Journal of molecular biology [journal]
- 3D structure and interaction of p24β and p24δ Golgi dynamics domains: Implication for p24 complex formation and cargo transport. [JOURNAL ARTICLE]
- J Mol Biol 2016 Aug 25.
The p24 family consists of four subfamilies (p24α, p24β, p24γ and p24δ) and the proteins are thought to form hetero-oligomeric complexes for efficient transport of cargo proteins from the endoplasmic reticulum to the Golgi apparatus. The proteins possess a conserved luminal Golgi dynamics (GOLD) domain, whose functions are largely unknown. Here we present structural and biochemical studies of p24β1 and p24δ1 GOLD domains. Use of GOLD domain-deleted mutants revealed that the GOLD domain of p24δ1 is required for proper p24 hetero-oligomeric complex formation and efficient transport of GPI-anchored proteins. The p24β1 and p24δ1 GOLD domains share a common β-sandwich fold with a characteristic intra-sheet disulfide bond. The GOLD domain of p24δ1 crystallized as dimers, allowing analysis of a homophilic interaction site. Surface plasmon resonance and solution nuclear magnetic resonance analyses revealed that p24β1 and p24δ1 GOLD domains interact weakly (Kd~10(-4)M). Bi-protein titration provided interaction site maps. We propose that the heterophilic interaction of p24 GOLD domains contributes to formation of the p24 hetero-oligomeric complex as well as efficient cargo transport.
- The kinetic stability of a full-length antibody light chain dimer determines whether endoproteolysis can release amyloidogenic variable domains. [JOURNAL ARTICLE]
- J Mol Biol 2016 Aug 25.
Light chain amyloidosis (AL amyloidosis) appears to be caused by the aggregation of an antibody light chain (LC) or fragment thereof, and is fatal if untreated. LCs are secreted from clonally expanded plasma cells as disulfide-linked dimers, with each monomer comprising one constant and one variable domain. The energetic contribution of each domain and the role of endoproteolysis in AL amyloidosis remain unclear. To investigate why only some LCs form amyloid and cause organ toxicity, we measured the aggregation propensity and kinetic stability of LC dimers and associated variable domains from AL amyloidosis patients and non-patients. All the variable domains studied readily form amyloid fibrils, whereas none of the full-length LC dimers, even those from AL amyloidosis patients, are amyloidogenic. Kinetic stability-that is, the free energy difference between the native state and the unfolding transition state-dictates the LC's unfolding rate. Full-length LC dimers derived from AL amyloidosis patients unfold more rapidly than other full-length LC dimers and can be readily cleaved into their component domains by proteases, whereas non-amyloidogenic LC dimers are more kinetically stable and resistant to endoproteolysis. Our data suggest that amyloidogenic LC dimers are kinetically unstable (unfold faster) and thus are susceptible to endoproteolysis that results in the release amyloidogenic LC fragments, whereas other LCs are not as amenable to unfolding and endoproteolysis and are therefore aggregation resistant. Pharmacologic kinetic stabilization of the full-length LC dimer could be a useful strategy to treat AL amyloidosis.
- Structure and function of the RING domains of RNF20 and RNF40, dimeric E3 ligases that monoubiquitylate histone H2B. [JOURNAL ARTICLE]
- J Mol Biol 2016 Aug 25.
Monoubiquitylation of histone H2B is a post-translational mark that plays key roles in transcription regulation and genome stability. In humans, attachment of ubiquitin to lysine 120 of histone H2B depends on the activity of the E2 ubiquitin-conjugating enzyme, Ube2B, and the RING E3 ligases, RNF20 and RNF40. To better understand the molecular basis of this modification, we have solved the crystal structure of the RNF20 RING domain and show that it is a homodimer that specifically interacts with the Ube2B~Ub conjugate. By mutating residues at the E3-E2 and E3-ubiquitin interfaces we identify key contacts required for interaction of the RNF20 RING domain with the Ube2B~Ub conjugate. These mutants were used to generate a structure-based model of the RNF20-Ube2B~Ub complex that reveals differences from other RING-E2~Ub complexes, and suggests how the RNF20-Ube2B~Ub complex might interact with its nucleosomal substrate. Additionally, we show that the RING domains of RNF20 and RNF40 can form a stable heterodimer that is active. Our studies provide new insights into the mechanisms that regulate RNF20-mediated ubiquitin transfer from Ube2B.
- Structural Conservation and E2F Binding Specificity within the Retinoblastoma Pocket Protein Family. [JOURNAL ARTICLE]
- J Mol Biol 2016 Aug 24.
The human pocket proteins Rb, p107 and p130 are critical negative regulators of the cell cycle and contribute to tumor suppression. While strong structural conservation within the pocket protein family provides for some functional redundancy, important differences have been observed and may underlie why Rb is a uniquely potent tumor suppressor. It has been proposed that different E2F transcription factor binding partners mediate distinct pocket protein activities. In humans, Rb binds E2F1-5, whereas p107 and p130 almost exclusively associate with E2F4 and E2F5. To identify the molecular determinants of this specificity, we compared the crystal structures of Rb and p107 pocket domains and identified several key residues that contribute to E2F selectivity in the pocket family. Mutation of these residues in p107 to match the analogous residue in Rb results in an increase in affinity for E2F1 and E2F2 and an increase in the ability of p107 to inhibit E2F2 transactivation. Additionally, we investigated how phosphorylation by Cyclin dependent kinase (Cdk) on distinct residues regulates p107 affinity for the E2F4 transactivation domain. We found that phosphorylation of residues S650 and S975 weakens E2F4 transactivation domain binding. Our data reveal molecular features of pocket proteins that are responsible for their similarities and differences in function and regulation.
- Non-coding RNA molecules connect calorie restriction and lifespan. [REVIEW, JOURNAL ARTICLE]
- J Mol Biol 2016 Aug 22.
Calorie restriction is a broadly effective environmental intervention that extends life by operating through numerous biological processes. Here we discuss how non-coding RNA molecules act as mediators and targets of lifespan-extending calorie restriction. We also highlight how these RNA molecules connect calorie restriction to its effects on genome stability, cell metabolism, programmed cell death, senescence, cancer, and neurodegeneration. We anticipate that an advanced understanding of the connections between calorie restriction and non-coding RNA will provide unique insights into aging mechanisms while pointing to novel approaches aimed at modulating aging and age-related diseases.
- abYsis: Integrated Antibody Sequence and Structure - Management, Analysis and Prediction. [JOURNAL ARTICLE]
- J Mol Biol 2016 Aug 22.
abYsis is a web-based antibody research system that includes an integrated database of antibody sequence and structure data. The system can be interrogated in numerous ways - from simple text and sequence searches to sophisticated queries that apply 3D structural constraints. The publicly available version includes pre-analysed sequence data from EMBL-ENA and Kabat as well as structure data from the PDB. A researcher's own sequences can also be analysed through the web interface. A defining characteristic of abYsis is that sequences are automatically numbered with a series of popular schemes such as Kabat and Chothia and then annotated with key information such as CDRs and potential post-translational modifications. A unique aspect of abYsis is a set of residue frequency tables for each position in an antibody, allowing 'unusual residues' (those rarely seen at a particular position) to be highlighted and decisions to be made on which mutations may be acceptable. This is especially useful when comparing antibodies from different species. abYsis is useful for any researcher specializing in antibody engineering, especially those developing antibodies as drugs. abYsis is available at www.abysis.org.
- A small number of residues can determine if linker histones are bound on or off dyad in the chromatosome. [JOURNAL ARTICLE]
- J Mol Biol 2016 Aug 21.
Linker histones bind to the nucleosome and regulate the structure and function of chromatin. We have previously shown that the globular domains of chicken H5 and Drosophila H1 linker histones bind to the nucleosome with on- or off-dyad modes, respectively. To explore the determinant for the distinct binding modes, we investigated the binding of a mutant globular domain of H5 to the nucleosome. This mutant, termed GH5_pMut, includes substitutions of five globular domain residues of H5 with the corresponding residues in the globular domain of Drosophila H1. The residues at these five positions play important roles in nucleosome binding by either H5 or Drosophila H1. NMR and spin-labeling experiments showed that GH5_pMut bound to the nucleosome off the dyad. We further found that the nucleosome array condensed by either GH5_pMut or the globular domain of Drosophila H1 displayed a similar sedimentation coefficient, whereas the same nucleosome array condensed by the wild type globular domain of H5 showed a much larger sedimentation coefficient. Moreover, NMR and spin-labeling results from the study of the nucleosome in complex with the full-length human linker histone H1.0, whose globular domain shares high sequence conservation with the corresponding globular domain of H5, are consistent with an on-dyad binding mode. Taken together, our results suggest that a small number of residues in the globular domain of a linker histone can control its binding location on the nucleosome and higher-order chromatin structure.
- The HMGB1 C-terminal tail regulates DNA bending. [JOURNAL ARTICLE]
- J Mol Biol 2016 Aug 21.
HMGB1 (high mobility group box protein 1) is an architectural protein that facilitates formation of protein-DNA assemblies involved in transcription, recombination, DNA repair, and chromatin remodeling. Important to its function is the ability of HMGB1 to bend DNA non-sequence specifically. HMGB1 contains two HMG boxes that bind and bend DNA (the A box and the B box) and a C-terminal acidic tail. We investigated how these domains contribute to DNA bending by HMGB1 using single molecule FRET, which enabled us to resolve heterogeneous populations of bent and unbent DNA. We found that full length HMGB1 bent DNA more than the individual A and B boxes. Removing the C-terminal tail resulted in a protein that bent DNA to a greater extent than the full length protein. These data suggest that the A and B boxes simultaneously bind DNA in the absence of the C-terminal tail, but the tail modulates DNA binding and bending by one of the HMG boxes in the full length protein. Indeed, a construct composed of the B box and the C-terminal tail only bent DNA at higher protein concentrations. Moreover, in the context of the full length protein, mutating the A box such that it could not bend DNA resulted in a protein that bent DNA similarly to a single HMG box and only at higher protein concentrations. We propose a model in which the HMGB1 C-terminal tail serves as an intramolecular damper that modulates the interaction of the B box with DNA.
- Pathogenic mutations within the disordered palindromic region of the prion protein induce structure therein and accelerate the formation of misfolded oligomers. [JOURNAL ARTICLE]
- J Mol Biol 2016 Aug 18.
Little is understood about how the intrinsically disordered N-terminal region (NTR) of the prion protein modulates its misfolding and aggregation, which lead to prion disease. In this study, two pathogenic mutations, G113 V and A116 V in the palindromic region of the NTR, are shown to have no effect on the structure, stability or dynamics of native mouse prion protein (moPrP), but to nevertheless accelerate misfolding and oligomerization. For wt moPrP, misfolding and oligomerization appear to occur concurrently, while for both mutant variants, oligomerization is shown to precede misfolding. Kinetic hydrogen deuterium exchange-mass spectrometry experiments show that sequence segment 89-132 from the NTR becomes structured, albeit weakly, during the oligomerization of both mutant variants. Importantly, this structure formation occurs prior to structural conversion in the CTD, and appears to be the reason why the formation of misfolded oligomers is accelerated by the pathogenic mutations.
- Interaction Analyses of the Integrin β2 Cytoplasmic Tail with the F3 FERM Domain of Talin and 14-3-3ζ Reveal a Ternary Complex with Phosphorylated Tail. [JOURNAL ARTICLE]
- J Mol Biol 2016 Aug 18.
Integrins, hetero-dimeric (α and β subunits) signal-transducer proteins, are essential for cell adhesion and migration. Beta cytosolic tails (β-CTs) of integrins interact with a number of cytosolic proteins including talin, Dok1, and 14-3-3ζ. The formation of multi-protein complexes with β-CTs is involved in the activation and regulation of integrins. The leukocyte-specific β2 integrins are essential for leukocyte trafficking, phagocytosis, antigen-presentation and proliferation. In this study, we examined the binding interactions between integrin β2-CT and T758 phosphorylated β2-CT (pT758β2-CT) with positive regulators talin and 14-3-3ζ and negative regulator Dok1. Residues of the F3 domain of talin belonging to the C-terminal helix, β-strand 5 and the adjacent loop were found to be involved in binding interactions with β2-CT. The binding affinity between talin F3 and β2-CT was reduced when β2 T758 was phosphorylated but this modification promoted 14-3-3ζ binding. However, we were able to detect stable ternary complex formation of pT758β2-CT, talin F3 and 14-3-3ζ that involved the repositioning of talin F3 on β2-CT.We showed that Dok1 binding to β2-CT was reduced when β2 T758 was phosphorylated and in the presence of 14-3-3ζ. Based on these data, we propose a sequential model of β2 integrin activation involving these molecules. Our study provides for the first time insights towards β2 integrin activation that involves a multi-protein complex.