Structural features associated with movement and ‘catch’ of sea-urchin spines

Spine catch ligaments of a sea urchin Arbacia punctulata were extended under constant load. Ligaments from an undisturbed animal may show any extension rate from zero (catch state) to rapid extension to failure. Replacing the preparation bath with Ca²⁺- and Mg²⁺-free sea water reversibly abolishes the catch state. The fine structure of the outer muscle layer and inner ligament cone associated with the spine base is described. The unstriated paramyosin muscles bear thin flanges and form compact interlocking rows. Subsurface cisternae are associated with the plasma membrane. The muscles are innervated by glia-free axons ending in bulbous terminals containing lucent synaptic vesicles. The ligament comprises cylindrical bundles of collagen fibrils: one or more minute muscle fibers (paramyosin) lie parallel with and closely adjoining each bundle. The mean diameter of these muscles is 0.3 μg and they occupy 2–3 % of the ligament's cross-sectional area. Axons containing electronopaque secretory droplets accompany the muscles between the collagen bundles: the cell bodies of these neurones generally lie on the outer surface of the ligament. When an urchin points a spine, the ligament on the side of the contracting spine muscle shortens but does not buckle. A function of the intraligamental muscles is to effect this non-buckled shortening. The catch mechanism (which resides entirely within the ligament) may be due either to the intraligamental muscles and/or to a locked polymer mechanism in which matrix molecules between collagen fibrils are reversibly crosslinked by divalent cations.     

Structural features of the tmRNA–ribosome interaction

Trans-translation is a process which switches the synthesis of a polypeptide chain encoded by a nonstop messenger RNA to the mRNA-like domain of a transfer-messenger RNA (tmRNA). It is used in bacterial cells for rescuing the ribosomes arrested during translation of damaged mRNA and directing this mRNA and the product polypeptide for degradation. The molecular basis of this process is not well understood. Earlier, we developed an approach that allowed isolation of tmRNA–ribosomal complexes arrested at a desired step of tmRNA passage through the ribosome. We have here exploited it to examine the tmRNA structure using chemical probing and cryo-electron microscopy tomography. Computer modeling has been used to develop a model for spatial organization of the tmRNA inside the ribosome at different stages of trans-translation.     

Structural features of peptoid–peptide hybrids in lipid–water interfaces

The inclusion of peptoid monomers into antimicrobial peptides (AMPs) increases their proteolytic resistance, but introduces conformational flexibility (reduced hydrogen bonding ability and cis/trans isomerism). We here use NMR spectroscopy to answer how the insertion of a peptoid monomer influences the structure of a regular α-helical AMP upon interaction with a dodecyl phosphocholine (DPC) micelle. Insertion of [(2-methylpropyl)amino]acetic acid in maculatin-G15 shows that the structural change and conformational flexibility depends on the site of insertion. This is governed by the micelle interaction of the amphipathic helices flanking the peptoid monomer and the side chain properties of the peptoid and its preceding residue.     

Structural and functional mapping of α-fetoprotein

Alpha-fetoprotein (AFP) is a major mammalian oncofetal protein, which is also present in small quantities in adults. It is a member of the albuminoid gene superfamily, which consists of AFP, serum albumin, vitamin D binding protein, and alpha-albumin (afamin). Although physicochemical and immunological properties of AFP have been well-studied, its biological role in embryo- and carcinogenesis and in adult organisms as well as mechanisms underlying its functioning remain unclear. During the recent decades, the biological role of AFP has been evaluated by identification of its functionally important sites. Comparison of primary structure of AFP and some physiologically active proteins revealed similarity of some polypeptide regions. This has been used for prediction of AFP functions (i.e., its multifunctionality). Localization of functionally important sites followed by determination of their amino acid composition and type of biological activity has provided valuable information for structural-functional mapping of AFP. Some peptide fragments of AFP have been synthesized and tested for biological activity. This review summarizes data on structural-functional interrelationships. We also describe functionally important AFP sites found by various groups during the last decade of structural-functional mapping of AFP with experimentally confirmed and putative biologically active sites.     

Unique Combinations of βαβ-Units and Π-Like Modules in Proteins and Specific Features of Their Amino Acid Sequences

Possible combinations of βαβ-units and Π-like modules in proteins in both right- and left-handed forms have been analyzed in detail. The correlation between the mutual arrangement of the structural elements in the polypeptide chain and their handedness has been shown. In the βαβΠ combinations, which is encountered most frequently in proteins, the Π-module follows the βαβ unit along the chain and both elements are right-handed. In the Πβαβ combinations, where the Π-module is located at the N end and the βαβ-unit follows it, the former is left-handed and the latter is right-handed. In relatively rare combinations of the left-handed βαβ-units and right-handed Π-modules, the βαβ-unit follows Π-module in the chain. The combinations of left-handed Π-modules and the left-handed βαβ-units are unobservable in proteins. It has also been shown that the Π-modules with a β-strand—α-helix—arch—β-strand structure are observed in proteins only in a right-handed form and half of them (51%) contains cis-prolines in their arches. These arches of nonhomologous proteins, as well as the positions of cis-prolines, nearly coincide when superimposed. The superimposed Π-modules also demonstrate that their overall folds are very similar. Structural alignment of their amino acid sequences has shown that the Π-modules have very similar sequence patterns of the key hydrophobic, hydrophilic, glycine, and cis-proline residues.     

Structural features of influenza A virus panhandle RNA enabling the activation of RIG-I independently of 5′-triphosphate

Retinoic acid-inducible gene I (RIG-I) recognizes specific molecular patterns of viral RNAs for inducing type I interferon. The C-terminal domain (CTD) of RIG-I binds to double-stranded RNA (dsRNA) with the 5′-triphosphate (5′-PPP), which induces a conformational change in RIG-I to an active form. It has been suggested that RIG-I detects infection of influenza A virus by recognizing the 5′-triphosphorylated panhandle structure of the viral RNA genome. Influenza panhandle RNA has a unique structure with a sharp helical bending. In spite of extensive studies of how viral RNAs activate RIG-I, whether the structural elements of the influenza panhandle RNA confer the ability to activate RIG-I signaling has been poorly explored. Here, we investigated the dynamics of the influenza panhandle RNA in complex with RIG-I CTD using NMR spectroscopy and showed that the bending structure of the panhandle RNA negates the requirement of a 5′-PPP moiety for RIG-I activation.     

Structural features for α-galactomannan binding to galectin-1

Galectins have a highly conserved carbohydrate-binding domain to which a variety of galactose-containing saccharides, both β- and α-galactosides, can interact with varying degrees of affinity. Recently, we demonstrated that the relatively large α(1 → 6)-D-galacto-β(1 → 4)-D-mannan (Davanat) binds galectin-1 (gal-1) primarily at an alternative carbohydrate-binding domain. Here, we used a series of α-galactomannans (GMs) that vary in their mannose-to-galactose ratios for insight into an optimal structural signature for GM binding to gal-1. Heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopy with (15)N-labeled gal-1 and statistical modeling suggest that the optimal signature consists of α-D-galactopyranosyl doublets surrounded by regions of about four or more "naked" mannose residues. These relatively large and complex GMs all appear to interact with varying degrees at essentially the same binding surface on gal-1 that includes the Davanat alternative binding site and elements of the canonical β-galactoside-binding region. The use of two small, well-defined GMs [6(1)-α(1 → 6)-D-galactosyl-β-D-mannotriaose and 6(3),6(4)-di-α(1 → 6)-D-galactosyl-β-D-mannopentaose] helped characterize how GMs, in general, interact in part with the canonical site. Overall, our findings contribute to better understanding interactions of gal-1 with larger, complex polysaccharides and to the development of GM-based therapeutics for clinical use.     

Structural and mechanistic commonalities of amyloid-β and the prion protein

Amyloid beta (Aβ) is a major causative agent of Alzheimer disease (AD). This neurotoxic peptide is generated as a result of the cleavage of the Amyloid-Precursor-Protein (APP) by the action of β-secretase and γ-secretase. The neurotoxicity was previously thought to be the result of aggregation. However, recent studies suggest that the interaction of Aβ with numerous cell surface receptors such as N-methyl-D-aspartate (NMDA), receptor for advanced glycosylation end products (RAGE), P75 neurotrophin receptor (P75^NTR) as well as cell surface proteins such as the cellular prion protein (PrP^c) and heparan sulfate proteoglycans (HSPG) strongly enhances Aβ induced apoptosis and thereby contributes to neurotoxicity. This review focuses on the molecular mechanism resulting in Aβ-shedding as well as Aβ-induced apoptotic processes, genetic risk factors for familial AD and interactions of Aβ with cell surface receptors and proteins, with particular emphasis on the cellular prion protein. Furthermore, comparisons are drawn between AD and prion disorders and the role of laminin, an extracellular matrix protein, glycosaminoglycans and the 37 kDa/67 kDa laminin receptor (LRP/LR) have been highlighted with regards to both neurodegenerative diseases.Key words: Alzheimer disease, amyloid β, apoptosis, 37 kDa/67 kDa laminin receptor, prion proteinsAlzheimer disease (AD), primarily defined by psychiatrist Alois Alzheimer in 1906, is a neurodegenerative disorder and currently exhibits a prevalence that “doubles approximately every five years from 0.5% at the common age of onset-65 years old.”¹ This disease is the most common form of dementia afflicting the elderly and at present affects in excess of 37 million people globally² and it is predicted that 100 million people will be living with the disease by 2050.³AD has received mounting scientific interest and has stimulated tireless research endeavours not only due to the complex mechanism by which it is caused; the multitude of contributing factors and contradictions which have arisen between hypotheses and acquired results, but also due to the rise in life expectancies⁴ owing to the advent of modern medicine, which has socio-economic implications particularly in terms of strain placed upon national health systems.     

Structural features of cold-adapted dimeric GH2 β-D-galactosidase from Arthrobacter sp. 32cB

Crystal structures of cold-adapted β-d-galactosidase (EC 3.2.1.23) from the Antarctic bacterium Arthrobacter sp. 32cB (ArthβDG) have been determined in an unliganded form resulting from diffraction experiments conducted at 100 K (at resolution 1.8 Å) and at room temperature (at resolution 3.0 Å). A detailed comparison of those two structures of the same enzyme was performed in order to estimate differences in their molecular flexibility and rigidity and to study structural rationalization for the cold-adaptation of the investigated enzyme. Furthermore, a comparative analysis with structures of homologous enzymes from psychrophilic, mesophilic, and thermophilic sources has been discussed to elucidate the relationship between structure and cold-adaptation in a wider context. The performed studies confirm that the structure of cold-adapted ArthβDG maintains balance between molecular stability and structural flexibility, which can be observed independently on the temperature of conducted X-ray diffraction experiments. Obtained information about proper protein function under given conditions provide a guideline for rational engineering of proteins in terms of their temperature optimum and thermal stability.     

Structural relatedness ofκ-casein and fibrinogenγ-chain

Summary -Caseins, involved in the milk clotting process, and human fibrinogen-chain, involved in the blood clotting process, show structural similarities. Several long-casein sections, together corresponding to 80% of the whole protein molecule, have their counterparts in the-chain of fibrinogen, in that 31-42% of the amino acid residues occupy identical positions. The section of-casein which contains the chymosin-sensitive bond has a counterpart not only in the but also in the B-chain of fibrinogen. Furthermore, the secondary structures of the-caseins and of the-chain predicted according to the method of Chou and Fasman present several common features.41th communication on caseins.     

Caenorhabditis elegans galectins LEC-1–LEC-11: Structural features and sugar-binding properties

Galectins form a large family of β-galactoside-binding proteins in metazoa and fungi. This report presents a comparative study of the functions of potential galectin genes found in the genome database of Caenorhabditis elegans. We isolated full-length cDNAs of eight potential galectin genes (lec-2–5 and 8–11) from a λZAP cDNA library. Among them, lec-2–5 were found to encode 31–35-kDa polypeptides containing two carbohydrate-recognition domains similar to the previously characterized lec-1, whereas lec-8–11 were found to encode 16–27-kDa polypeptides containing a single carbohydrate-recognition domain and a C-terminal tail of unknown function. Recombinant proteins corresponding to lec-1–4, -6, and 8–10 were expressed in Escherichia coli, and their sugar-binding properties were assessed. Analysis using affinity adsorbents with various β-galactosides, i.e., N-acetyllactosamine (Galβ1-4GlcNAc), lacto-N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4Glc), and asialofetuin, demonstrated that LEC-1–4, -6, and -10 have a significant affinity for β-galactosides, while the others have a relatively lower affinity. These results indicate that the integrity of key amino acid residues responsible for recognition of lactose (Galβ1-4Glc) or N-acetyllactosamine in vertebrate galectins is also required in C. elegans galectins. However, analysis of their fine oligosaccharide-binding properties by frontal affinity chromatography suggests their divergence towards more specialized functions.     

Quantifying sequence and structural features of protein–RNA interactions

Increasing awareness of the importance of protein–RNA interactions has motivated many approaches to predict residue-level RNA binding sites in proteins based on sequence or structural characteristics. Sequence-based predictors are usually high in sensitivity but low in specificity; conversely structure-based predictors tend to have high specificity, but lower sensitivity. Here we quantified the contribution of both sequence- and structure-based features as indicators of RNA-binding propensity using a machine-learning approach. In order to capture structural information for proteins without a known structure, we used homology modeling to extract the relevant structural features. Several novel and modified features enhanced the accuracy of residue-level RNA-binding propensity beyond what has been reported previously, including by meta-prediction servers. These features include: hidden Markov model-based evolutionary conservation, surface deformations based on the Laplacian norm formalism, and relative solvent accessibility partitioned into backbone and side chain contributions. We constructed a web server called aaRNA that implements the proposed method and demonstrate its use in identifying putative RNA binding sites.     

Structural and mechanistic commonalities of amyloid-β and the prion protein

Amyloid β (Aβ) is a major causative agent of Alzheime disease. This neurotoxic peptide is generated as a result of the cleavage of the Amyloid-Precursor-Protein (APP) by the action of beta secretase and gamma secretase. The neurotoxicity was previously thought to be the result of aggregation. However, recent studies suggest that the interaction of Aβ with numerous cell surface receptors such as N-methyl-D-aspartate (NMDA), receptor for advanced glycosylation end products (RAGE), P75 neurotrophin receptor (P75NTR) as well as cell surface proteins such as the cellular prion protein (PrP^c) and heparan sulfate proteoglycans (HSPG) strongly enhances Aβ induced apoptosis and thereby contributes to neurotoxicity. This review focuses on the molecular mechanism resulting in Aβ-shedding as well as Aβ-induced apoptotic processes, genetic risk factors for familial Alzheimer disease and interactions of Aβ with cell surface receptors and proteins, with particular emphasis on the cellular prion protein. Furthermore, comparisons are drawn between Alzheimer disease and prion disorders and the role of laminin, an extracellular matrix protein, glycosaminoglycans and the 37 kDa/67 kDa laminin receptor (LRP/LR) have been highlighted with regards to both neurodegenerative diseases.     

Effect of Trifluoroethanol on the Structural and Functional Properties of α-Crystallin

Alpha crystallin is an eye lens protein with a molecular weight of approximately 800 kDa. It belongs to the class of small heat shock proteins. Besides its structural role, it is known to prevent the aggregation of β- and γ-crystallins and several other proteins under denaturing conditions and is thus believed to play an important role in maintaining lens transparency. In this communication, we have investigated the effect of 2,2,2-trifluoroethanol (TFE) on the structural and functional features of the native α-crystallin and its two constituent subunits. A conformational change occurs from the characteristic β-sheet to the α-helix structure in both native α-crystallin and its subunits with the increase in TFE levels. Among the two subunits, αA-crystallin is relatively stable and upon preincubation prevents the characteristic aggregation of αB-crystallin at 20% and 30% (v/v) TFE. The hydrophobicity and chaperone-like activity of the crystallin subunits decrease on TFE treatment. The ability of αA-crystallin to bind and prevent the aggregation of αB-crystallin, despite a conformational change, could be important in protecting the lens from external stress. The loss in chaperone activity of αA-crystallin exposed to TFE and the inability of peptide chaperone—the functional site of αA-crystallin—to stabilize αB-crystallin at 20–30% TFE suggest that the site(s) involved in subunit interaction and chaperone-like function are quite distinct.     

Structural features underlying raloxifene’s biophysical interaction with bone matrix

Raloxifene, a selective estrogen receptor modulator (SERM), reduces fracture risk at least in part by improving the mechanical properties of bone in a cell- and estrogen receptor-independent manner. In this study, we determined that raloxifene directly interacts with the bone tissue. Through the use of multiple and complementary biophysical techniques including nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR), we show that raloxifene interacts specifically with the organic component or the organic/mineral composite, and not with hydroxyapatite. Structure–activity studies reveal that the basic side chain of raloxifene is an instrumental determinant in the interaction with bone. Thus, truncation of portions of the side chain reduces bone binding and also diminishes the increase in mechanical properties. Our results support a model wherein the piperidine interacts with bone matrix through electrostatic interactions with the piperidine nitrogen and through hydrophobic interactions (van der Waals) with the aliphatic groups in the side chain and the benzothiophene core. Furthermore, in silico prediction of the potential binding sites on the surface of collagen revealed the presence of a groove with sufficient space to accommodate raloxifene analogs. The hydroxyl groups on the benzothiophene nucleus, which are necessary for binding of SERMs to the estrogen receptor, are not required for binding to the bone surface, but mediate a more robust binding of the compound to the bone powder. In conclusion, we report herein a novel property of raloxifene analogs that allows them to interact with the bone tissue through potential contacts with the organic matrix and in particular collagen.     

Structural polymorphism of Alzheimer Aβ and other amyloid fibrils

Deposits of amyloid fibrils characterize a diverse group of human diseases that includes Alzheimer’s disease, Creutzfeldt-Jakob disease and type II diabetes. Amyloid fibrils formed from different polypeptides contain a common cross-β spine. Nevertheless, amyloid fibrils formed from the same polypeptide can occur in a range of structurally different morphologies. The heterogeneity of amyloid fibrils reflects different types of polymorphism: (i) variations in the protofilament number, (ii) variations in the protofilament arrangement and (iii) different polypeptide conformations. Amyloid fibril polymorphism implies that fibril formation can lead, for the same polypeptide sequence, to many different patterns of inter- or intra-residue interactions. This property differs significantly from native, monomeric protein folding reactions that produce, for one protein sequence, only one ordered conformation and only one set of inter-residue interactions.