Determination of the Agretopic Residues of a Peptide Co-Restricted to Different Class II Isotypes,I-Au and I-Eu,and Its Application for Preparation of a Synthetic Peptide Vaccine against Influenza Virus A/Aichi/2/68

We have defined that residues 46 and 54 on a synthetic peptide composed of residues 43–58 of pigeon cytochrome c (p43–58) work as agretopes (sites bound to an MHC molecule) in I-A^b mice. Substitution of amino acid residues on these positions altered the peptide to bind with the other MHC molecules. Furthermore, by substituting the agretopic residues with a variety of amino acids, we could determine the class II binding motif for each MHC molecule. In the present study, immunogenicity of a peptide, 46R50V54A, carrying valine (V) at epitopic (site bound to TCR) position 50, arginine (R) and alanine (A) at agretopic positions 46 and 54 of the p43–58, respectively has been analyzed in B10.PL (H-2^u) mice. We found that this peptide bound to two different class II isotypes, I-A^u and I-E^u. Arginine at position 46 or alanine at position 54 of the 46R50V54A was shown to be critical for binding to I-A^u or I-E^u, respectively. Further, on the basis of this class II binding motif we could prepare potent peptide vaccines against influenza A/Aichi/2/68 virus in B10. PL mice.     

Liddle综合征触发蛋白ENaC的PY模体突变型的结合肽段的筛选

Liddle综合征是一种常染色体显性、盐敏感型的高血压综合征,其分子发病机制研究认为是上皮钠离子通道(epithelial Na+channel,ENaC)的β亚基或γ亚基的胞质侧羧基端区域低频率的点突变或缺失突变导致肾远曲小管钠离子重吸收增加.本研究提出了一种从分子水平上治疗Liddle综合征的设想,即构建一种可识别Liddle综合征患者中ENaC蛋白突变的PY模体的人工泛素连接酶E3,使其结合并降解突变的ENaC蛋白,从而使肾远曲小管上皮细胞膜上ENaC的表达数量和活性恢复.而识别PY突变体的E3,可通过用患者中的PY突变体筛选随机多肽文库获得与之结合的随机肽段,用其替换PY模体天然配体蛋白Nedd4的WW结构域,从而得到一个新的人工E3.本研究中以一种Liddle综合征突变型Y620H为诱饵蛋白,筛选新型随机多肽文库,获得了1个至少能与2种PY突变体(Y620H和P618L)特异性结合的随机肽段,为进一步构建可降解ENaC突变体的人工E3积累了重要的实验数据.     

The Role of the Circulation in Processing pro-Brain Natriuretic Peptide (proBNP) to Amino-Terminal BNP and BNP-32

Hunt, P. J., E. A. Espiner, G. M. Nicholls, A. M. Richards and T. G. Yandle. The role of the circulation in processing pro-brain natriuretic peptide (proBNP) to amino-terminal BNP and BNP-32. Peptides 18(10) 1475–1481, 1997.—Human proBNP (purified from cardiac tissue) was incubated at 37°C in whole blood, serum and plasma and the products analyzed by size exclusion high pressure liquid chromatography and radioimmunoassay (RIA). In addition to RIAs for BNP-32 and NT-proBNP(1-13), a newly developed RIA for proBNP(62-76) was also used to identify the peptides. Incubation with serum resulted in the formation of a 9 kDa and a 3 kDa peptide, consistent with the N-terminal and the C-terminal peptides of the propeptide. Minimal processing of proBNP was seen in blood or plasma, suggesting that the circulation does not play a major role in the activation of proBNP. Analysis of degradation products of human proBNP using site directed specific antisera indicates that removal of N-terminal amino acids from proBNP occurs in serum. These findings support the view that the “high molecular weight BNP-32” previously identified in human plasma comprises amino-terminal deleted forms, and is unlikely to be intact proBNP(1-108).     

Structure of Amantadine-Bound M2 Transmembrane Peptide of Influenza A in Lipid Bilayers from Magic-Angle-Spinning Solid-State NMR: The Role of Ser31 in Amantadine Binding

The M2 proton channel of influenza A is the target of the antiviral drugs amantadine and rimantadine, whose effectiveness has been abolished by a single-site mutation of Ser31 to Asn in the transmembrane domain of the protein. Recent high-resolution structures of the M2 transmembrane domain obtained from detergent-solubilized protein in solution and crystal environments gave conflicting drug binding sites. We present magic-angle-spinning solid-state NMR results of Ser31 and a number of other residues in the M2 transmembrane peptide (M2TMP) bound to lipid bilayers. Comparison of the spectra of the membrane-bound apo and complexed M2TMP indicates that Ser31 is the site of the largest chemical shift perturbation by amantadine. The chemical shift constraints lead to a monomer structure with a small kink of the helical axis at Gly34. A tetramer model is then constructed using the helix tilt angle and several interhelical distances previously measured on unoriented bilayer samples. This tetramer model differs from the solution and crystal structures in terms of the openness of the N-terminus of the channel, the constriction at Ser31, and the side-chain conformations of Trp41, a residue important for channel gating. Moreover, the tetramer model suggests that Ser31 may interact with amantadine amine via hydrogen bonding. While the apo and drug-bound M2TMP have similar average structures, the complexed peptide has much narrower linewidths at physiological temperature, indicating drug-induced changes of the protein dynamics in the membrane. Further, at low temperature, several residues show narrower lines in the complexed peptide than the apo peptide, indicating that amantadine binding reduces the conformational heterogeneity of specific residues. The differences of the current solid-state NMR structure of the bilayer-bound M2TMP from the detergent-based M2 structures suggest that the M2 conformation is sensitive to the environment, and care must be taken when interpreting structural findings from non-bilayer samples.     

The C-Domain of the NAC Transcription Factor ANAC019 Is Necessary for pH-Tuned DNA Binding through a Histidine Switch in the N-Domain

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The Second Extracellular Loop of Pore-Forming Subunits of ATP-Binding Cassette Transporters for Basic Amino Acids Plays a Crucial Role in Interaction with the Cognate Solute Binding Protein(s)

In the thermophile Geobacillus stearothermophilus, the uptake of basic amino acids is mediated by an ABC transporter composed of the substrate binding protein (receptor) ArtJ and a homodimer each of the pore-forming subunit, ArtM, and the nucleotide-binding subunit, ArtP. We recently identified two putative binding sites in ArtJ that might interact with the Art(MP)₂ complex, thereby initiating the transport cycle (A. Vahedi-Faridi et al., J. Mol. Biol. 375:448-459, 2008). Here we investigated the contribution of charged amino acid residues in the second extracellular loop of ArtM to contact with ArtJ. Our results demonstrate a crucial role for residues K177, R185, and E188, since mutations to oppositely charged amino acids or glutamine led to a complete loss of ArtJ-stimulated ATPase activity of the complex variants in proteoliposomes. The defects could not be suppressed by ArtJ variants carrying mutations in site I (K39E and K152E) or II (E163K and D170K), suggesting a more complex interplay than that by a single salt bridge. These findings were supported by cross-linking assays demonstrating physical proximity between ArtJ(N166C) and ArtM(E182C). The importance of positively charged residues for receptor-transporter interaction was underscored by mutational analysis of the closely related transporter HisJ/LAO-HisQMP₂ of Salmonella enterica serovar Typhimurium. While transporter variants with mutated positively charged residues in HisQ displayed residual ATPase activities, corresponding mutants of HisM could no longer be stimulated by HisJ/LAO. Interestingly, the ATPase activity of the HisQM(K187E)P₂ variant was inhibited by l- and d-histidine in detergent, suggesting a role of the residue in preventing free histidine from gaining access to the substrate binding site within HisQM.ATP-binding cassette (ABC) transporters are found in all three kingdoms of life and form one of the largest protein families. Members of the family catalyze the uptake or export across biological membranes of a large variety of substrates, ranging from small inorganic ions to proteins, at the expense of ATP (16).ABC transporters display a modular architecture composed of two nucleotide-binding domains (NBDs) that contain all conserved sequence motifs, such as Walker A and B sites and the ABC signature (LSGGQ motif) (31), and of two transmembrane domains (TMDs), which can be arranged in any possible combination (7, 11).Structural and biochemical data suggest that alternating access of the translocation pore to the intra- and extracellular spaces achieves a net transport of substrate. The key feature of this model is that the alternation of the TMDs between outward-facing and inward-facing conformations is energized by the NBDs'' catalytic cycle. The latter comprises NBD dimer closure upon ATP binding, hydrolysis of ATP in the closed conformer, and reopening toward a semiclosed, ADP-bound state (reviewed in reference 11).Canonical ABC import systems that are involved in diverse cellular functions and thus far confined to prokaryotes require an additional module, either an extracellular solute binding protein (SBP) or a receptor, for functionality (27, 37, 38). While in Gram-negative bacteria SBPs are found as soluble proteins in the periplasm, in Gram-positive bacteria the binding proteins are anchored to the outer leaflet of the cytoplasmic membrane by fatty acids that are covalently linked to the N-terminal cysteine residue (34). SBPs typically consist of two lobes that are connected by a linker region. The interface between the two lobes forms the substrate binding site. Upon binding of ligand, the proteins undergo a conformational change from an open toward a closed state (27, 38), which initiates the transport process by interaction with extracytoplasmic peptide regions of TMDs of the cognate ABC transporter (12). Receptor-transporter interactions are highly specific, but the molecular details are still poorly understood. Three X-ray structures of complete ABC importers cocrystallized with their cognate solute binding proteins and mutational analyses have shed some light on the molecular basis of these interactions (5, 17, 19, 26). Accordingly, charged residues were found to contribute to the interface between liganded binding proteins and extracellular loops of the transmembrane subunits. However, whether salt bridges are a general feature of receptor-transporter interfaces, as implied by these structures, remains to be established.To address this question, we studied two well-characterized members of the PAAT (polar amino acid transporter) subfamily of ABC transporters (7) for which crystal structures are still elusive (1, 3, 30).The ArtJ-Art(MP)₂ transporter of Geobacillus stearothermophilus is composed of the solute receptor ArtJ, a homodimer of the membrane-integral subunit, ArtM, predicted to span the membrane five times, and a homodimer of the ATPase subunit, ArtP (14). Recently, we reported the crystal structures of ArtJ in complex with arginine, lysine, and histidine and identified residues in ArtJ that are crucial for interaction with the Art(MP)₂ complex. These cluster in two putative binding sites, at the N- and C-terminal lobes of the protein (36) (Fig. ​(Fig.1A),1A), as revealed by superimposition of ArtJ with the complex-bound receptor ModA of the molybdate/tungstate transporter (17). In particular, residue E163 from site II is of interest, as its replacement with glutamine completely abolished the capability of the variant to stimulate ATPase activity of Art(MP)₂ in proteoliposomes. We speculated that E163 might form a salt bridge with a positively charged residue in ArtM. In fact, close inspection of the predicted extracytoplasmic loop regions of ArtM revealed two positively charged residues in loop E2, but there were also acidic residues present (Fig. ​(Fig.1B).1B). Moreover, basic residues were also found in the corresponding peptide regions of close relatives of the ArtMP transporter with experimentally proven substrate specificities (Fig. ​(Fig.1C),1C), suggesting common functions.Open in a separate windowFIG. 1.(A) Crystal structure of ArtJ complexed with l-arginine. The figure was drawn with DS ViewerPro 6.0 (Accelrys, Cambridge, United Kingdom), using the coordinates from protein sequence 2Q2A in the Brookhaven Protein Data Bank. l-Arginine is shown in space-filling representation. Residues mutated in this study are indicated. Site I and site II denote putative interaction sites with Art(MP)₂ (36). (B) Topological model of ArtM. Membrane-spanning helices were predicted using the program TMHMM 2.0 (http://www.cbs.dtu.dk). Residues mutated in this study are indicated. E1 and E2, extracellular loop regions; C1 and C2, cytoplasmic loop regions. (C) Sequence alignment of the extended loop region E2 of ArtM and its close relatives. The proteins considered are as follows: ArtQ_EC and ArtM_EC, membrane-spanning subunits of the E. coli arginine transporter (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"CAA60103","term_id":"769792","term_text":"CAA60103"}}CAA60103 and {"type":"entrez-protein","attrs":{"text":"CAA60104","term_id":"769793","term_text":"CAA60104"}}CAA60104); HisQ_ST and HisM_ST, membrane-spanning subunits of the S. enterica serovar Typhimurium histidine transporter (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"NP_461295","term_id":"16765680","term_text":"NP_461295"}}NP_461295 and {"type":"entrez-protein","attrs":{"text":"NP_461294","term_id":"16765679","term_text":"NP_461294"}}NP_461294); and ArtM_GST, membrane-spanning subunit of the arginine transporter of G. stearothermophilus (currently within contig 298 of the incomplete genomic sequence of strain DSMZ 13240 [http://www.genome.ou.edu/bstearo.html]; the protein is 100% identical with the sequence {"type":"entrez-protein","attrs":{"text":"ZP_03556510","term_id":"221086970","term_text":"ZP_03556510"}}ZP_03556510 of the sequenced genome from Geobacillus sp. strain Y412MC61). Charged residues in each sequence are underlined, and those that were mutated in this study are given in bold.In this study, we investigated the putative role of the E2 loop in ArtM in transporter function by mutational analysis and site-specific cross-linking assays. In a comparative effort, we additionally studied the related but more complex histidine transporter of Salmonella enterica serovar Typhimurium. This system is composed of the membrane-spanning subunits HisM and HisQ, a homodimer of the ATPase subunit, HisP, and as a rare exception from the rule, two solute binding proteins, HisJ and LAO, which are ∼70% identical (1, 3). While HisJ displays its highest affinity for histidine (25), the LAO protein (product of the argT gene) prefers lysine, arginine, and ornithine over histidine (24).Our results demonstrate that site II of ArtJ is presumably in close contact with loop E2 of ArtM and that positively charged residues of the latter are crucial for receptor-transporter interaction. These results were underscored by the finding that positively charged residues of the corresponding loops of HisQ and HisM are likewise required for histidine transporter function. Notably, a K187E mutation of HisM caused sensitivity of the transporter to inhibition by l- and d-histidine.     

Thermo-optically Induced Reorganizations in the Main Light Harvesting Antenna of Plants. II. Indications for the Role of LHCII-only Macrodomains in Thylakoids

We have investigated the circular dichroism spectral transients associated with the light-induced reversible reorganizations in chirally organized macrodomains of pea thylakoid membranes and loosely stacked lamellar aggregates of the main chlorophyll a/b light harvesting complexes (LHCII) isolated from the same membranes. These reorganizations have earlier been assigned to originate from a thermo-optic effect. According to the thermo-optic mechanism, fast local thermal transients due to dissipation of the excess excitation energy induce elementary structural changes in the close vicinity of the dissipation [Cseh et al. (2000) Biochemistry 39: 15250–15257]. Here we show that despite the markedly different CD spectra in the dark, the transient (light-minus-dark) CD spectra associated with the structural changes induced by high light in thylakoids and LHCII are virtually indistinguishable. This, together with other close similarities between the two systems, strongly suggests that the gross short-term, thermo-optically induced structural reorganizations in the membranes occur mainly, albeit probably not exclusively, in the LHCII-only domains [Boekema et al. (2000) J Mol Biol 301: 1123–1133]. Hence, LHCII-only domains might play an important role in light adaptation and photoprotection of plants.     

The effects of ultraviolet irradiation on P680+ reduction in PS II core complexes measured for individual S-states and during repetitive cycling of the oxygen-evolving complex

Flash-induced absorbance measurements at 830 nm on both nanosecond and microsecond timescales have been used to characterise the effect of ultraviolet light on Photosystem II core particles. A combination of UV-A and UV-B, closely simulating the spectrum of sunlight below 350 nm, was found to have a primary effect on the donor side of P680. Repetitive measurements indicated reductions in the nanosecond components of the absorbance decay with a concomitant appearance and increase in the amplitude of a component with a 10 s time constant attributed to slow reduction of P680⁺ by Tyr_z when the function of the oxygen evolving complex is inhibited. Single-flash measurements show that the nanosecond components have amplitudes which vary with S-state. Increasing UV irradiation inhibited the amplitude of these components without changing their S-state dependence. In addition, UV irradiation resulted in a reduction in the total amplitude, with no change in the proportion of the 10 s contribution.Abbreviations BBY- PS II membrane fragments - P680- primary electron donor of PS II - PS II- Photosystem II - Q_A and Q_B– primary and secondary quinone electron acceptors of PS II - S-state- redox state of the oxygenevolving complex - Tyr_z– tyrosine residue in PS II - UV-A- ultraviolet radiation 320–400 nm - UV-B- ultraviolet radiation 280–320 nm     

Studies on the functional organization of Photosystem II. The effect of protein-modifying procedures and of ADRY agents on the reaction pattern of photosystem II in Tris-washed chloroplasts

The role of the protein matrix embedding the functionally active redox components of Photosystem II reaction centers has been studied by investigating the effects of procedures which modify the structure of proteins. In order to reduce the influence of the electron transport involving secondary donor and acceptor components, Triswashed chloroplasts were used which are completely deprived of their oxygen-evolving capacity. The functional activity was detected via absorption changes, reflecting at 334 and 690 or 834 nm the turnover of the primary plastoquinone acceptor, X320, and of the photochemically active chlorophyll a complex, Chl a_II, respectively, and at 520 nm the transient formation of a transmembrane electric potential gradient. Under repetitive flash excitation of Tris-washed chloroplasts it was found that: (a) The relaxation kinetics at 690 nm become significantly accelerated in the presence of external electron donors. (b) Trypsin treatment blocks to a high degree the turnover of Chl a_II and X320 unless exogenous acceptors are present, which directly oxidize X320^?, such as K₃Fe(CN)₆. (c) In the presence of K₃Fe(CN)₆ the recovery kinetics of Chl a_II and X320 are retarded markedly by trypsin, followed by a progressive decline in the extent thereof. (d) 2-(3-Chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p), known to reduce the lifetime of S₂ and S₃ in normal chloroplasts, significantly accelerates the recovery of Chl a_II. 10 μs kinetics are observed which correspond with the electron-transfer rate from D₁ to Chl a⁺_II. ANT 2p simultaneously retards the decay kinetics of X320^? and of the electrochromic absorption changes. (e) The kinetic pattern of the electrochromic absorption changes is also affected by the salt content of the suspension. Under dark-adapted conditions, the 10 μs relaxation kinetics of the 834 nm absorption change due to the first flash are hardly affected by mild trypsinization of 5–10 min duration, whereas the amplitude decreases by approx. 30%. The data obtained in Tris-washed chloroplasts could consistently be interpreted as a modification of the back reaction between X320^? and Chl a⁺_II which is caused solely by a change in the reactivity of X320 due to trypsin-induced degradation of the native X320-B apoprotein. Furthermore, ADRY agents are inferred to stimulate cyclic electron flow, which leads to reduction of D⁺₁ between the flashes. A simplified scheme is discussed which describes the functional organization of the reaction center complex.     

The type II Ca/calmodulin-dependent protein kinases are involved in the regulation of cell wall integrity and oxidative stress response in Candida albicans

The type II Ca²⁺/calmodulin-dependent protein kinases (CaMKs) are thought to play a vital role in cellular regulation in mammalian cells. Two genes CMK1 and CMK2 in the Candida albicans genome encode homologues of mammalian CaMKs. In this work, we constructed the cmk1Δ/Δ, the cmk2Δ/Δ and the cmk1Δ/Δcmk2Δ/Δ mutants and found that CaMKs function in cell wall integrity (CWI) and cellular redox regulation. Loss of either CMK1 or CMK2, or both resulted in increased expression of CWI-related genes under Calcofluor white (CFW) treatment. Besides, CaMKs are essential for the maintenance of cellular redox balance. Disruption of either CMK1 or CMK2, or both not only led to a significant increase of intracellular ROS levels, but also led to a decrease of the mitochondrial membrane potential (MMP), suggesting the important roles that CaMKs play in the maintenance of the mitochondrial function.