C-terminal Alpha Helix Research Articles

Structural modeling and characterization of the Mycobacterium tuberculosis MmpL3 C-terminal domain.

The Mycobacterium tuberculosis (Mtb) cell envelope provides a protective barrier against the immune response and antibiotics. The mycobacterial membrane protein large (MmpL) family of proteins export cell envelope lipids and siderophores; therefore, these proteins are important for the basic biology and pathogenicity of Mtb. In particular, MmpL3 is essential and a known drug target. Despite interest in MmpL3, the structural data in the field are incomplete. Utilizing homology modeling, AlphaFold, and biophysical techniques, we characterized the cytoplasmic C-terminal domain (CTD) of MmpL3 to better understand its structure and function. Our in silico models of the MmpL11TB and MmpL3TB CTD reveal notable features including a long unstructured linker that connects the globular domain to the last transmembrane (TM) in each transporter, charged lysine and arginine residues facing the membrane, and a C-terminal alpha helix. Our predicted overall structure enables a better understanding of these transporters.

Cdc14 phosphatases use an intramolecular pseudosubstrate motif to stimulate and regulate catalysis

Cdc14 phosphatases are related structurally and mechanistically to protein tyrosine phosphatases (PTPs) but evolved a unique specificity for phosphoSer-Pro-X-Lys/Arg sites primarily deposited by cyclin-dependent kinases. This specialization is widely conserved in eukaryotes. The evolutionary reconfiguration of the Cdc14 active site to selectively accommodate phosphoSer-Pro likely required modification to the canonical PTP catalytic cycle. While studying Saccharomyces cerevisiae Cdc14, we discovered a short sequence in the disordered C terminus, distal to the catalytic domain, which mimics an optimal substrate. Kinetic analyses demonstrated this pseudosubstrate binds the active site and strongly stimulates rate-limiting phosphoenzyme hydrolysis, and we named it "substrate-like catalytic enhancer" (SLiCE). The SLiCE motif is found in all Dikarya fungal Cdc14 orthologs and contains an invariant glutamine, which we propose is positioned via substrate-like contacts to assist orientation of the hydrolytic water, similar to a conserved active site glutamine in other PTPs that Cdc14 lacks. AlphaFold2 predictions revealed vertebrate Cdc14 orthologs contain a conserved C-terminal alpha helix bound to the active site. Although apparently unrelated to the fungal sequence, this motif also makes substrate-like contacts and has an invariant glutamine in the catalytic pocket. Altering these residues in human Cdc14A and Cdc14B demonstrated that it functions by the same mechanism as the fungal motif. However, the fungal and vertebrate SLiCE motifs were not functionally interchangeable, illuminating potential active site differences during catalysis. Finally, we show that the fungal SLiCE motif is a target for phosphoregulation of Cdc14 activity. Our study uncovered evolution of an unusual stimulatory pseudosubstrate motif in Cdc14 phosphatases.

Characterization of Three Different Endolysins Effective against Gram-Negative Bacteria.

Genes encoding endolysins were identified and cloned from three different Escherichia coli bacteriophages, 10-24(13), PBEC30, and PBEC56. Putative antimicrobial peptide (AMP)-like C-terminal alpha helix structures with amphipathic natures were predicted from the three endolysins. Each gene was cloned and expressed as hexahistidine-tagged forms, and the products were purified and characterized. The purified endolysins exhibited antibacterial activities against a variety of Gram-negative bacteria including Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumonia. Their antibacterial activities were improved by N-terminal fusion with an antimicrobial peptide, cecropin A. Minimum inhibitory concentrations (MIC) were as low as 4 μg/mL, depending on the targeted strain. The endolysins' enzymatic activities were not affected by changes in pH at ranges from 5 to 10 and were stable at temperatures between 4 and 65 °C. The in vivo efficacies of the three endolysins were also demonstrated using Galleria melonella for infection models.

Structure of PDE3A-SLFN12 complex reveals requirements for activation of SLFN12 RNase

DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies.

Identification and structural analysis of the Schizosaccharomyces pombe SMN complex.

The macromolecular SMN complex facilitates the formation of Sm-class ribonucleoproteins involved in mRNA processing (UsnRNPs). While biochemical studies have revealed key activities of the SMN complex, its structural investigation is lagging behind. Here we report on the identification and structural determination of the SMN complex from the lower eukaryote Schizosaccharomyces pombe, consisting of SMN, Gemin2, 6, 7, 8 and Sm proteins. The core of the SMN complex is formed by several copies of SMN tethered through its C-terminal alpha-helices arranged with alternating polarity. This creates a central platform onto which Gemin8 binds and recruits Gemins 6 and 7. The N-terminal parts of the SMN molecules extrude via flexible linkers from the core and enable binding of Gemin2 and Sm proteins. Our data identify the SMN complex as a multivalent hub where Sm proteins are collected in its periphery to allow their joining with UsnRNA.

Structural and molecular dynamic studies of Pseudomonas aeruginosa OdaA reveal the regulation role of a C-terminal hinge element

BackgroundCrotonase superfamily members exhibit great catalytic diversity towards various acyl-CoA substrates. A common CoA moiety binding pattern is usually observed in this family, understanding the substrate-binding mechanism would facilitate the rational engineering of crotonases for improved properties. MethodsWe applied X-ray crystallography to investigate a putative enoyl-CoA hydratase/isomerase OdaA in Pseudomonas aeruginosa. Thermal shift assay (TSA) were performed to explore the binding of OdaA with CoA thioester substrates. Furthermore, we performed molecular dynamics (MD) simulations to elucidate the dynamics of its CoA-binding site. ResultsWe solved the crystal structures of the apo and CoA-bound OdaA. Thermal shift assay (TSA) showed that CoA thioester substrates bind to OdaA with a different degree. MD simulations demonstrated that the C-terminal alpha helix underwent a structural transition and a hinge region would associate with this conformational change. ConclusionsTSA in combination with MD simulations elucidate that the dynamics of C-terminal alpha helix in CoA-binding, and a hinge region play an important role in conformational change. General significanceThose results help to extend our knowledge about the nature of crotonases and would be informative for future mechanistic studies and industry applications.

Abstract 5873: Identification of novel BIRC5/survivin splice variants in cancer

Abstract Background: BIRC5/survivin is highly expressed in cancer cells and represents a promising therapeutic target. In order to complete a comprehensive survey of its splice variants, we applied a direct RT-PCR approach as well as next generation sequencing (NGS) to tumor cell cDNAs. We have identified 10 novel splice junctions and three novel splice variants of survivin. Methods: Survivin transcripts were amplified from A1207 (glioblastoma) HeLa (cervical adenocarcinoma) and U87 (glioblastoma) cDNAs. For manual identification of splice forms, individual products were subcloned into TOPO vector and sequenced. For NGS analysis, RT-PCR products used to generate libraries which were subjected to sequencing, after which resulting reads were mapped against the human reference genome and assembled by Trinity. Results: Survivin RT-PCR products from A1207 cDNA were isolated and selected for TOPO cloning/sequencing. Of seventeen clones, the canonical isoform was represented by 4, and known variants including Δex3, 2B and 3B were represented by 5. Two novel sequences were identified: 1) 2B/Δex3, which contains a 68 bp shuffled exon 4/3 segment between exons 3 and 4 plus the 2B insert, and 2) 4B, in which exon 4 is extended by 27 bp at its 5' end. Both of these transcripts are predicted to encode full-length proteins: 2B/Δex3 contains the 23 aa 2B insert plus Δex3's unique C-terminus; 4B contains a novel 9 aa motif embedded in survivin's C-terminal alpha helix. RT-PCR products from A1207, HeLa and U87 templates subjected to NGS yielded five full-length BIRC5 contigs, including the canonical isoform, Δex3, 2B, 3B, and the predicted variant 2B/3B. To identify novel splice junctions, sequences were searched excluding canonical exon boundaries. The resulting 151 reads represented 10 novel exon junctions, among which the greatest proportion consisted of exon 1 and 3 (71 reads, 47%), or an extension of exon 2B by 32 bp at its 3' end (35 reads, 23%). Two variants contained the extended version of exon 4 seen in isoform 4B. A primer annealing within exon 4B's unique 27 bp was subsequently used for RT-PCR to validate the expression of the full-length isoform in HeLa, U87, A1207 and HEK293T cells. Conclusions: We have identified multiple novel splice variants of survivin, including Δex3/2B, 4B, and Δex3/4B. Proteins encoded by these transcripts potentially have pro- or anti-oncogenic functions, and further studies are needed to delineate their role in survivin biologies. Citation Format: Sheila Figel, Meaghan Birkemeier, Jesse Luce, Prashant Singh, Jianmin Wang, Cheryl Frank, Sanam Dharma, Emma Steinmetz, Michael Ciesielski, Robert Fenstermaker. Identification of novel BIRC5/survivin splice variants in cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5873.

Structural Conservation and Diversity of PilZ-Related Domains.

The widespread bacterial second messenger cyclic diguanylate (c-di-GMP) regulates a variety of processes, including protein secretion, motility, cell development, and biofilm formation. c-di-GMP-dependent responses are often mediated by its binding to the cytoplasmic receptors that contain the PilZ domain. Here, we present comparative structural and sequence analysis of various PilZ-related domains and describe three principal types of them: (i) the canonical PilZ domain, whose structure includes a six-stranded beta-barrel and a C-terminal alpha helix, (ii) an atypical PilZ domain that contains two extra alpha helices and forms stable tetramers, and (iii) divergent PilZ-related domains, which include the eponymous PilZ protein and PilZN (YcgR_N) and PilZNR (YcgR_2) domains. We refine the second c-di-GMP binding motif of PilZ as [D/N]hSXXG and show that the hydrophobic residue h of this motif interacts with a cluster of conserved hydrophobic residues, helping maintain the PilZ domain fold. We describe several novel PilZN-type domains that are fused to the canonical PilZ domains in specific taxa, such as spirochetes, actinobacteria, aquificae, cellulose-degrading clostridia, and deltaproteobacteria. We propose that the evolution of the three major groups of PilZ domains included (i) fusion of pilZ with other genes, which produced Alg44, cellulose synthase, and other multidomain proteins; (ii) insertion of an ∼200-bp fragment, which resulted in the formation of tetramer-forming PilZ proteins; and (iii) tandem duplication of pilZ genes, which led to the formation of PilZ dimers and YcgR-like proteins.IMPORTANCE c-di-GMP is a ubiquitous bacterial second messenger that regulates motility, biofilm formation, and virulence of many bacterial pathogens. The PilZ domain is a widespread c-di-GMP receptor that binds c-di-GMP through its RXXXR and [D/N]hSXXG motifs; some PilZ domains lack these motifs and are unable to bind c-di-GMP. We used structural and sequence analysis to assess the diversity of PilZ-related domains and define their common features. We show that the hydrophobic residue h in the second position of the second motif is highly conserved; it may serve as a readout for c-di-GMP binding. We describe three principal classes of PilZ-related domains, canonical, tetramer-forming, and divergent PilZ domains, and propose the evolutionary pathways that led to the emergence of these PilZ types.

Two new ene-reductases from photosynthetic extremophiles enlarge the panel of old yellow enzymes: CtOYE and GsOYE

Looking for new ene-reductases with uncovered features beneficial for biotechnological applications, by mining genomes of photosynthetic extremophile organisms, we identified two new Old Yellow Enzyme homologues: CtOYE, deriving from the cyanobacterium Chroococcidiopsis thermalis, and GsOYE, from the alga Galdieria sulphuraria. Both enzymes were produced and purified with very good yields and displayed catalytic activity on a broad substrate spectrum by reducing α,β-unsaturated ketones, aldehydes, maleimides and nitroalkenes with good to excellent stereoselectivity. Both enzymes prefer NADPH but demonstrate a good acceptance of NADH as cofactor. CtOYE and GsOYE represent robust biocatalysts showing high thermostability, a wide range of pH optimum and good co-solvent tolerance. High resolution X-ray crystal structures of both enzymes have been determined, revealing conserved features of the classical OYE subfamily as well as unique properties, such as a very long loop entering the active site or an additional C-terminal alpha helix in GsOYE. Not surprisingly, the active site of CtOYE and GsOYE structures revealed high affinity toward anions caught from the mother liquor and trapped in the anion hole where electron-withdrawing groups such as carbonyl group are engaged. Ligands (para-hydroxybenzaldehyde and 2-methyl-cyclopenten-1-one) added on purpose to study complexes of GsOYE were detected in the enzyme catalytic cavity, stacking on top of the FMN cofactor, and support the key role of conserved residues and FMN cofactor in the catalysis.

TfVPS32 Regulates Cell Division in the Parasite Tritrichomonas foetus.

The flagellated protist Tritrichomonas foetus is a parasite that causes bovine trichomonosis, a major sexually transmitted disease in cattle. Cell division has been described as a key player in controlling cell survival in other cells, including parasites but there is no information on the regulation of this process in T.foetus. The regulation of cytokinetic abscission, the final stage of cell division, is mediated by members of the ESCRT (endosomal sorting complex required for transport) machinery. VPS32 is a subunit within the ESCRTIII complex and here, we report that TfVPS32 is localized on cytoplasmic vesicles and a redistribution of the protein to the midbody is observed during the cellular division. In concordance with its localization, deletion of TfVPS32 C-terminal alpha helices (α5 helix and/or α4-5 helix) leads to abnormal T.foetus growth, an increase in the percentage of multinucleated parasites and cell cycle arrest at G2/M phase. Together, these results indicate a role of this protein in controlling normal cell division.

The C-terminal α-helix of YsxC is essential for its binding to 50S ribosome and rRNAs

YsxC is an essential P-loop GTPase that interacts with the 50S subunit of the ribosome. The putative implication in ribosome binding of two basic clusters of YsxC, a conserved positively charged patch including R31, R116, H117 and K146 lying adjacent to the nucleotide-binding site, and the C-terminal alpha helix, was investigated. C-terminal truncation variants of YsxC were unable to bind to both ribosome and rRNAs, whereas mutations in the other cluster did not affect YsxC binding. Our results indicate that the basic C-terminal region of YsxC is required for its binding to the 50S ribosomal subunit.

Signal-sequence induced conformational changes in the signal recognition particle.

Co-translational protein targeting is an essential, evolutionarily conserved pathway for delivering nascent proteins to the proper cellular membrane. In this pathway, the signal recognition particle (SRP) first recognizes the N-terminal signal sequence of nascent proteins and subsequently interacts with the SRP receptor. For this, signal sequence binding in the SRP54 M domain must be effectively communicated to the SRP54 NG domain that interacts with the receptor. Here we present the 2.9 Å crystal structure of unbound- and signal sequence bound SRP forms, both present in the asymmetric unit. The structures provide evidence for a coupled binding and folding mechanism in which signal sequence binding induces the concerted folding of the GM linker helix, the finger loop, and the C-terminal alpha helix αM6. This mechanism allows for a high degree of structural adaptability of the binding site and suggests how signal sequence binding in the M domain is coupled to repositioning of the NG domain.

Identification and Characterization of a Yeast Iso-1-Cytochrome C C-Terminal Domain Swapped Dimer

Domain swapped protein dimers consist of a swapped domain linked by a hinge loop. They have been proposed as a means of achieving larger assemblies potentially contributing to biological cellular activity or conferring disease. Here we present a crystal structure of a C-terminal domain swapped dimer in yeast iso-1-cytochrome c. In this structure the C-terminal alpha helix from one monomer positions itself in the native position of the opposite monomer and vice versa. The highly dynamic heme crevice loop, the most highly conserved portion of the cytochrome c sequence, spans the gap acting as the hinge loop. Interestingly, conversion of the heme crevice loop to the hinge loop results in a loss of the native Met80-heme ligation. This produces an open heme coordination site on each subunit of the dimer. As cytochrome c requires an open heme coordination site to act as a peroxidase, to oxidize cardiolipin and initiate the intrinsic apoptotic pathway, this dimer structure could potentially be a structure particularly suited to function in oxidizing cardiolipin. In fact, a recently reported C-terminal domain swapped dimer of horse cytochrome c demonstrates increased peroxidase activity relative to the monomer. Although the yeast and horse dimer are similar, the hinge loop orientations differ. The hinge loop is two residues longer in the yeast dimer resulting in an increased distance between the heme groups and an altered angle of the hinge loops relative to the horse cytochrome c dimer. Even though they contain similar structure and sequence, the domain swapped yeast iso-1-cytochrome c dimer demonstrates decreased stability compared to the horse cytochrome c dimer.

Unfolding of beta‐lactoglobulin on the surface of polystyrene nanoparticles: Experimental and computational approaches

Structural changes ensuing from the non-covalent absorption of bovine beta-lactoglobulin (BLG) on the surface of polystyrene nanoparticles were investigated by using spectroscopic approaches, by assessing the reactivity of specific residues, and by limited proteolysis/mass spectrometry. Also, the immunoreactivity of absorbed and free BLG was compared. All these approaches indicated substantial rearrangements of the protein structure in the absorbed state, in spite of the reported structural rigidity of BLG. Changes made evident by experimental measurements were confirmed by computational approaches. These indicate that adsorption-related changes are most marked in the area between the main C-terminal alpha helix and the beta-barrel, and lead to full exposure of the thiol on Cys121 , consistent with experimental measurements. In the computational model of bound BLG, both Trp61 and Trp19 also move away from their neighboring quenchers and become solvent-exposed, as indicated by fluorescence measurement. Upon binding, the beta-barrel also loosens, with a substantial increase in immunoreactivity and with noticeable changes in the trypsinolytic pattern. The possible general significance of the structural changes reported here for non-covalently adsorbed BLG is discussed with respect to recognition events involving surface-bound proteins, as are aspects related to the carrier function(s) of BLG, and to its use as a common ingredient in many food systems.

Abstract LB-426: Potent inhibitors of RAS pathways that bind directly to Ras proteins

Abstract RAS genes are the most frequently mutated oncogenes in human cancer. Although they are believed to be among the most important drug targets in oncology, no synthetic molecules that directly target any member of the family and inhibit its function with appreciable in vivo or even in vitro activity have been identified. We have utilized a previously developed approach of peptide structural stabilization by membrane anchoring for the development of potent inhibitors of Ras proteins. We have tested lipopeptide analogs of different parts of Ras proteins and their interaction partners and have found lipopeptide analogs of hypervariable regions and of the C-terminal alpha helix (helix 6) of Ras proteins to be the most effective in interfering with Ras function and inhibiting Ras-dependent, but not Ras-independent growth of cancer cells. Direct binding of inhibitors to recombinant K-Ras (1-166) was detected by Microscale Thermophoresis (MST), and circular dichroism spectroscopy. NMR titration data revealed a localized interaction of helix 6 analogs with the Switch I region of K-Ras with the most significant chemical shift perturbations found in residues Q25, N26, S32, T35, and E37. Compounds inhibit the formation of active RasGTP and prevent Ras signaling to downstream effectors. Structural optimization of helix 6 analogs resulted in a metabolically stable, 12 amino acid residues long peptide made of all-D amino acids that inhibited the growth Ras-dependent cancer cell lines with GI50 below 1 nM. MST studies have shown that a fluorescently-labeled version of the compound binds to recombinant GDP-loaded K-Ras (1-166) with KD= 86 ±8 nM. Derivatives of K-Ras4B hypervariable region exert their activity by a significantly different mechanism: they cause degradation of Ras protein. Partially optimized compound of the series inhibit growth of cancer cells with GI 50 in submicromolar range and bind to recombinant K-Ras (1-166) with KD=1250±140nM (as determined in MST assay). CD spectroscopy data suggest that binding of compounds of both classes to K-Ras is accompanied by significant changes in protein secondary structure. In mice, inhibitors potently inhibited growth of H358 human lung tumor xenograft and Lewis lung carcinoma when injected subcutaneously every second day. The compounds are promising drug candidates for the treatment of many tumor types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-426. doi:1538-7445.AM2012-LB-426

Differential Effects of Crowding Reagents on the Interaction of Profilin and Thymosin Beta 4 with Actin

Actin polymerization in vivo is spatially organized, with dynamic actin polymerization occurring at the leading edge of a motile cell, and occurs in the context of molecular crowding effects, including osmolyte concentration, that also vary with location. The current study tests the hypothesis that molecular crowding could influence interactions of actin with actin-regulatory proteins so as to shift the pool of actin monomers to a polymerization competent state within the active polymerization zone. In support of this hypothesis, we found that a naturally occurring osmolyte trimethylamine N-oxide (TMAO) dramatically increases affinity, KT, of the actin sequestering protein thymosin b4 (TB4) to monomeric actin and significantly decreases barbed end actin critical concentration Ac in presence of ATP. The N- and C-terminal alpha helices in TB4 helices are known to be unstructured in aqueous solution and to adopt helical conformation in organic solvents or upon binding to actin. The effects of TMAO are consistent with a decrease in free energy of the actin-TB4 interaction through facilitating alpha helix formation. The effect of TMAO on the affinity, KP, of ATP-G-actin to a globular actin regulating protein, profilin, is much weaker, as might be expected based on structural considerations. Unlike TMAO, polyethylene glycol (PEG) affects Ac, KT, and KP by similar extents. We also found that the TMAO facilitates ternary complex formation between actin, profilin, and TB4 which may very significantly increase the amount of unpolymerized actin in presence of profilin and TB4, and therefore inhibit actin polymerization. We conclude that spatial variation in osmolytes could, in principle, explain how actin sheds TB4 prior to polymerization.

Structure–function studies of chemokine-derived carboxy-terminal antimicrobial peptides

Recent reports which show that several chemokines can act as direct microbicidal agents have drawn renewed attention to these chemotactic signalling proteins. Here we present a structure-function analysis of peptides derived from the human chemokines macrophage inflammatory protein-3alpha (MIP-3alpha/CCL20), interleukin-8 (IL-8), neutrophil activating protein-2 (NAP-2) and thrombocidin-1 (TC-1). These peptides encompass the C-terminal alpha-helices of these chemokines, which have been suggested to be important for the direct antimicrobial activities. Far-UV CD spectroscopy showed that the peptides are unstructured in aqueous solution and that a membrane mimetic solvent is required to induce a helical secondary structure. A co-solvent mixture was used to determine solution structures of the peptides by two-dimensional (1)H-NMR spectroscopy. The highly cationic peptide, MIP-3alpha(51-70), had the most pronounced antimicrobial activity and displayed an amphipathic structure. A shorter version of this peptide, MIP-3alpha(59-70), remained antimicrobial but its structure and mechanism of action were unlike that of the former peptide. The NAP-2 and TC-1 proteins differ in their sequences only by the deletion of two C-terminal residues in TC-1, but intact TC-1 is a very potent antimicrobial while NAP-2 is inactive. The corresponding C-terminal peptides, NAP-2(50-70) and TC-1(50-68), had very limited and no bactericidal activity, respectively. This suggests that other regions of TC-1 contribute to its bactericidal activity. Altogether, this work provides a rational structural basis for the biological activities of these peptides and proteins and highlights the importance of experimental characterization of peptide fragments as distinct entities because their activities and structural properties may differ substantially from their parent proteins.

Characterization of an Unusual LOV Domain Protein in the α‐Proteobacterium Rhodobacter sphaeroides

The facultatively phototrophic purple bacterium Rhodobacter sphaeroides 2.4.1 harbors a LOV (light, oxygen and voltage) domain protein, which shows a particular structure. LOV domains perceive blue light by a noncovalently bound flavin and transmit the signal to various coupled output domains. Proteins, that harbor a LOV core, function e.g. as phototropins or circadian clock regulators. Jalpha helices, which act as linker between the LOV core and the output domain, were shown to be involved in the light-dependent activation of the output domain. Like PpSB2 from Pseudomonas putida, the LOV domain protein of R. sphaeroides is not coupled to an effector domain and harbors an extended C-terminal alpha helix. We expressed the R. sphaeroides LOV domain recombinantly in Escherichia coli. The protein binds an FMN as a cofactor and shows a photocycle typical for LOV domain containing proteins. In R. sphaeroides, we detected the protein as well in the cytoplasm as in the membrane fraction, which was not reported for other bacterial LOV domain proteins.

Structure and Insight into Blue Light-Induced Changes in the BlrP1 BLUF Domain,

BLUF domains (sensors of blue light using flavin adenine dinucleotide) are a group of flavin-containing blue light photosensory domains from a variety of bacterial and algal proteins. While spectroscopic studies have indicated that these domains reorganize their interactions with an internally bound chromophore upon illumination, it remains unclear how these are converted into structural and functional changes. To address this, we have solved the solution structure of the BLUF domain from Klebsiella pneumoniae BlrP1, a light-activated c-di-guanosine 5'-monophosphate phosphodiesterase which consists of a sensory BLUF and a catalytic EAL (Glu-Ala-Leu) domain [Schmidt et. al. (2008) J. Bacteriol. 187, 4774-4781]. Our dark state structure of the sensory domain shows that it adopts a standard BLUF domain fold followed by two C-terminal alpha helices which adopt a novel orientation with respect to the rest of the domain. Comparison of NMR spectra acquired under dark and light conditions suggests that residues throughout the BlrP1 BLUF domain undergo significant light-induced chemical shift changes, including sites clustered on the beta(4)beta(5) loop, beta(5) strand, and alpha(3)alpha(4) loop. Given that these changes were observed at several sites on the helical cap, over 15 A from chromophore, our data suggest a long-range signal transduction process in BLUF domains.

Les protéines à domaine START, des trafiquants intracellulaires de lipides

Fifteen START domain-containing proteins exist in mammals. On the basis of their structural homology, this family is divided into several sub-families consisting mainly of non-vesicular intracellular lipid carriers. With the exception of the Thioesterase-START subfamily, the other subfamilies are represented among invertebrates. The START domain is always located in the C-terminus of the protein. It is a module of about 210 residues that binds lipids, including sterols. Cholesterol, 25-hydroxycholesterol, phosphatidylcholine, phosphatidylethanolamine and ceramides are ligands for STARD1/STARD3-6, STARD5, STARD2/STARD10, STARD10 and STARD11, respectively. The lipids or sterols bound by the remaining 7 START proteins are unknown. The START domain can be regarded as a lipid-exchange and/or a lipid-sensing domain. The START domain consists in a deep lipid-binding pocket--that shields the hydrophic ligand from the external aqueous environment--covered by a lid formed by a C-terminal alpha helix. Within the same subgroup, such as the sterols-carriers subgroup, different START domains have similar biochemical properties; however, their expression profile and their subcellular localization distinguish them and are critical for their different biological functions. START proteins act in a variety of distinct physiological processes, such as lipid transfer between intracellular compartments, lipid metabolism and modulation of signaling events. Mutation or misexpression of START proteins is linked to pathological processes, including genetic disorders, autoimmune diseases and cancers.