Structural variability of sunflower gene for methionine-rich albumin SFA8

Background. The 2S albumins of sunflower and other oilseed plants possess a high nutritional quality, the defense activity against fungi diseases casual gents and also valuable functional properties. The major component of albumin fraction, the SFA8 protein consists of 103 amino acid residues among which methionine constitutes 15 Mole %. In the cultivated sunflower gene pool the SFA8 structural gene is represented by the two alleles the products of which have different isoelectric points and differ by the electrophoretic mobility, however molecular mechanisms of the polymorphism are still unknown. Results. The amplified sequences of the SFA8 gene from seven Helianthus annuus L. accessions and three accessions of wild Helianthus L. species from VIR collection were sequences. The intron of 258-303 bp length depending on the genotype was firstly found in the central part of the gene. The length of the first exon constitutes 99 bp, the second exon is of 210 bp length. The nucleotide and translated amino acid sequences are polymorphic among different genotypes. The line VIR 130 in which the two expressing SFA8 proteins, the normal polypeptide with isoelectric point (pI) approximately 6.0 (normal SFA8) and its allelic variant with pI 6.5 (variant SFA8) have been earlier revealed possesses two types of the SFA8 encoding sequence. In one sequence the substitution 108С—G is present that results in the substitution of the polar uncharged amino acid serine for the positively charged arginine and respectively in alteration of the protein charge and isoelectric point. The intron sequence is also polymorphic and characterized by the presence of indels of approximately 45 bp. The intron sequences of all accessions contain dinucleotides GT at the 5΄ end and AG at the 3΄ end which are characteristic for consensus sequences of splicing sites in the U2-type introns. The variants of the secondary structure of the SFA8 intron sequences of H. argophyllus Torr. & A. Gray and all the analyzed H. annuus genotypes are similar and differ from those of H. petiolaris Nutt. and H. giganteus L. Conclusions. The data on the SFA8 gene sequence polymorphism are important understanding the molecular mechanisms of genotypic differences in biochemical and functional properties of the protein, and he revealed differences in the intron secondary structure can be important for understanding expression patterns of the protein

Observation of the Early Structural Changes Leading to the Formation of Protein Superstructures.

Formation of superstructures in protein aggregation processes has been indicated as a general pathway for several proteins, possibly playing a role in human pathologies. There is a severe lack of knowledge on the origin of such species in terms of both mechanisms of formation and structural features. We use equine lysozyme as a model protein, and by combining spectroscopic techniques and microscopy with X-ray fiber diffraction and ab initio modeling of Small Angle X-ray Scattering data, we isolate the partially unfolded state from which one of these superstructures (i.e., particulate) originates. We reveal the low-resolution structure of the unfolded state and its mechanism of formation, highlighting the physicochemical features and the possible pathway of formation of the particulate structure. Our findings provide a novel detailed knowledge of such a general and alternative aggregation pathway for proteins, this being crucial for a basic and broader understanding of the aggregation phenomena.This is the author's accepted manuscript and will be under embargo until the 3rd of September 2015. The final version is published by ACS in The Journal of Physical Chemistry Letters here: http://pubs.acs.org/doi/abs/10.1021/jz501614e

Lectin-like bacteriocins from pseudomonas spp. utilise D-rhamnose containing lipopolysaccharide as a cellular receptor

Lectin-like bacteriocins consist of tandem monocot mannose-binding domains and display a genus-specific killing activity. Here we show that pyocin L1, a novel member of this family from Pseudomonas aeruginosa, targets susceptible strains of this species through recognition of the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide that is predominantly a homopolymer of d-rhamnose. Structural and biophysical analyses show that recognition of CPA occurs through the C-terminal carbohydrate-binding domain of pyocin L1 and that this interaction is a prerequisite for bactericidal activity. Further to this, we show that the previously described lectin-like bacteriocin putidacin L1 shows a similar carbohydrate-binding specificity, indicating that oligosaccharides containing d-rhamnose and not d-mannose, as was previously thought, are the physiologically relevant ligands for this group of bacteriocins. The widespread inclusion of d-rhamnose in the lipopolysaccharide of members of the genus Pseudomonas explains the unusual genus-specific activity of the lectin-like bacteriocins

Solution-Based Structural Analysis of the Decaheme Cytochrome, MtrA, by Small-Angle X-ray Scattering and Analytical Ultracentrifugation

The potential exploitation of metal-reducing bacteria as a means for environmental cleanup or alternative fuel is an exciting prospect; however, the cellular processes that would allow for these applications need to be better understood. MtrA is a periplasmic decaheme c-type cytochrome from Shewanella oneidensis involved in the reduction of extracellular iron oxides and therefore is a critical element in Shewanella ability to engage in extracellular charge transfer. As a relatively small 333-residue protein, the heme content is surprisingly high. MtrA is believed to obtain electrons from the inner membrane-bound quinol oxidoreductase, CymA, and shuttle them across the outer membrane to MtrC, another decaheme cytochrome that directly interacts with insoluble metal oxides. How MtrA is able to perform this task is a question of interest. Here through the use of two solution-based techniques, small-angle X-ray scattering (SAXS) and analytical ultracentrifugation (AUC), we present the first structural analysis of MtrA. Our results establish that between 0.5 and 4 mg/mL, MtrA exists as a monomeric protein that is shaped like an extended molecular “wire” with a maximum protein dimension (D[subscript max]) of 104 Å and a rod-like aspect ratio of 2.2 to 2.5. This study contributes to a greater understanding of how MtrA fulfills its role in the redox processes that must occur before electrons reach the outside of the cell.National Science Foundation (U.S.). (0546323)National Institutes of Health (U.S.) (Grant Number F32GM904862)Howard Hughes Medical Institute. InvestigatorNational Science Foundation (U.S.) (Award DMR- 0936384

Low-Resolution Molecular Models Reveal the Oligomeric State of the PPAR and the Conformational Organization of Its Domains in Solution

The peroxisome proliferator-activated receptors (PPARs) regulate genes involved in lipid and carbohydrate metabolism, and are targets of drugs approved for human use. Whereas the crystallographic structure of the complex of full length PPARγ and RXRα is known, structural alterations induced by heterodimer formation and DNA contacts are not well understood. Herein, we report a small-angle X-ray scattering analysis of the oligomeric state of hPPARγ alone and in the presence of retinoid X receptor (RXR). The results reveal that, in contrast with other studied nuclear receptors, which predominantly form dimers in solution, hPPARγ remains in the monomeric form by itself but forms heterodimers with hRXRα. The low-resolution models of hPPARγ/RXRα complexes predict significant changes in opening angle between heterodimerization partners (LBD) and extended and asymmetric shape of the dimer (LBD-DBD) as compared with X-ray structure of the full-length receptor bound to DNA. These differences between our SAXS models and the high-resolution crystallographic structure might suggest that there are different conformations of functional heterodimer complex in solution. Accordingly, hydrogen/deuterium exchange experiments reveal that the heterodimer binding to DNA promotes more compact and less solvent-accessible conformation of the receptor complex

A homologue of the Parkinson's disease-associated protein LRRK2 undergoes a monomer-dimer transition during GTP turnover.

Mutations in LRRK2 are a common cause of genetic Parkinson's disease (PD). LRRK2 is a multi-domain Roco protein, harbouring kinase and GTPase activity. In analogy with a bacterial homologue, LRRK2 was proposed to act as a GTPase activated by dimerization (GAD), while recent reports suggest LRRK2 to exist under a monomeric and dimeric form in vivo. It is however unknown how LRRK2 oligomerization is regulated. Here, we show that oligomerization of a homologous bacterial Roco protein depends on the nucleotide load. The protein is mainly dimeric in the nucleotide-free and GDP-bound states, while it forms monomers upon GTP binding, leading to a monomer-dimer cycle during GTP hydrolysis. An analogue of a PD-associated mutation stabilizes the dimer and decreases the GTPase activity. This work thus provides insights into the conformational cycle of Roco proteins and suggests a link between oligomerization and disease-associated mutations in LRRK2

Structure and catalytic regulatory function of ubiquitin specific protease 11 N-terminal and ubiquitin-like domains

The ubiquitin specific protease 11 (USP11) is implicated in DNA repair, viral RNA replication, and TGFβ signaling. We report the first characterization of the USP11 domain architecture and its role in regulating the enzymatic activity. USP11 consists of an N-terminal "domain present in USPs" (DUSP) and "ubiquitin-like" (UBL) domain, together referred to as DU domains, and the catalytic domain harboring a second UBL domain. Crystal structures of the DU domains show a tandem arrangement with a shortened β-hairpin at the two-domain interface and altered surface characteristics compared to the homologues USP4 and USP15. A conserved VEVY motif is a signature feature at the two-domain interface that shapes a potential protein interaction site. Small angle X-ray scattering and gel filtration experiments are consistent with the USP11DU domains and full-length USP11 being monomeric. Unexpectedly, we reveal, through kinetic assays of a series of deletion mutants, that the catalytic activity of USP11 is not regulated through intramolecular autoinhibition or activation by the N-terminal DU or UBL domains. Moreover, ubiquitin chain cleavage assays with all eight linkages reveal a preference for Lys(63)-, Lys(6)-, Lys(33)-, and Lys(11)-linked chains over Lys(27)-, Lys(29)-, and Lys(48)-linked and linear chains consistent with USP11's function in DNA repair pathways that is mediated by the protease domain. Our data support a model whereby USP11 domains outside the catalytic core domain serve as protein interaction or trafficking modules rather than a direct regulatory function of the proteolytic activity. This highlights the diversity of USPs in substrate recognition and regulation of ubiquitin deconjugation

Altering APP Proteolysis: Increasing sAPPalpha Production by Targeting Dimerization of the APP Ectodomain

One of the events associated with Alzheimer's disease is the dysregulation of α- versus β-cleavage of the amyloid precursor protein (APP). The product of α-cleavage (sAPPα) has neuroprotective properties, while Aβ1-42 peptide, a product of β-cleavage, is neurotoxic. Dimerization of APP has been shown to influence the relative rate of α- and β- cleavage of APP. Thus finding compounds that interfere with dimerization of the APP ectodomain and increase the α-cleavage of APP could lead to the development of new therapies for Alzheimer's disease. Examining the intrinsic fluorescence of a fragment of the ectodomain of APP, which dimerizes through the E2 and Aβ-cognate domains, revealed significant changes in the fluorescence of the fragment upon binding of Aβ oligomers—which bind to dimers of the ectodomain— and Aβ fragments—which destabilize dimers of the ectodomain. This technique was extended to show that RERMS-containing peptides (APP695 328–332), disulfiram, and sulfiram also inhibit dimerization of the ectodomain fragment. This activity was confirmed with small angle x-ray scattering. Analysis of the activity of disulfiram and sulfiram in an AlphaLISA assay indicated that both compounds significantly enhance the production of sAPPα by 7W-CHO and B103 neuroblastoma cells. These observations demonstrate that there is a class of compounds that modulates the conformation of the APP ectodomain and influences the ratio of α- to β-cleavage of APP. These compounds provide a rationale for the development of a new class of therapeutics for Alzheimer's disease