Structural and Functional Characterization of the α-Tubulin Acetyltransferase MEC-17

Tubulin protomers undergo an extensive array of post-translational modifications to tailor microtubules to specific tasks. One such modification, the acetylation of lysine 40 of α-tubulin, located in the lumen of microtubules, is associated with stable, long-living microtubule structures. MEC-17 was recently identified as the acetyltransferase that mediates this event. We have determined the crystal structure of the catalytic core of human MEC-17 in complex with its cofactor acetyl-CoA at 1.7 Å resolution. The structure reveals that the MEC-17 core adopts a canonical Gcn5-related N-acetyltransferase (GNAT) fold that is decorated with extensive surface loops. An enzymatic analysis of 33 MEC-17 surface mutants identifies hot-spot residues for catalysis and substrate recognition. A large, evolutionarily conserved hydrophobic surface patch that is critical for enzymatic activity is identified, suggesting that specificity is achieved by interactions with the α-tubulin substrate that extend outside of the modified surface loop. An analysis of MEC-17 mutants in Caenorhabditis elegans shows that enzymatic activity is dispensable for touch sensitivity.     

Truncated α-amylase: an improved candidate for textile processing

Abstract

Enzymes are indispensable biocatalysts required in various steps of textile processing to minimize various chemical-induced hazards. The present work focuses on the applications of the truncated α-amylase in textile industry for desizing of fabrics by starch hydrolysis. The multiple sequence alignment was performed to find homology and the possible truncation region in Bacillus subtilis MTCC 121 α-amylase with same bacilli family α-amylase. Two constructs were generated for α-amylase gene of Bacillus subtilis MTCC 121 (Amy_F, full-length and Amy_T, C-terminal truncated) were cloned, overexpressed, purified, and characterized. Results revealed that activity of Amy_T was found to be 2.87-fold better than Amy_F. Further, the optimum temperature of Amy_F and Amy_T was obtained at 45?°C and 55?°C, respectively, whereas optimum pH was recorded at pH 7 and pH 8, respectively. Improved thermostability of Amy_T was further confirmed through thermal shift assay. Subsequently, starch-coated fabrics were tested for starch removal using the α-amylases. Comparative analysis revealed that Amy_T performed better in starch removal from polystyrene (85%), silk (75%), and cotton (70%) fabrics. The removal of starch from the fabrics was further confirmed by FESEM. Conclusively, this work presents one truncated α-amylase as an improved candidate over its full-length counterpart for textile desizing.     

Structural basis for α-conotoxin potency and selectivity

Parkinson’s disease is a debilitating movement disorder characterized by altered levels of α₆β₂1 (1 indicates the possible presence of additional subunits) nicotinic acetylcholine receptors (nAChRs) localized on presynaptic striatal catecholaminergic neurons. α-Conotoxin MII (α-CTx MII) is a highly useful ligand to probe α₆β₂ nAChRs structure and function, but it does not discriminate among closely related α₆1 nAChR subtypes. Modification of the α-CTx MII primary sequence led to the identification of α-CTx MII[E11A], an analog with 500–5300-fold discrimination between α₆1 subtypes found in both human and non-human primates. α-CTx MII[E11A] binds most strongly (femtomolar dissociation constant) to the high affinity α₆ nAChR, a subtype that is selectively lost in Parkinson’s disease. Here, we present the three-dimensional solution structure for α-CTx MII[E11A] as determined by two-dimensional ¹H NMR spectroscopy to 0.13 ± 0.09 ? backbone and 0.45 ± 0.08 ? heavy atom root-mean-square deviation from mean structure. Structural comparisons suggest that the increased hydrophobic area of α-CTx MII[E11A] relative to other members of the α-CTx family may be responsible for its exceptionally high affinity for α6α4β21 nAChR as well as discrimination between α₆β₂ and α₃β₂ containing nAChRs. This finding may enable the rational design of novel peptide analogs that demonstrate enhanced specificity for α₆1 nAChR subunit interfaces and provide a means to better understand nAChR structural determinants that modulate brain dopamine levels and the pathophysiology of Parkinson’s disease.     

Structural and Functional Studies of the Modulator NS9283 Reveal Agonist-like Mechanism of Action at α4β2 Nicotinic Acetylcholine Receptors

Modulation of Cys loop receptor ion channels is a proven drug discovery strategy, but many underlying mechanisms of the mode of action are poorly understood. We report the x-ray structure of the acetylcholine-binding protein from Lymnaea stagnalis with NS9283, a stoichiometry selective positive modulator that targets the α4-α4 interface of α4β2 nicotinic acetylcholine receptors (nAChRs). Together with homology modeling, mutational data, quantum mechanical calculations, and pharmacological studies on α4β2 nAChRs, the structure reveals a modulator binding mode that overlaps the α4-α4 interface agonist (acetylcholine)-binding site. Analysis of contacts to residues known to govern agonist binding and function suggests that modulation occurs by an agonist-like mechanism. Selectivity for α4-α4 over α4-β2 interfaces is determined mainly by steric restrictions from Val-136 on the β2-subunit and favorable interactions between NS9283 and His-142 at the complementary side of α4. In the concentration ranges where modulation is observed, its selectivity prevents NS9283 from directly activating nAChRs because activation requires coordinated action from more than one interface. However, we demonstrate that in a mutant receptor with one natural and two engineered α4-α4 interfaces, NS9283 is an agonist. Modulation via extracellular binding sites is well known for benzodiazepines acting at γ-aminobutyric acid type A receptors. Like NS9283, benzodiazepines increase the apparent agonist potency with a minimal effect on efficacy. The shared modulatory profile along with a binding site located in an extracellular subunit interface suggest that modulation via an agonist-like mechanism may be a common mechanism of action that potentially could apply to Cys loop receptors beyond the α4β2 nAChRs.     

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.     

The Human α-Lactalbumin Molten Globule: Comparison of Structural Preferences at pH 2 and pH 7

Structural investigations of molten globules provide an important contribution towards understanding protein folding pathways. A close similarity between equilibrium molten globule states and kinetic species observed during refolding has been reported for several proteins. However, the experimental conditions, and in particular the pH, under which the equilibrium and kinetic species are studied often differ significantly. For human α-lactalbumin (α-LA), the equilibrium molten globule is most often studied at pH 2, the so-called A-state, while kinetic refolding experiments are performed at neutral pH. α-LA contains a large number of acidic amino acid residues that may influence the properties of the molten globule differently at low and neutral pH. In this study, we investigate the structural preferences of the α-LA molten globule at pH 7 at the level of individual residues using nuclear magnetic resonance spectroscopy and compare these data with previous results obtained at pH 2. We show that differences exist in the conformational ensemble that describes the α-LA molten globule at these two pH values. The molten globule at pH 7 is generally less stable than that at the low pH A-state. Most notable are differences in the stability of structure for the C-helix and the calcium-binding loop that precedes it and differences in the contribution of long-range hydrophobic contacts between the N-terminal and C-terminal regions of the α-domain to the stability of the molten globule. Our results are discussed in the context of previous studies of the α-LA molten globule and can be used to reconcile apparent discrepancies in published data relating to the C-helix. In the light of our results, the low pH A-state may not be the best model for the kinetic molten globule observed during refolding of α-LA. The pH-dependent effects reported here for α-LA may be of relevance in comparisons of equilibrium and kinetic molten globules of other proteins.     

Food-Derived Carbohydrates — Structural Complexity and Functional Diversity

ABSTRACT:?

Carbohydrates are biomolecules abundantly available in nature. They are found in bewildering types ranging from simple sugars through oligo- and polysaccharides to glycoconjugates and saccharide complexes, each exhibiting characteristic bio-physiological and/or nutritional functions both in in vivo and in vitro systems. For example, their presence or inclusion in food dictates the texture (body) and gives desirable customer appeal (satisfaction), or their inclusion in the diet offers beneficial effects of great therapeutic value. Thus, carbohydrates are integrally involved in a multitude of biological functions such as regulation of the immune system, cellular signaling (communication), cell malignancy, antiinfection responses, host-pathogen interactions, etc. If starch is considered the major energy storage carbohydrate, the gums/mucilages and other non-starch carbohydrates are of structural significance. The most investigated properties of starch are its gelatinization and melting behavior, especially during food processing. This has led to the development of the food polymer science approach, which has enabled a new interpretive and experimental frame work for the study of the plasticizing influence of simple molecules such as water, sugars, etc. on food systems that are kinetically constrained. Starch, although considered fully digestible, has been challenged, and starch is found to be partly indigestible in the GI tract of humans. This fraction of starch-resisting digestion in vivo is known as resistant starch (RS). The latter, due to its excellent fermentative capacity in the gut, especially yielding butyric acid is considered a new tool for the creation of fiber-rich foods, which are of nutraceutical importance. By a careful control of the processing conditions the content of RS, a man-made fiber, can be increased to as high as 30%. Arabinoxylans are the major endospermic cell wall polysaccharides of cereals. In wheat they are found complexed with ferulic acid esters, which after oxidative coupling in vivo mediated by H₂O₂ and peroxidases or even by photochemical means give cross linked diferuloyl derivatives. The latter confer strength and extensibility to the cell wall and offer resistance for digestibility by ruminants. They also help blocking of the ingress of pathogens. The ester bound ferulic acid after oxidation in vivo generates reactive oxygen species that contribute to the fragmentation of non-starch polysaccharides (hemicelluloses), and thereby reduces the product viscosity, a property seen during long-term storage of rice. In plant tissues, the arabinogalactans are implicated in such diverse functions as cell-cell adhesion, nutrition of growing pollen tubes, response to microbial infections, and also as markers of identity expressed in the terminal sequences of saccharide chains.     

Structural Basis and Catalytic Mechanism for the Dual Functional Endo-β-N-Acetylglucosaminidase A

Endo-β-N-acetylglucosaminidases (ENGases) are dual specificity enzymes with an ability to catalyze hydrolysis and transglycosylation reactions. Recently, these enzymes have become the focus of intense research because of their potential for synthesis of glycopeptides. We have determined the 3D structures of an ENGase from Arthrobacter protophormiae (Endo-A) in 3 forms, one in native form, one in complex with Man₃GlcNAc-thiazoline and another in complex with GlcNAc-Asn. The carbohydrate moiety sits above the TIM-barrel in a cleft region surrounded by aromatic residues. The conserved essential catalytic residues – E173, N171 and Y205 are within hydrogen bonding distance of the substrate. W216 and W244 regulate access to the active site during transglycosylation by serving as “gate-keepers”. Interestingly, Y299F mutation resulted in a 3 fold increase in the transglycosylation activity. The structure provides insights into the catalytic mechanism of GH85 family of glycoside hydrolases at molecular level and could assist rational engineering of ENGases.     

Inhibition of Human Pepsin and Gastricsin by α2-Macroglobulin

The inhibitory effects of human α₂-macroglobulin (α₂-M), a major plasma proteinase inhibitor, on human pepsin and gastricsin were investigated. The activities of pepsin and gastricsin towards a protein substrate (reduced and carboxymethylated ribonuclease A) were significantly inhibited by α₂-M at pH 5.5, whereas those towards a peptide substrate (oxidized insulin B-chain) were scarcely inhibited. Under these conditions at pH 5.5, pepsin and gastricsin cleaved α₂-M mainly at the His⁶⁹⁴-Ala⁶⁹⁵ bond and Leu⁶⁹⁷-Val⁶⁹⁸ bond, respectively, in the bait regions sequence of α₂-M. The conformation of α₂-M was also shown to be markedly altered upon inhibition of these enzymes as examined by native polyacrylamide gel electrophoresis and electron microscopy. These results show the entrapment and concomitant inhibition of those proteinases by α₂-M.     

Preparation of Monoclonal Antibodies against Human Ventricular Myosin Light Chain 1 （HVMLC1） for Functional Studies

Using purified recombinant human ventricular myosin light chain 1 (HVMLC 1) as the antigen,three monoclonal antibodies,designated C8,C9 and B 12,were prepared.Immunoblot experiments demonstratedthat all monoclonal antibodies could react with the ventricular myosin light chain 1 isolated from differentsources,such as human,rat or pig.It was also demonstrated that C8 was directed against the NN part of theN-fragment (amino acid 1-40) of HVMLC1,and both C9 and B12 against the C-fragment (amino acid 99-195).The affinity constants of C8,C9 and B12 were 3.20×10~8,8.60×10~7 and 1.77×10~8 M~(-1),respectively,determined by non-competitive ELISA.The isotype of B12 was determined as lgG2a,whereas the isotype ofboth C8 and C9 were IgG1.In the presence of C9 or B12,the actin-activated Mg~(2 )ATPase activity of myosinwas greatly inhibited,but there was almost no effect on the Mg~(2 )ATPase activity for C8.B12 and C9 alsoinhibited the superprecipitation of porcine cardiac native actomyosin (myosin B) and reconstituted actomyosin,but C8 did not.The results indicate that all three monoclonal antibodies could bind the intact myosin molecule,but B12 and C9 might more easily react with epitopes located in the C-fragment of HVMLC1.The inhibitoryeffects of B 12 and C9 on ATPase activity and superprecipitation assays show that light chain 1,particularlythe C-fragment domain,is involved in the modulation of the actin-activated Mg~(2 )ATPase activity of myosinand,as a consequence,plays an essential role in the interaction of actin and myosin.     

Structural and Functional Integration of the PLCγ Interaction Domains Critical for Regulatory Mechanisms and Signaling Deregulation

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Highlights? Domains from PLCγ regulatory region show structural and functional integration ? Only cSH2 domain interacts with the PLC-core forming a high affinity surface ? Activation involves removal of autoinhibition and dissociation from the receptor ? Disease-linked mutations map to the autoinhibitory interface