular cysteine protease calpain cleaves the 16E1^E4 protein after amino acid 17 to generate species that lack the N terminus. These C-terminal fragments are able to multimerize and form amyloid-like fibers. This can lead to accumulation of 16E1^E4 and disruption of the normal dynamics of the keratin networks. The cleavage of E1^E4 proteins by calpain may be a common strategy used by α-group viruses, since we show that cleavage of type 18 E1^E4 in raft culture is also dependent on calpain. Interestingly, the cleavage of 16E1^E4 by calpain appears to be highly regulated as differentiation of HPV genome-containing cells by methylcellulose is insufficient to induce cleavage. We hypothesize that this is important since it ensures that the formation of the amyloid fibers is not prematurely triggered in the lower layers and is restricted to the upper layers, where calpain is active and where disruption of the keratin networks may aid virus release.

E1--E4-mediated keratin phosphorylation and ubiquitylation: a mechanism for keratin depletion in HPV16-infected epithelium.

The keratin IF network of epidermal keratinocytes provides a protective barrier against mechanical insult, it is also a major player in absorbing stress in these cells. The human papilloma virus (HPV) type 16 E1--E4 protein accumulates in the upper layers of HPV16-infected epithelium and is known to associate with and reorganise the keratin IF network in cells in culture. Here, we show that this function is conserved amongst a number of HPV alpha-group E1--E4 proteins and that the differentiation-dependent keratins are also targeted. Using time-lapse microscopy, HPV16 E1--E4 was found to effect a dramatic cessation of keratin IF network dynamics by associating with both soluble and insoluble keratin. Network disruption was accompanied by keratin hyperphosphorylation at several sites, including K8 S73, which is typically phosphorylated in response to stress stimuli. Keratin immunoprecipitated from E1--E4-expressing cells was also found to be ubiquitylated, indicating that it is targeted for proteasomal degradation. Interestingly, the accumulation of hyperphosphorylated, ubiquitylated E1--E4-keratin structures was found to result in an impairment of proteasomal function. These observations shed new light on the mechanism of keratin IF network reorganisation mediated by HPV16 E1--E4 and provide an insight into the depletion of keratin co-incident with E1--E4 accumulation observed in HPV-infected epithelium.

Phosphorylation of the human papillomavirus type 16 E1--E4 protein at T57 by ERK triggers a structural change that enhances keratin binding and protein stability.

The E1--E4 protein of human papillomavirus type 16 (HPV16) causes cytokeratin reorganization in the middle and upper epithelial layers and is thought to contribute to multiple facets of the virus life cycle. Although little is known as to how HPV16 E1--E4 (16E1--E4) functions are controlled following the first expression of this protein, the finding that low-risk E1--E4 proteins can be phosphorylated in vivo suggests an important role for kinases. Here, we show that 16E1--E4 is phosphorylated by cyclin-dependent kinase 1 (CDK1) and CDK2, extracellular signal-regulated kinase (ERK), protein kinase A (PKA), and PKC alpha, with CDK1/2 serine 32 and ERK threonine 57 phosphorylations representing the two primary events seen in cells in cycle. Interestingly, T57 phosphorylation was found to trigger a structural change in the 16E1--E4 protein that compacts the central fold region, leading to an increase in 16E1--E4 stability and overall abundance in the cell. When compared to wild-type 16E1--E4, a T57D phosphomimic was found to have greatly enhanced keratin-binding ability and an ability to modulate the binding of the unphosphorylated form, with keratin binding protecting the T57-phosphorylated form of 16E1--E4 from proteasomal degradation. In HPV16 genome-containing organotypic rafts, the T57-phosphorylated form was specifically detected in the intermediate cell layers, where productive infection occurs, suggesting that T57 phosphorylation may have a functional role at this stage of the viral life cycle. Interestingly, coexpression with 16E5 and ERK activation enhanced T57 phosphorylation, suggesting that E1--E4 and E5 may work together in vivo. Our data suggest a model in which the expression of 16E5 from the major E1--E4-E5 mRNA promotes T57 phosphorylation of E1--E4 and keratin binding, with dephosphorylation occurring following the switch to late poly(A) usage. Other forms of E1--E4, with alternative functional roles, may then increase in prevalence in the upper layers of the epithelium.

Structural analysis reveals an amyloid form of the human papillomavirus type 16 E1--E4 protein and provides a molecular basis for its accumulation.

The abundant human papillomavirus (HPV) type 16 E4 protein exists as two distinct structural forms in differentiating epithelial cells. Monomeric full-length 16E1--E4 contains a limited tertiary fold constrained by the N and C termini. N-terminal deletions facilitate the assembly of E1--E4 into amyloid-like fibrils, which bind to thioflavin T. The C-terminal region is highly amyloidogenic, and its deletion abolishes amyloid staining and prevents E1--E4 accumulation. Amyloid-imaging probes can detect 16E1--E4 in biopsy material, as well as 18E1--E4 and 33E1--E4 in monolayer cells, indicating structural conservation. Our results suggest a role for fibril formation in facilitating the accumulation of E1--E4 during HPV infection.

NMR structure of the DNA-binding domain of the cell cycle protein Mbp1 from Saccharomyces cerevisiae.

The three-dimensional solution structure of the DNA-binding domain of Mlu-1 box binding protein (Mbp1) has been determined by multidimensional NMR spectroscopy. Mbp1 is a cell cycle transcription factor from Saccharomyces cerevisiae and consists of an N-terminal DNA-binding domain, a series of ankyrin repeats, and a heterodimerization domain at the C-terminus. A set of conformers comprising 19 refined structures was calculated via a molecular dynamics simulated annealing protocol using distance, dihedral angle, and residual dipolar coupling restraints derived from either double or triple resonance NMR experiments. The solution structure consists of a six-stranded beta-sheet segment folded against two pairs of alpha-helices in the topology of the winged helix-turn-helix family of proteins and is in agreement with the X-ray structures. In addition, the solution structure shows that the C-terminal tail region of this domain folds back and makes specific interactions with the N-terminal beta-strand of the protein. This C-terminal region is essential for full DNA-binding activity but appears in the X-ray structure to be disordered. The fold-back structure extends the region of positive electrostatic potential, and this may enhance the nonspecific contribution to binding by favorable electrostatic interactions with the DNA backbone.