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1.
Kezuka Y  Yoshida Y  Nonaka T 《Proteins》2012,80(10):2447-2458
Hydrogen sulfide (H2S) is a causative agent of oral malodor and may play an important role in the pathogenicity of oral bacteria such as Streptococcus anginosus. In this microorganism, H2S production is associated with βC‐S lyase (Lcd) encoded by lcd gene, which is a pyridoxal 5′‐phosphate (PLP)‐dependent enzyme that catalyzes the α,β‐elimination of sulfur‐containing amino acids. When Lcd acts on L ‐cysteine, H2S is produced along with pyruvate and ammonia. To understand the H2S‐producing mechanism of Lcd in detail, we determined the crystal structures of substrate‐free Lcd (internal aldimine form) and two reaction intermediate complexes (external aldimine and α‐aminoacrylate forms). The formation of intermediates induced little changes in the overall structure of the enzyme and in the active site residues, with the exception of Lys234, a PLP‐binding residue. Structural and mutational analyses highlighted the importance of the active site residues Tyr60, Tyr119, and Arg365. In particular, Tyr119 forms a hydrogen bond with the side chain oxygen atom of L ‐serine, a substrate analog, in the external aldimine form suggesting its role in the recognition of the sulfur atom of the true substrate (L ‐cysteine). Tyr119 also plays a role in fixing the PLP cofactor at the proper position during catalysis through binding with its side chain. Finally, we partly modified the catalytic mechanism known for cystalysin, a βC‐S lyase from Treponema denticola, and proposed an improved mechanism, which seems to be common to the βC‐S lyases from oral bacteria. Proteins 2012;. © 2012 Wiley Periodicals, Inc.  相似文献   

2.
Protein-protein interactions play fundamental roles in physiological and pathological biological processes. The characterization of the structural determinants of protein-protein recognition represents an important step for the development of molecular entities able to modulate these interactions. We have recently found that IκB-α (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) blocks the HIV-1 expression and replication in a NF-κB-independent manner by directly binding to the virus-encoded Tat transactivator. Here, we report the evaluation of the entity of binding of IκB-α to Tat through in vitro Surface Plasmon Resonance assay. Moreover, by designing and characterizing a set of peptides of the C-terminus region of IκB-α, we show that the peptide corresponding to the IκB-α sequence 262-287 was able to bind to Tat with high affinity (300 nM). The characterization of a number of IκB-α-based peptides also provided insights into their intrinsic folding properties. These findings have been corroborated by mutagenesis studies on the full-length IκB-α, which unveil that different IκB-α residues are involved in NF-κB or Tat recognition.  相似文献   

3.
In muscle, excitation-contraction coupling is defined as the process linking depolarization of the surface membrane with Ca2+ release from cytoplasmic stores, which activates contraction of striated muscle. This process is primarily controlled by interplay between two Ca2+ channels—the voltage-gated L-type Ca2+ channel (dihydropyridine receptor, DHPR) localized in the t-tubule membrane and the Ca2+-release channel (ryanodine receptor, RyR) of the sarcoplasmic reticulum membrane. The structures of both channels have been extensively studied by several groups using electron cryomicroscopy and single particle reconstruction techniques. The structures of RyR, determined at resolutions of 22–30 Å, reveal a characteristic mushroom shape with a bulky cytoplasmic region and the membrane-spanning stem. While the cytoplasmic region exhibits a complex structure comprising a multitude of distinctive domains with numerous intervening cavities, at this resolution no definitive statement can be made about the location of the actual pore within the transmembrane region. Conformational changes associated with functional transitions of the Ca2+ release channel from closed to open states have been characterized. Further experiments determined localization of binding sites for various channel ligands. The structural studies of the DHPR are less developed. Although four 3D maps of the DHPR were reported recently at 24–30 Å resolution from studies of frozen-hydrated and negatively stained receptors, there are some discrepancies between reported structures with respect to the overall appearance and dimensions of the channel structure. Future structural studies at higher resolution are needed to refine the structures of both channels and to substantiate a proposed molecular model for their interaction.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1506–1514.Original Russian Text Copyright © 2004 by Serysheva.  相似文献   

4.
The atypical PKC isoforms(ζandι)play essential roles in regulating various cellular processes.Both the hetero-interaction between PKCζand p62 through their N-terminal PB1 domains and the homo-oligomerization of p62 via its PB1 domain are critical for the activation of NF-B signaling;however,the molecular mechanisms concerning the formation and regulation of these homotypic complexes remain unclear.Here we determined the crystal structure of PKCζ-PB1 in complex with a monomeric p62-PB1 mutant,where the massive electrostatic interactions between the acidic OPCA motif of PKCζ-PB1 and the basic surface of p62-PB1,as well as additional hydrogen bonds,ensure the formation of a stable and specific complex.The PKCζ-p62 interaction is interfered with the modification of a specific Cys of PKCζby the antiarthritis drug aurothiomalate,though all four cysteine residues in the PKCζ-PB1 domain can be modified in in vitro assay.In addition,detailed structural and biochemical analyses demonstrate that the PB1 domains of aPKCs belong to the type I group,which can depolymerize the high-molecular-weight p62 aggregates into homo-oligomers of lower order.These data together unravel the molecular mechanisms of the homo-or hetero-interactions between p62 and PKCζand provide the basis for designing inhibitors of NF-B signaling.  相似文献   

5.
The chemokine receptor 5 (CCR5) belongs to the superfamily of serpentine G protein-coupled receptors (GPCRs). The DRY motif (Asp, Arg, Tyr) of the intracellular loop 2 (ICL2), which is highly conserved in the GPCRs has been shown to be essential for the stability of folding of CCR5 and the interaction with β-arrestin. But the molecular mechanism by which it recognizes and interacts with β-arrestin has not been elucidated. In the present study, we described the active state of the β-arrestin structure using normal mode analysis and characterized the binding cleft of CCR5-ICL2 with β-arrestin using SABRE© docking tool and molecular dynamics simulation. Based on our computational results, we proposed a mode of binding between the ICL2 loop of CCR5 and β-arrestin structure, and modeled the energetically stable β-arrestin/CCR5 complex. In view of CCR5’s importance as a therapeutic target for the treatment of HIV, this observation provides novel insight into the β-arrestin/CCR5 pathway. As a result, the current computational study of the detailed β-arrestin/CCR5 binding complex could provide the rationale for the development of next generation of HIV peptide inhibitors as therapeutic agents.  相似文献   

6.
The main pathogenic process underlying dialysis-related amyloidosis is the accumulation of β-2-microglobulin (β2m) as amyloid fibrils in the musculoskeletal system, and some evidence suggests that Cu(II) may play a role in β2m amyloid formation. Cu(II)-induced β2m fibril formation is preceded by the formation of discrete, oligomeric intermediates, including dimers, tetramers, and hexamers. In this work, we use selective covalent labeling reactions combined with mass spectrometry to investigate the amino acids responsible for mediating tetramer formation in wild-type β2m. By comparing the labeling patterns of the monomer, dimer, and tetramer, we find evidence that the tetramer interface is formed by the interaction of D strands from one dimer unit and G strands from another dimer unit. These covalent labeling data along with molecular dynamics calculations allow the construction of a tetramer model that indicates how the protein might proceed to form even higher-order oligomers.  相似文献   

7.
This Letter describes the results of two combined approaches: homology modeling and molecular docking studies, in order to propose the molecular basis of IKKβ inhibition by staurosporine and quercetin as ATP-competitive inhibitors. The results provides a rationale and structural frameworks for designing potent ATP binding-site inhibitors of IKKβ, which is an attractive drug target for inflammatory diseases and has been found to be responsible for some of the already observed pharmacological effects for marketed drugs.  相似文献   

8.
RNase J is a conserved ribonuclease that belongs to the β-CASP family of nucleases. It possesses both endo- and exo-ribonuclease activities, which play a key role in pre-rRNA maturation and mRNA decay. Here we report high-resolution crystal structures of Deinococcus radiodurans RNase J complexed with RNA or uridine 5′-monophosphate in the presence of manganese ions. Biochemical and structural studies revealed that RNase J uses zinc ions for two-metal-ion catalysis. One residue conserved among RNase J orthologues (motif B) forms specific electrostatic interactions with the scissile phosphate of the RNA that is critical for the catalysis and product stabilization. The additional manganese ion, which is coordinated by conserved residues at the dimer interface, is critical for RNase J dimerization and exonuclease activity. The structures may also shed light on the mechanism of RNase J exo- and endonucleolytic activity switch.  相似文献   

9.
The Old World scorpion Androctonus australis hector (Aah) produces one of the most lethal venoms for humans. Peptidic α-toxins AahI to AahIV are responsible for its potency, with AahII accounting for half of it. All four toxins are high affinity blockers of the fast inactivation phase of mammalian voltage-activated Na(+) channels. However, the high antigenic polymorphism of α-toxins prevents production of a polyvalent neutralizing antiserum, whereas the determinants dictating their trapping by neutralizing antibodies remain elusive. From an anti-AahII mAb, we generated an antigen binding fragment (Fab) with high affinity and selectivity for AahII and solved a 2.3 ?-resolution crystal structure of the complex. Sequestering of the C-terminal region of the bound toxin within a groove formed by the Fab combining loops is associated with a toxin orientation and main and side chain conformations that dictate the AahII antigenic specificity and efficient neutralization. From an anti-AahI mAb, we also preformed and crystallized a high affinity AahI-Fab complex. The 1.6 ?-resolution structure solved revealed a Fab molecule devoid of a bound AahI and with combining loops involved in packing interactions, denoting expulsion of the bound antigen upon crystal formation. Comparative analysis of the groove-like combining site of the toxin-bound anti-AahII Fab and planar combining surface of the unbound anti-AahI Fab along with complementary data from a flexible docking approach suggests occurrence of distinctive trapping orientations for the two toxins relative to their respective Fab. This study provides complementary templates for designing new molecules aimed at capturing Aah α-toxins and suitable for immunotherapy.  相似文献   

10.
《BBA》2020,1861(3):148153
Complex I is the largest and most intricate redox-driven proton pump of the respiratory chain. The structure of bacterial and mitochondrial complex I has been determined by X-ray crystallography and cryo-EM at increasing resolution. The recent cryo-EM structures of the complex I-like NDH complex and membrane bound hydrogenase open a new and more comprehensive perspective on the complex I superfamily. Functional studies and molecular modeling approaches have greatly advanced our understanding of the catalytic cycle of complex I. However, the molecular mechanism by which energy is extracted from the redox reaction and utilized to drive proton translocation is unresolved and a matter of ongoing debate. Here, we review progress in structure determination and functional characterization of complex I and discuss current mechanistic models.  相似文献   

11.
The Cambrian Maotianshan-Shale lagerstätten have yielded fossils of a great variety of taxa, particularly arthropods. We report further information on two of these arthropods, Fuxianhuia protensa and Chengjiangocaris longiformis and present a new species, Shankouia zhenghei. Their morphologies shed new light on the early evolution of Arthropoda, which can be resolved into three major phases. In the first phase, new features such as segmental uniramous limbs were developed. The second phase is characterized by segmental sclerotizations of body and appendages. In this phase, the head included only the segments bearing lateral eyes and antennae. A shield-like structure is recognized as the expanded tergite of the second head segment. This tergite housed a pair of sac-like structures. Formerly interpreted as appendages in Fuxianhuia, they are regarded here as gut diverticula. Behind the head, all tergites free from the shield-like one are tripartite, made of an elevated middle region and pleurotergites. All post-antennal limbs comprise a multi-annulated rod and an exopod flap. The antenna is limb-shaped and was, most likely, the only structure suitable for food gathering. Remarkably, the two head segments are those anterior segments, which are not expressed by Hox genes. The third step is the development of euarthropod characteristics.  相似文献   

12.
Snakebite envenoming is an important public health problem in many tropical and subtropical countries, and is considered a neglected tropical disease by the World Health Organization. Most severe cases are inflicted by species of the families Elapidae and Viperidae, and lead to a number of systemic and local effects in the victim. One of the main problems regarding viperidic accidents is prominent local tissue damage whose pathogenesis is complex and involves the combined actions of a variety of venom components. Phospholipases A2 (PLA2s) are the most abundant muscle-damaging components of these venoms. Herein, we report functional and structural studies of PrTX-I, a Lys49-PLA2 from Bothops pirajai snake venom, and the influence of rosmarinic acid (RA) upon this toxin''s activities. RA is a known active component of some plant extracts and has been reported as presenting anti-myotoxic properties related to bothopic envenomation. The myotoxic activity of Lys49-PLA2s is well established in the literature and although no in vivo neurotoxicity has been observed among these toxins, in vitro neuromuscular blockade has been reported for some of these proteins. Our in vitro studies show that RA drastically reduces both the muscle damage and the neuromuscular blockade exerted by PrTX-I on mice neuromuscular preparations (by ∼80% and ∼90%, respectively). These results support the hypothesis that the two effects are closely related and lead us to suggest that they are consequences of the muscle membrane-destabilizing activity of the Lys49-PLA2. Although the C-terminal region of these proteins has been reported to comprise the myotoxic site, we demonstrate by X-ray crystallographic studies that RA interacts with PrTX-I in a different region. Consequently, a new mode of Lys49-PLA2 inhibition is proposed. Comparison of our results with others in the literature suggests possible new ways to inhibit bothropic snake venom myotoxins and improve serum therapy.  相似文献   

13.
RlmG is a specific AdoMet-dependent methyltransferase (MTase) responsible for N2-methylation of G1835 in 23S rRNA of Escherichia coli. Methylation of m2G1835 specifically enhances association of ribosomal subunits and provides a significant advantage for bacteria in osmotic and oxidative stress. Here, the crystal structure of RlmG in complex with AdoMet and its structure in solution were determined. The structure of RlmG is similar to that of the MTase RsmC, consisting of two homologous domains: the N-terminal domain (NTD) in the recognition and binding of the substrate, and the C-terminal domain (CTD) in AdoMet-binding and the catalytic process. However, there are distinct positively charged protuberances and a distribution of conserved residues contributing to the charged surface patch, especially in the NTD of RlmG for direct binding of protein-free rRNA. The RNA-binding properties of the NTD and CTD characterized by both gel electrophoresis mobility shift assays and isothermal titration calorimetry showed that NTD could bind RNA independently and RNA binding was achieved by the NTD, accomplished by a coordinating role of the CTD. The model of the RlmG-AdoMet-RNA complex suggested that RlmG may unfold its substrate RNA in the positively charged cleft between the NTD and CTD, and then G1835 disengages from its Watson-Crick pairing with C1905 and flips out to insert into the active site. Our structure and biochemical studies provide novel insights into the catalytic mechanism of G1835 methylation.  相似文献   

14.
In this study, the crystal structure of a novel endo-1,4-β-xylanase from Scytalidium acidophilum, XYL1, was solved at 1.9 Å resolution. This is one of the few solved crystal structures of acidophilic proteins. The enzyme has the overall fold typical to family 11 xylanases. Comparison of this structure with other homologous acidophilic, neutrophilic and alkalophilic xylanases provides additional insights into the general features involved in low pH adaptation (stability and activity). Several sequence and structure modifications appeared to be responsible for the acidophilic characteristic: (a) the presence of an aspartic acid H bonded to the acid/base catalyst (b) the nature of specifically conserved residues in the active site (c) the negative potential at the surface (d) the decreased number of salt bridges and H bonds in comparison with highly alkaline enzymes.  相似文献   

15.
Interactions of oligomeric aggregates of the intrinsically disordered protein α-synuclein with lipid membranes appear to play an important role in the development of Parkinson's disease. The permeabilization of cellular membranes by oligomers has been proposed to result in neuronal death. The detailed mechanisms by which α-synuclein oligomers permeabilize lipid bilayers remain unknown. Two different mechanisms are conceivable. Oligomers may either insert into membranes forming pores through which small molecules can cross the membrane or their interaction with the membrane may disorder the lipid packing, giving rise to membrane defects. Here we show, using kinetic leakage measurements, that α-synuclein oligomer induced impairment of membrane integrity is not limited to the formation of permanent membrane spanning pores. Fast membrane permeabilization could be observed in a fraction of the large unilamellar vesicles. We have also observed, for the first time, that α-synuclein oligomers cause an enhanced lipid flip-flop. In neuronal cells, most of the α-synuclein is not expected to be present in an oligomeric form, but as monomers. In our in vitro experiments, we find that membrane bound monomeric α-synuclein can only delay the onset of oligomer-induced membrane permeabilization, implying that α-synuclein monomers cannot counteract oligomer toxicity.  相似文献   

16.
Organisms embedded within food webs must balance arms races with their predators and prey. For venom users, venom may mediate each arms race, but the dynamical evolutionary changes in venom production in response to the two arms races are still poorly understood. Here, we use a simple model to evaluate the evolutionary response of a venomous consumer to the presence of an apex generalist predator and evolution of the consumer’s prey. We find that introduction of the apex predator can weaken the arms race between the two lower trophic levels. In addition, when consumer prey capture and predator defense venoms functionally overlap, a reduced evolutionary response in the prey population can drive investment in venom used for prey capture going beyond what is optimal for subduing prey. These dynamics suggest that interactions with multiple trophic levels can substantially alter the venom complexity in predatory venomous animals and may explain the paradox of the overkill hypothesis.  相似文献   

17.
The surface-exposed β-galactosidase BgaC from Streptococcus pneumoniae was reported to be a virulence factor because of its specific hydrolysis activity toward the β(1,3)-linked galactose and N-acetylglucosamine (Galβ(1,3)NAG) moiety of oligosaccharides on the host molecules. Here we report the crystal structure of BgaC at 1.8 Å and its complex with galactose at 1.95 Å. At pH 5.5–8.0, BgaC exists as a stable homodimer, each subunit of which consists of three distinct domains: a catalytic domain of a classic (β/α)8 TIM barrel, followed by two all-β domains (ABDs) of unknown function. The side walls of the TIM β-barrel and a loop extended from the first ABD constitute the active site. Superposition of the galactose-complexed structure to the apo-form revealed significant conformational changes of residues Trp-243 and Tyr-455. Simulation of a putative substrate entrance tunnel and modeling of a complex structure with Galβ(1,3)NAG enabled us to assign three key residues to the specific catalysis. Site-directed mutagenesis in combination with activity assays further proved that residues Trp-240 and Tyr-455 contribute to stabilizing the N-acetylglucosamine moiety, whereas Trp-243 is critical for fixing the galactose ring. Moreover, we propose that BgaC and other galactosidases in the GH-35 family share a common domain organization and a conserved substrate-determinant aromatic residue protruding from the second domain.  相似文献   

18.
Abstract

Primary tumor cells often spread to other organs by metastasis. Despite of it, primary tumor cells break their surrounding extra cellular matrix (ECM) proteins and reach the destination organ by the process of intravasation and extravasation. Metastasized tumor cells induce the process of angiogenesis, this highly regulated process involves several ECM proteins. However, integrins are primarily involved in the blood vessel growth and repair. Therefore, integrins are promising angiogenesis targets. Integrins are receptors on cell surface, involved in signal transduction and attachments in extra cellular matrix (ECM). IntegrinαVβ3 and αVβ5 are implicated in tumor angiogenesis, metastasis, inflammation and bone resorption. The crystal structure of integrinαvβ5 is not available in protein structural databases, therefore; molecular model of integrinβ5 structure was prepared and stereo chemical model quality was checked. Integrin β5 active sites were identified based on insilico analysis tools. Further, molecular level interactions between integrinβ5 and ECM proteins were predicted. In the present study ECM proteins such as focal adhesion kinase 1 (FAK1), annexin A5 and P21 activated kinase 4 (PAK4) were considered for protein-protein docking, to understand inter molecular interactions. The predicted model is conceived to be stereo chemically good and can be used for molecular interaction studies of angiogenic inhibitors.  相似文献   

19.
We compared root responses to spatial heterogeneity of Zn and Ni in Thlaspi caerulescens J. and C. Presl from normal (NM plants) and metalliferous soil (M plants). We investigated whether the strong metal accumulation capacity of NM plants (compared to M plants) was related to a greater capacity of roots to grow towards metal-enriched soil compartments. Two similar experiments were conducted in summer (slow growth) and spring (high growth), respectively. Our study is the first to show that NM plants of T. caerulescens have the ability to allocate more roots in the Zn-enriched compartment of soil. However, the positive response to Zn by roots of NM plants does not explain their higher Zn accumulation capacity as M plants express a similar level of root allocation in Zn-enriched compartment of soil. In M plants, root response to the Zn-rich compartment appears to be more susceptible to variations in growth conditions. Preferential root allocation in Ni-enriched compartment was consistently found in M plants only, suggesting that Ni supply is critical in their native metalliferous soil. Our study also illustrates bias in the interpretation of root allocation studies using two dimensional boxes, as interferences between root response to metal and root chirality have been highlighted.  相似文献   

20.
α-Synuclein (α-Syn) aggregation/fibrillation is a leading cause of neuronal death and is one of the major pathogenic factors involved in the progression of Parkinson's' disease (PD). Against this backdrop, discovering new molecules as inhibitors or modulators of α-Syn aggregation/fibrillation is a subject of enormous research. In this study, we have shown modulation, disaggregation, and neuroprotective potential of aloin and emodin against α-Syn aggregation/fibrillation. Thioflavin T (ThT) fluorescence assay showed an increase in lag phase from (51.14 ± 2) h to (68.58 ± 2) h and (74.14 ± 3) h in the presence of aloin and emodin respectively. ANS binding assay represents a modulatory effect of these molecules on hydrophobicity which is crucial for aggregates/fibril formation. NMR spectroscopy and tyrosine quenching studies reveal the binding of aloin/emodin with monomeric α-Syn. TEM and DLS micrographs illustrate the attenuating effect of aloin/emodin against the development of large aggregates/fibrils. Our seeding experiments suggest aloin/emodin generate seeding incompetent oligomers that direct the off-pathway aggregation/fibrillation. Also, aloin/emodin capably reduces the fibrils-induced cytotoxicity and disassembles the preexisting amyloid fibrils. These findings provide deep insight into the modulatory mechanism of α-Syn aggregation/fibrillation in the presence of aloin and emodin, thereby suggesting their potential roles as promising therapeutic molecules against aggregation/fibrillation related disorders.  相似文献   

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