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1.
Junhe Ma Frank Edlich Guillermo A. Bermejo Kristi L. Norris Richard J. Youle Nico Tjandra 《Proceedings of the National Academy of Sciences of the United States of America》2012,109(51):20901-20906
The human protein Bax sits at a critical regulatory junction of apoptosis, or programmed cell death. Bax exists in equilibrium between cytosolic and mitochondria-associated forms that shifts toward the latter when Bax is activated by proapoptotic proteins. Activated Bax changes conformation, inserts into the mitochondrial outer membrane (MOM), oligomerizes, and induces MOM permeabilization, causing the release of cytochrome c, which effectively commits the cell to die. Because apoptosis is also a basic defense mechanism against invading pathogens, many viruses have developed counteractive measures. Such is the case of human cytomegalovirus, the replication of which hinges on vMIA (viral mitochondria-localized inhibitor of apoptosis), a virus-encoded protein with a unique, albeit poorly understood antiapoptotic activity by which it binds and recruits Bax to mitochondria. Here we show, via the structure determination of the complex between Bax and a peptide comprising vMIA’s Bax-binding domain, that vMIA contacts Bax at a previously unknown regulatory site. Notably, using full-length vMIA, the structure is independently confirmed by assays in human cells that measure Bax subcellular localization and cytochrome c release. Mutants that disrupt key intermolecular interactions disfavor vMIA’s mitochondrial recruitment of Bax, and increase cytochrome c release upon apoptosis induction. In a more stringent test, an engineered binding interface that achieves wild-type–like charge complementarity, although in a reversed fashion, recovers wild-type behavior. The structure suggests that by stabilizing key elements in Bax needed to unravel for its MOM insertion and oligomerization, vMIA prevents these important steps in apoptosis.Apoptosis, or programmed cell death, is a process essential for organ development and tissue maintenance (1–3) that, when dysfunctional, can contribute to several diseases, including cancer and autoimmunity (4). The protein Bax plays a crucial role in apoptosis in that its activation defines the fate of the cell (5): Bax translocates from the cytosol to mitochondria, accompanied by dramatic conformational changes, oligomerization, and the permeabilization of the mitochondrial outer membrane (MOM), which, in turn causes the release of cytochrome c that activates the cell-demolition machinery. Bax’s function is modulated by both pro- and antiapoptotic proteins from the Bcl-2 family (6). Members of this family share at least one Bcl-2 homology (BH) domain, and can be classified into three different groups: (i) antiapoptotic proteins with four BH units (BH1234) (e.g., Bcl-2 and Bcl-xL), (ii) proapoptotic proteins with three BH units (BH123) (e.g., Bax and Bak), and (iii) proapoptotic BH3-only proteins (e.g., Bid and Bim). The latter transmit prodeath signals, which, unless neutralized by the antiapoptotic Bcl-2 members, activate Bax (or Bak).Many viruses have evolved mechanisms to inhibit apoptosis, used by the host organism to defend itself against infection (7). Indeed, the replication of human cytomegalovirus—a widely spread herpesvirus, pathogenic in immunocompromised individuals—relies on the encoded antiapoptotic protein vMIA (viral mitochondria-localized inhibitor of apoptosis) (8–10). In contrast with antiapoptotic Bcl-2 members, which inhibit Bax mitochondrial localization, vMIA recruits Bax to mitochondria as part of its apoptotic inhibitory activity (11). Although previous structural determination efforts have provided invaluable atomistic insights about the interactions of Bcl-2 proteins (see below), little is known with regards to that between Bax and vMIA.In solution, Bax is α-helical (Fig. 1) and displays its C-terminal helix, α9, lodged within a hydrophobic pocket (12). Experimental truncation of this helix in homologous (but functionally opposite) Bcl-xL exposes the pocket, which binds peptides from BH3 domains of proapoptotic Bcl-2 members Bak, Bad, and Bim (5). Despite the existence of this canonical BH3 domain-binding site, structural studies on the complex of full-length Bax and a Bim-BH3 peptide have revealed a different binding target, involving Bax helices α1 and α6, antipodal to the hydrophobic pocket, still occupied by α9 (13) (Fig. 1). Helix α9 is believed to be part of a C-terminal membrane-anchoring region, and is crucial for mitochondrial localization of Bax in normal cells. In cytomegalovirus-infected cells Bax is also recruited to mitochondria by vMIA. This process, however, is independent of α9 (14), which suggests the canonical BH3-binding site excludes the vMIA–Bax interaction surface. Furthermore, assuming the aforementioned Bax Bim-BH3 binding site (13) is shared with other BH3-only proteins, such as tBid (truncated Bid), this site also excludes vMIA, as tBid and vMIA do not compete for Bax as a binding partner (11).Open in a separate windowFig. 1.Chemical-shift mapping of the vMIA-BBD binding site on Bax. The chemical-shift change of 1H and 15N signals of 15N-enriched Bax upon addition of vMIA-BBD, Δδ (Materials and Methods), is displayed against the structure of Bax (Upper) and versus residue number (Lower). α-Helical segments are indicated throughout. Prolines and residues with overlapped NMR signals are not represented in the bar plot. Residues with chemical shift changes larger than 0.017 ppm (horizontal dashed line) are denoted by yellow bars, the corresponding Cα atoms indicated by yellow spheres in the structure.Taken together, the above evidence suggests vMIA, with its unique antiapoptotic activity, binds Bax at a unique site. Indeed, this finding is confirmed here at atomic detail via the solution NMR structure determination of the complex between Bax and vMIA’s Bax-binding domain (vMIA-BBD). The structure is additionally supported by mutations of residues identified to be important for the stability of the complex. The effect of the mutations is assessed in solution on the binding affinity between Bax and vMIA-BBD, as well as in human cells (using full-length vMIA) by the quantification of both the subcellular localization of Bax and the release of cytochrome c upon apoptosis induction. The structure of the complex not only suggests the mechanism by which vMIA inhibits the activation of Bax, but also helps dissect even further the different interactions that converge on this crucial player in apoptosis. 相似文献
2.
Dana Westphal Grant Dewson Marie Menard Paul Frederick Sweta Iyer Ray Bartolo Leonie Gibson Peter E. Czabotar Brian J. Smith Jerry M. Adams Ruth M. Kluck 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(39):E4076-E4085
The pivotal step on the mitochondrial pathway to apoptosis is permeabilization of the mitochondrial outer membrane (MOM) by oligomers of the B-cell lymphoma-2 (Bcl-2) family members Bak or Bax. However, how they disrupt MOM integrity is unknown. A longstanding model is that activated Bak and Bax insert two α-helices, α5 and α6, as a hairpin across the MOM, but recent insights on the oligomer structures question this model. We have clarified how these helices contribute to MOM perforation by determining that, in the oligomers, Bak α5 (like Bax α5) remains part of the protein core and that a membrane-impermeable cysteine reagent can label cysteines placed at many positions in α5 and α6 of both Bak and Bax. The results are inconsistent with the hairpin insertion model but support an in-plane model in which α5 and α6 collapse onto the membrane and insert shallowly to drive formation of proteolipidic pores.Commitment of cells to apoptosis is determined primarily by interactions within the B-cell lymphoma-2 (Bcl-2) protein family on the mitochondrial outer membrane (MOM) (1–4). The proapoptotic members Bcl-2 antagonist/killer (Bak) and Bcl-2–associated X protein (Bax) mediate the pivotal step of MOM permeabilization, which releases proteins, such as cytochrome c, that promote the proteolytic demolition by caspases. Two other Bcl-2 subfamilies tightly control Bak and Bax activation. Their activation is promoted by the Bcl-2 homology domain 3 (BH3)-only proteins, such as BH3-interacting domain death agonist (Bid), the truncated form of which (tBid) can directly bind both. Conversely, prosurvival family members can bind and inhibit activated Bak and Bax, as well as the BH3-only proteins.Like their prosurvival relatives, Bak and Bax in healthy cells are globular monomers, comprising similar helical bundles with a hydrophobic α-helix (α5) surrounded by amphipathic helices (5, 6). Their C-terminal helix (α9) is a hydrophobic transmembrane (TM) domain that anchors them in the MOM. In healthy cells Bak is already anchored there, presumably solely by α9, whereas Bax is primarily cytosolic (5), accumulating at the MOM after an apoptotic signal and inserting its α9. Other major conformational changes in both Bak and Bax, reviewed in ref 4, include exposure of their BH3 (α2) and its reburial within the surface groove of another activated Bak or Bax molecule (7–10). These novel “symmetric” homo-dimers can multimerize via association of α6 helices (8, 11, 12).Although oligomeric Bak and Bax are highly implicated in MOM permeabilization, how they interact with the membrane to form pores remains a mystery. The first structure of a Bcl-2 family member, the prosurvival protein Bcl-xL (13), and later those of Bax (5) and Bak (6), provided a tantalizing clue: similarities with the pore-forming domains of bacterial toxins, such as diphtheria toxin or colicin A. To form pores, these toxins are thought to insert their two hydrophobic core helices as a hairpin across the membrane (14), suggesting that the central helices of Bak and Bax (α5 and α6) might penetrate the MOM similarly (reviewed in refs 3, 15, and 16). Consistent with this hairpin insertion model, α5 and α6 peptides can permeabilize membranes (17–19). More pertinently, Bax α5 and α6 were reported to insert into and span the MOM as a hairpin before oligomerization (20).This longstanding model, however, does not fit well with recent evidence on the structure of Bak and Bax oligomers, as recently reviewed (2, 4). Analysis of Bak oligomers in liposomes by electron paramagnetic resonance (EPR) suggests that α6 inserts only shallowly in the lipid bilayer (21). Additionaly, the first 3D structures of activated forms of Bax (10) suggest that, early in its activation, α5 and α6 separate. Moreover, a Bax core domain containing only helices α2 to α5 generated a BH3:groove symmetric dimer in which two α4 and two α5 helices form an aromatic face that might sit on the bilayer (10). These findings fit better with an “in-plane model” in which only α9 is a TM domain and other helices (including α5 and α6) insert only shallowly into the bilayer.The nature of the apoptotic pores remains uncertain. Some findings favor a proteinaceous pore (22), but studies with model membranes suggest that Bax oligomers can perturb the bilayer and produce lipidic pores (i.e., pores not bounded entirely by protein) (23–26).These important unresolved questions about the pivotal event in apoptosis prompted us to explore the membrane topology of Bak, before, during, and after an apoptotic signal, and to reinvestigate that of Bax. In accord with recent Bax structures (10) and recent EPR studies on Bak (21, 27), the results show that neither oligomeric Bak nor Bax inserts an α5–α6 hairpin across the MOM. We propose instead that the α5 and α6 helices lie in the bilayer plane and disrupt membrane integrity by imposing tension and curvature to the membrane that provoke its permeabilization. 相似文献
3.
Xian Jiang Hui Jiang Zhirong Shen Xiaodong Wang 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(41):14782-14787
Intrinsic apoptotic stimuli initiate mammalian cells’ apoptotic program by first activating the proteins that have only Bcl-2 homology domain 3 (BH3), such as Bcl-2 interacting mediator of cell death (Bim) and truncated BH3 interacting death domain agonist (tBid), which in turn trigger conformational changes in BCL2-associated X (Bax) and BCL2-antagonist/killer (Bak) proteins that enable oligomer formation on the mitochondria, causing cytochrome c and other apoptogenic proteins in the intermembrane space to leak out. Leaked cytochrome c then initiates apoptotic caspase activation through a well-defined biochemical pathway. However, how oligomerized Bax and Bak cause cytochrome c release from mitochondria remains unknown. We report here the establishment of cell lines in which Bim or tBid can be inducibly expressed to initiate apoptosis in a controlled, quantitative manner. We used these cell lines to examine apoptotic events after Bax and Bak oligomerization but before cytochrome c release. The mitochondrial metalloprotease OMA1 was activated in this system in a Bax- and Bak-dependent fashion. Activated OMA1 cleaved the dynamin-like GTPase, optical nerve atrophy 1, an event that is critical for remodeling of mitochondrial cristae. Knockdown or knockout of OMA1 in these cells attenuated cytochrome c release. Thus it is clear that oligomerized Bax and Bak trigger apoptosis by causing both the permeabilization of the mitochondrial outer membrane and activation OMA1.Mitochondria in mammalian cells fulfill multiple functions. They are cells’ bio-energetic center, where reducing agents generated through the Krebs cycle transfer their electrons to oxygen in a manner mediated by the electron transfer chain, a process that builds a proton gradient across the inner membrane of mitochondria. The energy of this gradient is transferred into the high-energy bond of ATP by oxidative phosphorylation of ADP through the F1/F0 ATP synthase. During apoptosis, the sole water-soluble component of the electron transfer chain, cytochrome c, is released from the intermembrane space of mitochondria to the cytosol (1). Cytosolic cytochrome c binds to the Apaf-1 protein to promote the assembly of a heptamer complex named an “apoptosome”; this complex subsequently recruits procaspase-9, which autoactivates once on the apoptosome. The activated caspase-9 then cleaves and activates downstream caspase-3 and caspase-7, which subsequently cleave many intracellular substrates for apoptosis execution (2).In addition to cytochrome c, other proteins that normally are located in the mitochondrial intermembrane space also function in apoptosis. One such protein is second mitochondria-derived activator of caspase (Smac). When Smac is released, it binds to the BIR domain of inhibitors of apoptosis proteins to relieve their inhibition of the caspases directly or to cause their degradation (3, 4). Thus controlling the permeability of mitochondria for these apoptogenic proteins constitutes a key regulatory step for apoptosis.The B-cell leukemia/lymphoma 2 (Bcl-2) family of proteins constitutes a protein network that regulates the release of proteins such as cytochrome c and Smac (5, 6). BCL2-associated X (Bax) and BCL2-antagonist/killer (Bak), the proapoptotic members of the family with multiple Bcl-2 homology (BH) domains, form the core of the mitochondrial membrane permeability machinery that is activated by the proapoptotic proteins that have only the BH3 domain, a process that is inhibited by the proteins whose function is similar to that of Bcl-2 itself (7, 8). In response to apoptotic stimuli, BH3-only proteins, such as Bcl-2 interacting mediator of cell death (Bim), Puma, and truncated BH3 interacting death domain agonist (tBid) directly activate Bax/Bak and lift the inhibition of Bcl-2/Bcl-xl by forming stable heterodimers to sequester them from binding Bak/Bak (7, 9–13). Activated Bax and Bak initially form homodimers and then oligomers on the mitochondrial membrane (14–18). Bax/Bak oligomers are believed to form proteinaceous or lipidic pores on the mitochondrial outer membrane that allow the passage of proteins such as cytochrome c and Smac. Although the results of in vitro liposome leakage experiments support this model, there is no direct in vivo evidence to validate such a straightforward model (19–23).Moreover, increasing evidence indicates that the majority of cytochrome c in the mitochondrial intermembrane space is locked inside cristae by the protein complex containing optical nerve atrophy 1 (OPA1). The cristae must undergo reconfiguration to open up the neck of cristae for the bulk of cytochrome c to be released from the mitochondria after the outer membrane becomes permeable (24–26). The mitochondrial inner membrane fusion factor OPA1, a dynamin-like GTPase, plays a critical role in the remodeling of cristae. OPA1 presents in several spliced and proteolytic forms in mitochondria, and the maintenance of the relative amounts of each of these forms is known to be critical for stabilizing the cristae (27, 28). The longest form, L-OPA1, is cleaved in response to a variety of mitochondrial stresses, leading to the disassembly of OPA1-containing complexes and remodeling of the cristae (24, 26). It has been proposed that several different proteins cleave OPA1; these include the mitochondrial AAA proteases, presenilin-associated rhomboid-like protein (PARL), and the zinc metalloprotease OMA1 (overlapping activity with m-AAA protease) (26, 27, 29–31). However, the relationship between the pore formation on the mitochondrial outer membrane and OPA1 cleavage-mediated cristae remodeling during apoptosis, as well as the precise roles of those mitochondrial proteases in apoptosis, remain to be clarified.To dissect the molecular details of cytochrome c release induced by BH3-only proteins, we generated cell lines in which Bim or tBid can be inducibly expressed by adding doxycycline (Dox) into the culture medium. The expression of these proteins triggers apoptosis in a controlled and synchronized fashion. We used this cell-based system to characterize the mitochondrial response to the induction of Bim and tBid and found that OMA1 activation is an important step for apoptosis induction. 相似文献
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5.
Joshua B. Brettmann Darya Urusova Marco Tonelli Jonathan R. Silva Katherine A. Henzler-Wildman 《Proceedings of the National Academy of Sciences of the United States of America》2015,112(50):15366-15371
Flux-dependent inactivation that arises from functional coupling between the inner gate and the selectivity filter is widespread in ion channels. The structural basis of this coupling has only been well characterized in KcsA. Here we present NMR data demonstrating structural and dynamic coupling between the selectivity filter and intracellular constriction point in the bacterial nonselective cation channel, NaK. This transmembrane allosteric communication must be structurally different from KcsA because the NaK selectivity filter does not collapse under low-cation conditions. Comparison of NMR spectra of the nonselective NaK and potassium-selective NaK2K indicates that the number of ion binding sites in the selectivity filter shifts the equilibrium distribution of structural states throughout the channel. This finding was unexpected given the nearly identical crystal structure of NaK and NaK2K outside the immediate vicinity of the selectivity filter. Our results highlight the tight structural and dynamic coupling between the selectivity filter and the channel scaffold, which has significant implications for channel function. NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating.Ion conduction through the pore domain of cation channels is regulated by two gates: an inner gate at the bundle crossing of the pore-lining transmembrane helices and an outer gate located at the selectivity filter (Fig. 1 B and C). These two gates are functionally coupled as demonstrated by C-type inactivation, in which channel opening triggers loss of conduction at the selectivity filter (1–4). A structural model for C-type inactivation has been developed for KcsA, with selectivity filter collapse occurring upon channel opening (4–10). In the reverse pathway, inactivation of the selectivity filter has been linked to changes at the inner gate (5–14). However, flux-dependent inactivation occurs in Na+ and Ca2+ channels as well and would likely require a structurally different mechanism to explain coupling between the selectivity filter and inner gate (7, 13–18).Open in a separate windowFig. 1.Crystal structures of the nonselective cation channel NaK and the potassium-selective NaK2K mutant show structural changes restricted to the area of the selectivity filter. Alignment of the WT NaK (gray; PDB 3E8H) and NaK2K (light blue; PDB 3OUF) selectivity filters shows a KcsA-like four-ion-binding-site selectivity filter is created by the NaK2K mutations (D66Y and N68D) (A), but no structural changes occur outside the vicinity of the selectivity filter (B). (C) Full-length NaK (green; PDB 2AHZ) represents a closed conformation. Alignment of this structure with NaK (gray) highlights the changes in the M2 hinge (arrow), hydrophobic cluster (residues F24, F28, and F94 shown as sticks), and constriction point (arrow; residue Q103 shown as sticks) upon channel opening. Two (A) or three monomers (B and C) from the tetramer are shown for clarity.This study provides experimental evidence of structural and dynamic coupling between the inner gate and selectivity filter in the NaK channel, a nonselective cation channel from Bacillus
cereus (19). These results were entirely unexpected given the available high-resolution crystal structures (20, 21). The NaK channel has the same basic pore architecture as K+ channels (Fig. 1 B and C) and has become a second model system for investigating ion selectivity and gating due to its distinct selectivity filter sequence (63TVGDGN68) and structure (19–23). Most strikingly, there are only two ion binding sites in the selectivity filter of the nonselective NaK channel (Fig. 1A) (21, 24). However, mutation of two residues in the selectivity filter sequence converts the NaK selectivity filter to the canonical KcsA sequence (63TVGYGD68; Fig. 1 A and B), leading to K+ selectivity and a KcsA-like selectivity filter structure with four ion binding sites (21, 23). This K+-selective mutant of NaK is called NaK2K. Outside of the immediate vicinity of the two mutations in the selectivity filter, high-resolution crystal structures of NaK and NaK2K are essentially identical (Fig. 1B) with an all-atom rmsd of only 0.24 Å.NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating because there is no evidence for any collapse or structural change in the selectivity filter. The NaK selectivity filter structure is identical in Na+ or K+ (22) and even in low-ion conditions (25), consistent with its nonselective behavior. Even the selective NaK2K filter appears structurally stable in all available crystal structures (25). Here we use NMR spectroscopy to study bicelle-solubilized NaK. Surprisingly, we find significant differences in the NMR spectra of NaK and NaK2K that extend throughout the protein and are not localized to the selectivity filter region. This, combined with NMR dynamics studies of NaK, suggests a dynamic pathway for transmembrane coupling between the inner gate and selectivity filter of NaK. 相似文献
6.
Scott T. Aoki Aaron M. Kershner Craig A. Bingman Marvin Wickens Judith Kimble 《Proceedings of the National Academy of Sciences of the United States of America》2016,113(5):1279-1284
Cellular RNA-protein (RNP) granules are ubiquitous and have fundamental roles in biology and RNA metabolism, but the molecular basis of their structure, assembly, and function is poorly understood. Using nematode “P-granules” as a paradigm, we focus on the PGL granule scaffold protein to gain molecular insights into RNP granule structure and assembly. We first identify a PGL dimerization domain (DD) and determine its crystal structure. PGL-1 DD has a novel 13 α-helix fold that creates a positively charged channel as a homodimer. We investigate its capacity to bind RNA and discover unexpectedly that PGL-1 DD is a guanosine-specific, single-stranded endonuclease. Discovery of the PGL homodimer, together with previous results, suggests a model in which the PGL DD dimer forms a fundamental building block for P-granule assembly. Discovery of the PGL RNase activity expands the role of RNP granule assembly proteins to include enzymatic activity in addition to their job as structural scaffolds.Cytoplasmic RNA-protein (RNP) granules are found in virtually all cells and are thought to be central to RNA metabolism (1, 2). These diverse organelles include P-bodies, stress granules, neuronal granules, and germ granules (2). RNP granules are not membrane-bound and display liquid–liquid phase-separation properties (3, 4). Many of their molecular components have been identified, including scaffold proteins: proteins that recruit other key granule components and are sufficient to induce RNP granule assembly. Major challenges now are to understand how RNP granules are assembled and how they control RNAs.Germ granules are exemplary RNP granules with a profound yet largely mysterious role in metazoan germ-line development. These granules possess common components across phyla (5) but use unique scaffold proteins, such as Drosophila Oskar (6), zebrafish Bucky Ball (7), and Caenorhabditis elegans paralogs PGL-1 and PGL-3 (8, 9), called PGL collectively. Germ granule scaffold proteins from different phyla have distinct amino acid sequences with no conserved domains. The importance of these scaffolds has been attributed to their function in germ granule assembly (for examples, see refs. 10–12). However, the molecular basis of that assembly and how it impacts RNA regulation remain unknown.Here we focus on the Caenorhabditid PGL scaffold proteins and their role in assembly of nematode germ granules, called P-granules (13). P-granules are required for germ-line survival (8, 9) and germ-line totipotency (14). A recent model proposes that P-granules capture selected mRNAs exiting the nucleus (15), an idea based on the finding that untranslated mRNAs are enriched in P-granules, but translated mRNAs are absent (15, 16). The consequences of that capture are unclear but may include mRNA repression.The PGL family comprises the closely related PGL-1 and PGL-3 proteins plus divergent PGL-2. PGL-1 and PGL-3 are required for adult germ cell development but the function of PGL-2 is unknown (8). All three PGL proteins interact with each other in vitro (8), but thus far only PGL-1 and PGL-3 are known to self-assemble into granules when expressed in nematode somatic cells or in mammalian cell culture (17, 18). By primary sequence prediction, PGLs have only one recognizable region, C-terminal RGG repeats (Fig. 1A) (8, 9), which are associated with protein turnover and RNA binding (17, 19). In non-PGL proteins, RGG repeats can recruit RNA binding proteins (20) and facilitate in vitro granule formation (21). Experiments in tissue culture cells implicate the N-terminal half of the PGL protein—but not the RGG repeats—in P-granule assembly (17). Whereas full-length PGL-3 self-assembled, a mutant lacking ∼160 residues from the PGL-3 N-terminal half (Fig. 1A) no longer formed granules (17). Thus, in-roads have been made but molecular detail about how PGL organizes itself into a granule is lacking.Open in a separate windowFig. 1.Identification of the PGL dimerization domain. (A) C. elegans PGL-1, PGL-3, and C. remanei PGL-1 protein schematic. The only recognizable motif by sequence prediction is the RGG repeat region (yellow). The region implicated in granule formation (17) is shown here below PGL-3. We first identified the DD (orange) in PGL-3 (see main text). (B) Size-exclusion chromatography of C. elegans PGL-3 recombinant protein “N-term + DD” (blue) and “DD” (red). Arrows indicate positions of the void volume, albumin (60 kDa), and MBP (37 kDa).Here we identify a PGL dimerization domain (DD) and propose that PGL dimers are a key building block for P-granule assembly. We determine the PGL-1 DD crystal structure and find a novel 13-helix fold that creates a positively charged channel as a homodimer. Testing the idea that this channel might bind RNA, we discovered that PGL DD is an RNase and determined PGL-1 DD’s specificity in vitro for guanosine in single-stranded RNAs. We suggest that PGL DD has a dual role in P-granules, as a domain essential for assembly and as an RNase. 相似文献
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Stephanie Brown Marc Clastre Vincent Courdavault Sarah E. O’Connor 《Proceedings of the National Academy of Sciences of the United States of America》2015,112(11):3205-3210
The monoterpene indole alkaloids are a large group of plant-derived specialized metabolites, many of which have valuable pharmaceutical or biological activity. There are ∼3,000 monoterpene indole alkaloids produced by thousands of plant species in numerous families. The diverse chemical structures found in this metabolite class originate from strictosidine, which is the last common biosynthetic intermediate for all monoterpene indole alkaloid enzymatic pathways. Reconstitution of biosynthetic pathways in a heterologous host is a promising strategy for rapid and inexpensive production of complex molecules that are found in plants. Here, we demonstrate how strictosidine can be produced de novo in a Saccharomyces cerevisiae host from 14 known monoterpene indole alkaloid pathway genes, along with an additional seven genes and three gene deletions that enhance secondary metabolism. This system provides an important resource for developing the production of more complex plant-derived alkaloids, engineering of nonnatural derivatives, identification of bottlenecks in monoterpene indole alkaloid biosynthesis, and discovery of new pathway genes in a convenient yeast host.Monoterpene indole alkaloids (MIAs) are a diverse family of complex nitrogen-containing plant-derived metabolites (1, 2). This metabolite class is found in thousands of plant species from the Apocynaceae, Loganiaceae, Rubiaceae, Icacinaceae, Nyssaceae, and Alangiaceae plant families (2, 3). Many MIAs and MIA derivatives have medicinal properties; for example, vinblastine, vincristine, and vinflunine are approved anticancer therapeutics (4, 5). These structurally complex compounds can be difficult to chemically synthesize (6, 7). Consequently, industrial production relies on extraction from the plant, but these compounds are often produced in small quantities as complex mixtures, making isolation challenging, laborious, and expensive (8–10). Reconstitution of plant pathways in microbial hosts is proving to be a promising approach to access plant-derived compounds as evidenced by the successful production of terpenes, flavonoids, and benzylisoquinoline alkaloids in microorganisms (11–19). Microbial hosts can also be used to construct hybrid biosynthetic pathways to generate modified natural products with potentially enhanced bioactivities (8, 20, 21). Across numerous plant species, strictosidine is believed to be the core scaffold from which all 3,000 known MIAs are derived (1, 2). Strictosidine undergoes a variety of redox reactions and rearrangements to form the thousands of compounds that comprise the MIA natural product family (Fig. 1) (1, 2). Due to the importance of strictosidine, the last common biosynthetic intermediate for all known MIAs, we chose to focus on heterologous production of this complex molecule (1). Therefore, strictosidine reconstitution represents the necessary first step for heterologous production of high-value MIAs.Open in a separate windowFig. 1.Strictosidine, the central intermediate in monoterpene indole alkaloid (MIA) biosynthesis, undergoes a series of reactions to produce over 3,000 known MIAs such as vincristine, quinine, and strychnine. 相似文献
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Marie F. Soulière Roger B. Altman Veronika Schwarz Andrea Haller Scott C. Blanchard Ronald Micura 《Proceedings of the National Academy of Sciences of the United States of America》2013,110(35):E3256-E3264
Structural and dynamic features of RNA folding landscapes represent critical aspects of RNA function in the cell and are particularly central to riboswitch-mediated control of gene expression. Here, using single-molecule fluorescence energy transfer imaging, we explore the folding dynamics of the preQ1 class II riboswitch, an upstream mRNA element that regulates downstream encoded modification enzymes of queuosine biosynthesis. For reasons that are not presently understood, the classical pseudoknot fold of this system harbors an extra stem–loop structure within its 3′-terminal region immediately upstream of the Shine–Dalgarno sequence that contributes to formation of the ligand-bound state. By imaging ligand-dependent preQ1 riboswitch folding from multiple structural perspectives, we reveal that the extra stem–loop strongly influences pseudoknot dynamics in a manner that decreases its propensity to spontaneously fold and increases its responsiveness to ligand binding. We conclude that the extra stem–loop sensitizes this RNA to broaden the dynamic range of the ON/OFF regulatory switch.A variety of small metabolites have been found to regulate gene expression in bacteria, fungi, and plants via direct interactions with distinct mRNA folds (1–4). In this form of regulation, the target mRNA typically undergoes a structural change in response to metabolite binding (5–9). These mRNA elements have thus been termed “riboswitches” and generally include both a metabolite-sensitive aptamer subdomain and an expression platform. For riboswitches that regulate the process of translation, the expression platform minimally consists of a ribosomal recognition site [Shine–Dalgarno (SD)]. In the simplest form, the SD sequence overlaps with the metabolite-sensitive aptamer domain at its downstream end. Representative examples include the S-adenosylmethionine class II (SAM-II) (10) and the S-adenosylhomocysteine (SAH) riboswitches (11, 12), as well as prequeuosine class I (preQ1-I) and II (preQ1-II) riboswitches (13, 14). The secondary structures of these four short RNA families contain a pseudoknot fold that is central to their gene regulation capacity. Although the SAM-II and preQ1-I riboswitches fold into classical pseudoknots (15, 16), the conformations of the SAH (17) and preQ1-II counterparts are more complex and include a structural extension that contributes to the pseudoknot architecture (14). Importantly, the impact and evolutionary significance of these “extra” stem–loop elements on the function of the SAH and preQ1-II riboswitches remain unclear.PreQ1 riboswitches interact with the bacterial metabolite 7-aminomethyl-7-deazaguanine (preQ1), a precursor molecule in the biosynthetic pathway of queuosine, a modified base encountered at the wobble position of some transfer RNAs (14). The general biological significance of studying the preQ1-II system stems from the fact that this gene-regulatory element is found almost exclusively in the Streptococcaceae bacterial family. Moreover, the preQ1 metabolite is not generated in humans and has to be acquired from the environment (14). Correspondingly, the preQ1-II riboswitch represents a putative target for antibiotic intervention. Although preQ1 class I (preQ1-I) riboswitches have been extensively investigated (18–28), preQ1 class II (preQ1-II) riboswitches have been largely overlooked despite the fact that a different mode of ligand binding has been postulated (14).The consensus sequence and the secondary structure model for the preQ1-II motif (COG4708 RNA) (Fig. 1A) comprise ∼80–100 nt (14). The minimal Streptococcus pneumoniae R6 aptamer domain sequence binds preQ1 with submicromolar affinity and consists of an RNA segment forming two stem–loops, P2 and P4, and a pseudoknot P3 (Fig. 1B). In-line probing studies suggest that the putative SD box (AGGAGA; Fig. 1) is sequestered by pseudoknot formation, which results in translational-dependent gene regulation of the downstream gene (14).Open in a separate windowFig. 1.PreQ1 class II riboswitch. (A) Chemical structure of 7-aminomethyl-7-deazaguanosine (preQ1); consensus sequence and secondary structure model for the COG4708 RNA motif (adapted from reference 14). Nucleoside presence and identity as indicated. (B) S. pneumoniae R6 preQ1-II RNA aptamer investigated in this study. (C) Schematics of an H-type pseudoknot with generally used nomenclature for comparison.Here, we investigated folding and ligand recognition of the S. pneumoniae R6 preQ1-II riboswitch, using complementary chemical, biochemical, and biophysical methods including selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), mutational analysis experiments, 2-aminopurine fluorescence, and single-molecule fluorescence resonance energy transfer (smFRET) imaging. In so doing, we explored the structural and functional impact of the additional stem–loop element in the context of its otherwise “classical” H-type pseudoknot fold (29–32) (Fig. 1C). Our results reveal that the unique 3′-stem–loop element in the preQ1-II riboswitch contributes to the process of SD sequestration, and thus the regulation of gene expression, by modulating both its intrinsic dynamics and its responsiveness to ligand binding. 相似文献
10.
Dou Yu Devang K. Thakor Inbo Han Alexander E. Ropper Hariprakash Haragopal Richard L. Sidman Ross Zafonte Steven C. Schachter Yang D. Teng 《Proceedings of the National Academy of Sciences of the United States of America》2013,110(8):E746-E755
Diverse mechanisms including activation of NMDA receptors, microglial activation, reactive astrogliosis, loss of descending inhibition, and spasticity are responsible for ∼40% of cases of intractable neuropathic pain after spinal cord injury (SCI). Because conventional treatments blocking individual mechanisms elicit only short-term effectiveness, a multimodal approach with simultaneous actions against major pain-related pathways may have value for clinical management of chronic pain. We hypothesize that [-]-huperzine A (HUP-A), an alkaloid isolated from the club moss Huperzia serrata, that is a potent reversible inhibitor of acetylcholinesterase and NMDA receptors, could mitigate pain without invoking drug tolerance or dependence by stimulating cholinergic interneurons to impede pain signaling, inhibiting inflammation via microglial cholinergic activation, and blocking NMDA-mediated central hypersensitization. We tested our hypothesis by administering HUP-A i.p. or intrathecally to female Sprague–Dawley rats (200–235 g body weight) after moderate static compression (35 g for 5 min) of T10 spinal cord. Compared with controls, HUP-A treatment demonstrates significant analgesic effects in both regimens. SCI rats manifested no drug tolerance following repeated bolus i.p. or chronic intrathecal HUP-A dosing. The pain-ameliorating effect of HUP-A is cholinergic dependent. Relative to vehicle treatment, HUP-A administration also reduced neural inflammation, retained higher numbers of calcium-impermeable GluR2-containing AMPA receptors, and prevented Homer1a up-regulation in dorsal horn sensory neurons. Therefore, HUP-A may provide safe and effective management for chronic postneurotrauma pain by reestablishing homeostasis of sensory circuits.Neuropathic pain is one of the most debilitating sequelae of neurotrauma and is an unmet clinical need for at least 40% of patients with spinal cord injury (SCI) (1). Administration of conventional drugs has shown only various degrees of short-term efficacy in reducing at- and/or below-injury-level hypersensitivity after SCI by acting on individual pathways to inhibit descending facilitation of pain-transmission neurons (2), activate inhibitory interneurons in the spinal cord (2, 3), mitigate firing of pain-transmission neurons (3–7), or impede inflammation triggered by the activation of astrocytes and microglial cells (8). The diversity of mechanisms and potential targets for intervention may be partially responsible for the difficulty in developing therapeutics that can provide long-term efficacy. Therefore we postulated that devising a multimodal treatment with potent, defined simultaneous effects on multiple pain-related pathways might be an effective concept for clinical management of neuropathic pain.Given the potential roles of cholinergic agonists (9, 10) and antagonists of NMDA-subtype glutamate receptors (10, 11) in the treatment of neuropathic pain, we hypothesized that [-]-huperzine A (HUP-A) (Fig. 1A), a naturally occurring Lycopodium alkaloid isolated from the Chinese club moss, Huperzia serrata (Fig. 1B) that has potent reversible inhibitory action on acetylcholinesterase (AChE) (Fig. 1C) (10, 12) and NMDA receptors (Fig. 1D) (13), might be an exceptional prospect for multimodal treatment of SCI-induced neuropathic pain. We tested whether HUP-A–derived NMDA blockade and the overall HUP-A–derived augmentation of cholinergic neurotransmission arising from AChE inhibition might be harnessed specifically to stage a multifocal intervention in post-SCI pain pathways because (i) antagonism of NMDA receptor prevents the post-SCI hyperexcitability of neurons in the dorsal horn (DH) of the spinal cord that feature a wide, dynamic range of pain transmission (7); (ii) activation of presynaptic α3β2 nicotinic ACh receptors (nAChR) minimizes release of glutamate from C-fiber terminals in the DH that are involved in nociceptive neurotransmission (14); (iii) activation of GABAergic interneurons via stimulation of their M2 and M4 muscarinic ACh receptors (mAChR), in turn, inhibits presynaptic release of glutamate from primary afferent axons (15, 16); (iv) activation of the α4β2 nAChR on GABAergic inhibitory interneurons mitigates the firing of secondary spinothalamic pain transmission neurons (17); and (v) stimulation of the α7 nAChR on microglial cells blocks their activation, ameliorating neuroinflammation (18, 19). The primary goal of our study is to develop a class of therapeutics for the management of chronic neuropathic pain that aims to restore homeostasis of the sensory neurocircuitry by simultaneous multimodal mechanisms without invoking drug tolerance and dependence or respiratory suppression. HUP-A has been used for centuries in herbal medicine to treat inflammatory and other diseases (10, 12), has demonstrated effectiveness in inhibiting hypersensitivity triggered by peripheral neuropathy (20), and has been studied in phase II clinical trials for Alzheimer’s disease in the United States (11). We tested whether HUP-A regimens might translate into an effective and safe therapy for neuropathic pain following SCI in adult female rats (21–23).Open in a separate windowFig. 1.HUP-A is a natural alkaloid extract. HUP-A''s molecular structure is presented in (A) from the club moss Huperzia serrata (B) that is a potent, highly specific, and reversible inhibitor of AChE (C) as well as a noncompetitive partial antagonist of the NMDA receptor (D). A reproducible moderate static-compression SCI model maintaining 35 g of static weight on the dorsal surface of T10 spinal cord for 5 min was used for the present study (E). 相似文献
11.
Krishna K. Midde Nicolas Aznar Melanie B. Laederich Gary S. Ma Maya T. Kunkel Alexandra C. Newton Pradipta Ghosh 《Proceedings of the National Academy of Sciences of the United States of America》2015,112(9):E937-E946
Environmental cues are transmitted to the interior of the cell via a complex network of signaling hubs. Receptor tyrosine kinases (RTKs) and trimeric G proteins are two such major signaling hubs in eukaryotes. Conventionally, canonical signal transduction via trimeric G proteins is thought to be triggered exclusively by G protein-coupled receptors. Here we used molecular engineering to develop modular fluorescent biosensors that exploit the remarkable specificity of bimolecular recognition, i.e., of both G proteins and RTKs, and reveal the workings of a novel platform for activation of G proteins by RTKs in single living cells. Comprised of the unique modular makeup of guanidine exchange factor Gα-interacting vesicle-associated protein (GIV)/girdin, a guanidine exchange factor that links G proteins to a variety of RTKs, these biosensors provide direct evidence that RTK–GIV–Gαi ternary complexes are formed in living cells and that Gαi is transactivated within minutes after growth factor stimulation at the plasma membrane. Thus, GIV-derived biosensors provide a versatile strategy for visualizing, monitoring, and manipulating the dynamic association of Gαi with RTKs for noncanonical transactivation of G proteins in cells and illuminate a fundamental signaling event regulated by GIV during diverse cellular processes and pathophysiologic states.The ability of cells to respond and adapt to external signals is achieved through the concerted action of several receptors and regulatory proteins. Receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) are the two most widely studied cell signaling hubs in eukaryotes. Canonical RTK signaling begins with ligand binding to the ectodomain of the receptor, leading to receptor dimerization followed by autophosphorylation of the tyrosine residues on the cytoplasmic tail and propagation of the signals to the interior of the cell via adaptor proteins (1). Canonical G protein signaling begins with ligand binding to GPCRs, which are seven transmembrane receptors with an intrinsic guanine nucleotide exchange factor (GEF) activity that enables G protein recruitment and subsequent activation through the exchange of GDP for the GTP nucleotide (2). For several decades these two pathways were believed to operate in a selective and discrete mode by transducing signals through their respective downstream intermediates. However, mounting evidence over time has unfolded a complex array of cross-talk between these two pathways, so that activated receptors from one pathway transactivate the other pathway either directly by activating the receptors (3) or indirectly by activating the downstream adaptor proteins (4). A well-documented and widely accepted phenomenon is transactivation of RTKs by GPCRs via scaffolding proteins such as β-arrestins (5). However, the reverse concept, i.e., transactivation of trimeric G proteins by RTKs, remains controversial. Despite numerous clues supporting the concept that growth factors trigger the activation of heterotrimeric G proteins (6), the fundamental question as to how such trigger occurs in cells remains poorly understood. This poorly understood concept is met with skepticism because there is no evidence that G proteins and ligand-activated RTKs come within close proximity in cells or that RTKs or any member of the growing family of signal-transducing adaptors used by RTKs can serve as GEFs. Some of these unanswered questions are being clarified by the discovery and characterization of Gα-interacting vesicle-associated protein (GIV; also known as “girdin”), an unusual signal transducer that can bind both RTKs and G proteins.GIV is a multimodular signal transducer (Fig. 1A) and a GEF for Gαi (7). Working downstream of a variety of growth factors [EGF (8, 9), IGF (10), VEGF (11), insulin (7, 12, 13), and PDGF receptor (14)], GIV enhances PI3K-Akt activity, links Akt signaling to actin cytoskeleton remodeling, and triggers cell migration, all via activation of Gαi (7). Because cells can modulate incoming growth factor signals from multiple RTKs by altering the cellular levels of GIV or selectively modulating its GEF function, we likened GIV to a rheostat by which cells tune incoming signals up or down (15). Consistent with its ability to signal downstream of a variety of receptors, GIV modulates growth factor signaling during diverse biological processes (15), e.g., cell migration, chemotaxis (13), invasion (16), development (17), self-renewal (18), apoptosis (19, 20), and autophagy (12). Additionally, evidence gathered by us and others has demonstrated the clinical significance of GIV-dependent signaling during diverse disease processes, e.g., pathologic angiogenesis (11), liver fibrosis (19), diabetes (21), nephrotic syndrome (20), vascular repair (22), and tumor metastasis across a variety of cancers [gastric (23), esophageal (24), prostate (16), breast (10, 25, 26), colon (27), and glioblastoma (18)].Fig. 1.Design and characterization of GIV-derived biosensors. (A, Upper) Schematic of various known functional domains of GIV. (Lower) The unique modular make-up of GIV''s CT with in-tandem coexistence of a GEF domain (red) that activates Gαi (gray), ...Despite the accumulating information on the biological and clinical significance of GIV, how it may couple to multiple RTKs remained unknown until recently. Protein interaction assays showed that GIV''s C terminus (CT) directly binds autophosphorylated cytoplasmic tails of multiple RTKs (9). Homology modeling, sequence analysis, side-chain substitution, and limited proteolysis showed that an ∼110-aa stretch within GIV''s CT folds into a Scr homology 2 (SH2)-like domain and is necessary and sufficient to recognize and bind phosphotyrosine peptides (28) (Fig. 1A) and recruit Gαi to RTKs. The discovery of coexisting SH2-like and GEF domains in tandem within the GIV CT (Fig. 1A) supported the idea that GIV''s CT has the necessary modular make-up to serve as a platform for linking G proteins to multiple RTKs. However, the in vitro and standard biochemical assays used thus far have failed to provide direct in cellulo evidence that GIV assembles RTK–GIV–Gαi ternary complexes, and if it does, when and where this assembly might occur, what might be the consequences of such assembly on G protein signaling, and how such signaling compares with the dynamics of canonical GPCR-driven G protein signaling. Such evidence would provide insights into the fundamental mechanisms that define GIV''s role at the cross-roads of RTK and G protein signaling pathways in diverse pathophysiologic processes. Such findings also will imply that the evolutionarily conserved CT of GIV serves as the long-sought modular platform for transactivation of G proteins downstream of growth factors, a phenomenon that has been observed and reported by several groups over the past few decades (6). 相似文献
12.
Shamim Naghdi Péter Várnai Gy?rgy Hajnóczky 《Proceedings of the National Academy of Sciences of the United States of America》2015,112(41):E5590-E5599
Voltage-dependent anion channel (VDAC) proteins are major components of the outer mitochondrial membrane. VDAC has three isoforms with >70% sequence similarity and redundant roles in metabolite and ion transport. However, only Vdac2−/− (V2−/−) mice are embryonic lethal, indicating a unique and fundamental function of VDAC2 (V2). Recently, a specific V2 requirement was demonstrated for mitochondrial Bak import and truncated Bid (tBid)-induced apoptosis. To determine the relevant domain(s) of V2 involved, VDAC1 (V1) and V2 chimeric constructs were created and used to rescue V2−/− fibroblasts. Surprisingly, the commonly cited V2-specific N-terminal extension and cysteines were found to be dispensable for Bak import and high tBid sensitivity. In gain-of-function studies, V2 (123–179) was the minimal sequence sufficient to render V1 competent to support Bak insertion. Furthermore, in loss-of-function experiments, T168 and D170 were identified as critical residues. These motifs are conserved in zebrafish V2 (zfV2) that also rescued V2-deficient fibroblasts. Because high-resolution structures of zfV2 and mammalian V1 have become available, we could superimpose these structures and recognized that the critical V2-specific residues help to create a distinctive open “pocket” on the cytoplasmic surface that could facilitate Bak recruitment.Voltage-dependent anion channel (VDAC) proteins provide the primary pathway for metabolite and ion flux through the outer mitochondrial membrane (OMM) (1, 2). The VDAC has three isoforms in vertebrates, which show >70% sequence similarity and have a broad and overlapping tissue distribution. The three isoforms can substitute for each other in the metabolite and ion flux (2). However, they also have nonredundant roles in cell function. Striking evidence of an isoform-specific role of VDAC2 (V2) is that embryos of homozygous deletion of Vdac2 alleles die during development (3), whereas Vdac1−/− and Vdac3−/− mice are viable and display mild phenotypes (4, 5). Thus, it is of great relevance to determine the specific interactions and the underlying structural arrangements of V2.Recent evidence suggests that V2 is involved in Ca2+ transport by cardiac mitochondria (6–8), and specifically interacts with several endogenous cytoplasmic and mitochondria-associated proteins, including steroidogenic acute regulatory protein (StAR) (9), glycogen synthase kinase 3 beta (GSK3β) (10), O-GlcNAc transferase (11), ryanodine receptor 2 (8), tubulin (12), viral protein 5, (13, 14), and drugs [erastin (15) and esfevin (7)]. These interactions facilitate mitochondrial targeting, transport, and signaling processes, and, interestingly, they seem to have striking effects on cell survival. However, the most investigated interaction of V2 is with Bak, a proapoptotic Bcl-2 family protein that is central to OMM permeabilization, allowing the release of intermembrane space proteins like cytochrome c (cyto c) and AIF to the cytosol to induce apoptosis (16, 17).OMM permeabilization can occur when death receptors like TNF-α receptors are engaged and trigger caspase-8 activation to cleave Bid, a BH3-only proapoptotic Bcl-2 family member. Relocation of truncated Bid (tBid) (18, 19) to the OMM and interaction with Bcl-2 family proteins (either proapoptotic and/or antiapoptotic) lead to a conformational change and subsequent homo-oligomerization or hetero-oligomerization of Bak (19, 20) and another proapoptotic Bcl-2 family protein, Bax (21), providing the means of exit for intermembrane space proteins across the OMM (22–24). At least in several cell types, the rapid kinetics of tBid-induced OMM permeabilization are primarily dependent on Bak (25), presumably because Bak usually resides in the OMM, whereas the mostly cytoplasmic Bax first needs to translocate to the mitochondria (26, 27). Distribution of Bak among individual mitochondria is uneven in cells lacking the OMM fusion proteins Mfn1/2, indicating that OMM fusion is required for Bak distribution (28). However, the availability of Bak in the OMM is primarily controlled by insertion of Bak from cytoplasm to the OMM.Bak is a tail-anchored protein, but it is different from other tail-anchored proteins like Bcl-2 or Bcl-xL in that unmasking of its C-terminal tail domain and insertion to the mitochondria require V2 (25, 29). In the OMM, Bak was also shown to associate with V2 and to be part of a large complex (3). The interplay between Bak and V2 is central to mitochondrial apoptosis and might contribute to the vital significance of V2 (3, 25, 30–32).Structural predictions of the VDAC family, primarily VDAC1 (V1) have been done using biochemical methods (33, 34) or biophysical methods (i.e., NMR, crystallography) (35–39). These studies uniformly suggest that VDACs are β-barrel–forming transmembrane channels. Based on the biochemical studies, the β-barrel is composed of one helix and 13 β-sheets with some relatively long loops between β-sheets. In this model, the α-helix is part of the wall of the channel. It had been proposed that the long loops might play a role in protein–protein interaction (1). Differently, however, the biophysical studies show a β-barrel formed by 19 β-sheets with the N-terminal α-helix lying inside the pore. The α-helix is highly flexible and is likely to be associated with VDAC channeling activity for the transfer of metabolites like ATP (40, 41). The literature about the orientation of the VDACs is divergent; however, a recent work by De Pinto and coworkers (42) shows that the C terminus faces the intermembrane space. The only crystallography study of V2 used zebrafish V2 (zfV2) that had >90% sequence similarity to mammalian V2. This work revealed few differences between V1 and V2, mostly confined to the cytoplasmic loops (37).In the present project, we have studied the domain(s) of V2 that are necessary and/or sufficient for Bak targeting to the OMM and Bak-mediated cyto c release. To this end, the few sequence differences between mammalian V1 and V2 were systematically analyzed by creating a series of chimeric constructs. The functional activity of each construct was tested by evaluating the rescue of Bak’s mitochondrial insertion and tBid-induced OMM permeabilization in Vdac2−/− (V2−/−) mouse embryonic fibroblasts (MEFs). Our results demonstrate that previous suspects like the N-terminal extension and high cysteine (C) content of V2 are irrelevant. Unexpectedly, the sequences of amino acids 168–171 and 123–179 represent the minimum domain of V2, which is necessary and sufficient to provide for Bak import and rapid tBid-induced apoptosis, respectively. In combination with the results of recent structural studies, our findings allow us to propose a mechanism of how the apparently minor sequence differences between V1 and V2 can result in stark functional differences. 相似文献
13.
Van V. Vu William T. Beeson Elise A. Span Erik R. Farquhar Michael A. Marletta 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(38):13822-13827
The recently discovered fungal and bacterial polysaccharide monooxygenases (PMOs) are capable of oxidatively cleaving chitin, cellulose, and hemicelluloses that contain β(1→4) linkages between glucose or substituted glucose units. They are also known collectively as lytic PMOs, or LPMOs, and individually as AA9 (formerly GH61), AA10 (formerly CBM33), and AA11 enzymes. PMOs share several conserved features, including a monocopper center coordinated by a bidentate N-terminal histidine residue and another histidine ligand. A bioinformatic analysis using these conserved features suggested several potential new PMO families in the fungus Neurospora crassa that are likely to be active on novel substrates. Herein, we report on NCU08746 that contains a C-terminal starch-binding domain and an N-terminal domain of previously unknown function. Biochemical studies showed that NCU08746 requires copper, oxygen, and a source of electrons to oxidize the C1 position of glycosidic bonds in starch substrates, but not in cellulose or chitin. Starch contains α(1→4) and α(1→6) linkages and exhibits higher order structures compared with chitin and cellulose. Cellobiose dehydrogenase, the biological redox partner of cellulose-active PMOs, can serve as the electron donor for NCU08746. NCU08746 contains one copper atom per protein molecule, which is likely coordinated by two histidine ligands as shown by X-ray absorption spectroscopy and sequence analysis. Results indicate that NCU08746 and homologs are starch-active PMOs, supporting the existence of a PMO superfamily with a much broader range of substrates. Starch-active PMOs provide an expanded perspective on studies of starch metabolism and may have potential in the food and starch-based biofuel industries.Polysaccharide monooxygenases (PMOs) are enzymes secreted by a variety of fungal and bacterial species (1–5). They have recently been found to oxidatively degrade chitin (6–8) and cellulose (8–14). PMOs have been shown to oxidize either the C1 or C4 atom of the β(1→4) glycosidic bond on the surface of chitin (6, 7) or cellulose (10–12, 14), resulting in the cleavage of this bond and the creation of new chain ends that can be subsequently processed by hydrolytic chitinases and cellulases. Several fungal PMOs were shown to significantly enhance the degradation of cellulose by hydrolytic cellulases (9), indicating that these enzymes can be used in the conversion of plant biomass into biofuels and other renewable chemicals.There are three families of PMOs characterized thus far: fungal PMOs that oxidize cellulose (9–12) (also known as GH61 and AA9); bacterial PMOs that are active either on chitin (6, 8) or cellulose (8, 13) (also known as CBM33 and AA10); and fungal PMOs that oxidize chitin (AA11) (7). Sequence homology between these three families is very low. Nevertheless, the available structures of PMOs from all three families reveal a conserved fold, including an antiparallel β-sandwich core and a highly conserved monocopper active site on a flat protein surface (Fig. 1A) (2, 6, 7, 9, 10, 15–17). Two histidine residues in a motif termed the histidine brace coordinate the copper center. The N-terminal histidine ligand binds in a bidentate mode, and its imidazole ring is methylated at the Nε position in fungal PMOs (Fig. 1A).Open in a separate windowFig. 1.(A) Representative overall and active site structures of fungal PMOs (PDB ID code 2YET) (10). (B) Structure of cellulose (18, 19). Chitin also contains β(1→4) linkages and has similar crystalline higher order structure to cellulose. (C) Model structure of amylopectin (23–25). Hydrogen bonds are shown with green dashed lines.Considering the conserved structural features, it is not surprising that the currently known PMOs act on substrates with similar structures. Cellulose and chitin contain long linear chains of β(1→4) linked glucose units and N-acetylglucosamine units, respectively (Fig. 1B). The polymer chains form extensive hydrogen bonding networks, which result in insoluble and very stable crystalline structures (18–21). PMOs are thought to bind to the substrate with their flat active site surface, which orients the copper center for selective oxidation at the C1 or C4 position (6, 16, 22). Some bacterial chitin-binding proteins are cellulose-active PMOs (8, 13, 14), further suggesting that the set of PMO substrates is restricted to β(1→4) linked polymers of glucose and glucose derivatives.Here, we report on the identification of new families of PMOs that contain several key features of previously characterized PMOs, but act on substrates different from cellulose or chitin. A member of one of these novel families of PMOs, NCU08746, was shown to oxidatively cleave amylose, amylopectin, and starch. We designate the NCU08746 family as starch-active PMOs. Both amylose and amylopectin contain linear chains of α(1→4) linked glucose, whereas the latter also contains α(1→6) glycosidic linkages at branch points in the otherwise α(1→4) linked polymer. Unlike cellulose and chitin, amylose and amylopectin do not form microcrystals; instead, they exist in disordered, single helical, and double helical forms (23–27) (see Fig. 1C for example). Starch exists partially in nanocrystalline form, but lacks the flat molecular surfaces as those found in chitin and cellulose. The discovery of starch-active PMOs shows that this oxidative mechanism of glycosidic bond cleavage is more widespread than initially expected. 相似文献
14.
Yufan He Mohammad Mahfuzul Haque Dennis J. Stuehr H. Peter Lu 《Proceedings of the National Academy of Sciences of the United States of America》2015,112(38):11835-11840
Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS.Although proteins adopt structures determined by their amino acid sequences, they are not static objects and fluctuate among ensembles of conformations (1). Transitions between these states can occur on a variety of length scales (Å to nm) and time scales (ps to s) and have been linked to functionally relevant phenomena such as allosteric signaling, enzyme catalysis, and protein–protein interactions (2–4). Indeed, protein conformational fluctuations and dynamics, often associated with static and dynamic inhomogeneity, are thought to play a crucial role in biomolecular functions (5–11). It is difficult to characterize such spatially and temporally inhomogeneous dynamics in bulk solution by an ensemble-averaged measurement, especially in proteins that undergo multiple-conformation transformations. In such cases, single-molecule spectroscopy is a powerful approach to analyze protein conformational states and dynamics under physiological conditions, and can provide a molecular-level perspective on how a protein’s structural dynamics link to its functional mechanisms (12–21).A case in point is the nitric oxide synthase (NOS) enzymes (22–24), whose nitric oxide (NO) biosynthesis involves electron transfer reactions that are associated with relatively large-scale movement (tens of Å) of the enzyme domains (Fig. 1A). During catalysis, NADPH-derived electrons first transfer into an FAD domain and an FMN domain in NOS that together comprise the NOS reductase domain (NOSr), and then transfer from the FMN domain to a heme group that is bound in a separate attached “oxygenase” domain, which then enables NO synthesis to begin (22, 25–27). The electron transfers into and out of the FMN domain are the key steps for catalysis, and they appear to rely on the FMN domain cycling between electron-accepting and electron-donating conformational states (28, 29) (Fig. 1B). In this model, the FMN domain is suggested to be highly dynamic and flexible due to a connecting hinge that allows it to alternate between its electron-accepting (FAD→FMN) or closed conformation and electron-donating (FMN→heme) or open conformation (Fig. 1 A and B) (28, 30–36). In the electron-accepting closed conformation, the FMN domain interacts with the NADPH/FAD domain (FNR domain) to receive electrons, whereas in the electron donating open conformation the FMN domain has moved away to expose the bound FMN cofactor so that it may transfer electrons to a protein acceptor like the NOS oxygenase domain, or to a generic protein acceptor like cytochrome c. In this way, the reductase domain structure cycles between closed and open conformations to deliver electrons, according to a conformational equilibrium that determines the movements and thus the electron flux capacity of the FMN domain (25, 28, 32, 34, 35, 37). A similar conformational switching mechanism is thought to enable electron transfer through the FMN domain in the related flavoproteins NADPH-cytochrome P450 reductase and methionine synthase reductase (38–42).Open in a separate windowFig. 1.(A) The nNOSr ribbon structure (from PDB: 1TLL) showing bound FAD (yellow) in FNR domain (green), FMN (orange) in FMN domain (yellow), connecting hinge (blue), and the Cy3–Cy5 label positions (pink) and distance (42 Å, dashed line). (B) Cartoon of an equilibrium between the FMN-closed and FMN-open states, with Cy dye label positions indicated. (C) Cytochrome c reductase activity of nNOSr proteins in their CaM-bound and CaM-free states. Color scheme of bar graphs: Black, WT nNOSr unlabeled; Red, Cys-lite (CL) nNOSr unlabeled; Blue, E827C/Q1268C CL nNOSr unlabeled; and Dark cyan, E827C/Q1268C CL nNOSr labeled.NOS enzymes also contain a calmodulin (CaM) binding domain that is located just before the N terminus of the FMN domain (Fig. 1B), and this provides an important layer of regulation (25, 27). CaM binding to NOS enzymes increases electron transfer from NADPH through the reductase domain and also triggers electron transfer from the FMN domain to the NOS heme as is required for NO synthesis (31, 32). The ability of CaM, or similar signaling proteins, to regulate electron transfer reactions in enzymes is unusual, and the mechanism is a topic of interest and intensive study. It has long been known that CaM binding alters NOSr structure such that, on average, it populates a more open conformation (43, 44). Recent equilibrium studies have detected a buildup of between two to four discreet conformational populations in NOS enzymes and in related flavoproteins, and in some cases, have also estimated the distances between the bound FAD and FMN cofactors in the different species (26, 36, 37, 39, 40), and furthermore, have confirmed that CaM shifts the NOS population distribution toward more open conformations (34, 36, 45). Although valuable, such ensemble-averaged results about conformational states cannot explain how electrons transfer through these enzymes, or how CaM increases the electron flux in NOS, because answering these questions requires a coordinate understanding of the dynamics of the conformational fluctuations. Indeed, computer modeling has indicated that a shift toward more open conformations as is induced by CaM binding to nNOS should, on its own, actually diminish electron flux through nNOS and through certain related flavoproteins (38). Despite its importance, measuring enzyme conformational fluctuation dynamics is highly challenging, and as far as we know, there have been no direct measures on the NOS enzymes or on related flavoproteins, nor studies on how CaM binding might influence the conformational fluctuation dynamics in NOS.To address this gap, we used single-molecule fluorescence energy resonance transfer (FRET) spectroscopy to characterize individual molecules of nNOSr that had been labeled at two specific positions with Cyanine 3 (Cy3) donor and Cyanine 5 (Cy5) acceptor dye molecules, regarding their conformational states distribution and the associated conformational fluctuation dynamics, which in turn enabled us to determine how CaM binding impacts both parameters. This work provides a unique perspective and a novel study of the NOS enzymes and within the broader flavoprotein family, which includes the mammalian enzymes methionine synthase reductase (MSR) and cytochrome P450 reductase (CPR), and reveals how CaM’s control of the conformational behaviors may regulate the electron transfer reactions of NOS catalysis. 相似文献
15.
Aaron C. Mason Robert P. Rambo Briana Greer Michael Pritchett John A. Tainer David Cortez Brandt F. Eichman 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(21):7618-7623
SMARCAL1, a DNA remodeling protein fundamental to genome integrity during replication, is the only gene associated with the developmental disorder Schimke immuno-osseous dysplasia (SIOD). SMARCAL1-deficient cells show collapsed replication forks, S-phase cell cycle arrest, increased chromosomal breaks, hypersensitivity to genotoxic agents, and chromosomal instability. The SMARCAL1 catalytic domain (SMARCAL1CD) is composed of an SNF2-type double-stranded DNA motor ATPase fused to a HARP domain of unknown function. The mechanisms by which SMARCAL1 and other DNA translocases repair replication forks are poorly understood, in part because of a lack of structural information on the domains outside of the common ATPase motor. In the present work, we determined the crystal structure of the SMARCAL1 HARP domain and examined its conformation and assembly in solution by small angle X-ray scattering. We report that this domain is conserved with the DNA mismatch and damage recognition domains of MutS/MSH and NER helicase XPB, respectively, as well as with the putative DNA specificity motif of the T4 phage fork regression protein UvsW. Loss of UvsW fork regression activity by deletion of this domain was rescued by its replacement with HARP, establishing the importance of this domain in UvsW and demonstrating a functional complementarity between these structurally homologous domains. Mutation of predicted DNA-binding residues in HARP dramatically reduced fork binding and regression activities of SMARCAL1CD. Thus, this work has uncovered a conserved substrate recognition domain in DNA repair enzymes that couples ATP-hydrolysis to remodeling of a variety of DNA structures, and provides insight into this domain’s role in replication fork stability and genome integrity.Accurate DNA replication is essential for genomic stability (1). Aberrant DNA structures, protein barriers, and chemically modified DNA thwart progression of the replication fork and lead to mutations, cytotoxicity, and increased chromosomal rearrangements (2–5). Uncoupling of polymerase and helicase activities at a stalled fork (6) results in an accumulation of single-stranded (ss) DNA (Fig. 1A), rendering the genome susceptible to nuclease cleavage and thereby increasing the probability of genetic rearrangements (7–9). Failure to stabilize stalled forks can lead to replisome dissociation and fork degradation or collapse. DNA damage response (DDR) pathways maintain genomic integrity by rectifying or protecting stalled or collapsed forks and regulating DNA repair (9–11). In humans, accumulation of the ssDNA-binding protein Replication Protein A (RPA) at stalled forks signals recruitment of the S-phase checkpoint kinase ATR (12–15), consequently triggering recruitment of proteins that promote sister chromatid cohesion and stabilize the histone/chromatin structure (16).Open in a separate windowFig. 1.Structure of the SMARCAL1 HARP1 domain. (A) SMARCAL1 catalyzes regression of a stalled replication fork into a four-stranded “chicken foot” DNA structure. (B) SMARCAL1 domain organization. Patient mutations are marked by x (sense) and o (nonsense). HARP domains are shaded green, the RPA-binding domain (RBD) is purple, RecA domains are dark blue and red, and SNF2-specific inserted helical domains (HD) are light blue and pink. The HARP2-ATPase catalytic domain is denoted by a gold bar. (C) Two protomers (green and silver) of mouse HARP1 as observed in the crystal structure. The position of the twofold noncrystallographic symmetry axis is shown in orange. (D) SAXS model of monomeric HARP1 (green) superimposed on the GASBOR ab initio molecular envelope (gray). (E) Superposition of HARP1 (green) and the Archaeoglobus fulgidus XPB DRD (PDB ID code 2FWR; silver). (F) AfXPB (ATPase, blue/red; DRD, green). (G) T4 phage UvsW (PDB ID code 2OCA; ATPase, blue/red; HARP-like, green).Stabilization, repair, and restart of stalled forks can involve regression of the fork into a four-stranded “chicken foot” structure, in which the nascent DNA strands are unwound from their respective leading and lagging strand templates and reannealed to one another (Fig. 1A) (17). SMARCAL1 (SWI/SNF-related, matrix associated, actin-dependent regulator of chromatin, subfamily a-like 1), also known as HARP (HepA-related protein), is one of several ATP-dependent motor proteins capable of fork regression and important for genetic stability, including Rad54, RecQ paralogs, BLM, WRN, FANCM, ZRANB3, HLTF/Rad5, T4 bacteriophage UvsW, archaeal HelQ/Hel308/Hjm, and Escherichia coli RecG (18–26). SMARCAL1 is a distant SNF2 family member of dsDNA translocating chromatin remodeling proteins (27) with a binding preference for branched DNA structures, and has been shown to catalyze ATP-dependent regression of model replication forks (Fig. 1A), branch migration of Holliday junctions, and reannealing of RPA-coated plasmids (28–30). RPA binding recruits SMARCAL1 to stalled replication forks in cells and regulates its specificity to promote regression of stalled forks caused by stalling of the leading strand polymerase as well as the reverse reaction to restore normal replication forks with lagging-strand ssDNA (31).Depletion of SMARCAL1 in cells leads to MUS81-dependent double-strand (ds) breaks and cellular sensitivity to replication stress agents (29). Inherited, biallelic loss of function mutations in SMARCAL1 result in the disease Schimke immunoosseous dysplasia (SIOD), characterized by pleiotropic developmental problems and cancer predisposition (32–35). Mutations associated with SIOD are localized to the N-terminal RPA-binding domain, two HARP domains (HARP1 and HARP2), and the SNF2 (DEXD/HELICc) ATPase region (Fig. 1B).Despite the conservation of the SMARCAL1 ATPase domain to the SNF2 family of chromatin remodelers (27, 36), the HARP sequences are unique, and thus how these accessory domains contribute to DNA remodeling activities of SMARCAL1 is unclear. Here we show that the HARP domain is structurally homologous to the substrate recognition domains of MutS and XPB and the putative DNA specificity motif of UvsW. We demonstrate the importance of this domain to catalytic activity by showing that loss of UvsW activity by deleting this domain is restored by a HARP-UvsW chimera, and that mutations in SMARCAL1 HARP2 abrogate fork regression activity by preferentially reducing SMARCAL1 binding to DNA junctions that resemble replication forks. Together with SAXS analysis of the protein–DNA complex, these data illustrate how the HARP-ATPase catalytic core binds DNA junctions to promote stabilization of stalled replication forks. 相似文献
16.
17.
Kun Yue Priyanka Sandal Elisabeth L. Williams Evan Murphy Elisabeth Stes Natalia Nikonorova Priya Ramakrishna Nathan Czyzewicz Laura Montero-Morales Robert Kumpf Zhefeng Lin Brigitte van de Cotte Mudassar Iqbal Michiel Van Bel Eveline Van De Slijke Matthew R. Meyer Astrid Gadeyne Cyril Zipfel Geert De Jaeger Marc Van Montagu Dani?l Van Damme Kris Gevaert A. Gururaj Rao Tom Beeckman Ive De Smet 《Proceedings of the National Academy of Sciences of the United States of America》2016,113(5):1447-1452
18.
Shaodong Zhang Hao-Jan Sun Andrew D. Hughes Ralph-Olivier Moussodia Annabelle Bertin Yingchao Chen Darrin J. Pochan Paul A. Heiney Michael L. Klein Virgil Percec 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(25):9058-9063
A constitutional isomeric library synthesized by a modular approach has been used to discover six amphiphilic Janus dendrimer primary structures, which self-assemble into uniform onion-like vesicles with predictable dimensions and number of internal bilayers. These vesicles, denoted onion-like dendrimersomes, are assembled by simple injection of a solution of Janus dendrimer in a water-miscible solvent into water or buffer. These dendrimersomes provide mimics of double-bilayer and multibilayer biological membranes with dimensions and number of bilayers predicted by the Janus compound concentration in water. The simple injection method of preparation is accessible without any special equipment, generating uniform vesicles, and thus provides a promising tool for fundamental studies as well as technological applications in nanomedicine and other fields.Most living organisms contain single-bilayer membranes composed of lipids, glycolipids, cholesterol, transmembrane proteins, and glycoproteins (1). Gram-negative bacteria (2, 3) and the cell nucleus (4), however, exhibit a strikingly special envelope that consists of a concentric double-bilayer membrane. More complex membranes are also encountered in cells and their various organelles, such as multivesicular structures of eukaryotic cells (5) and endosomes (6), and multibilayer structures of endoplasmic reticulum (7, 8), myelin (9, 10), and multilamellar bodies (11, 12). This diversity of biological membranes inspired corresponding biological mimics. Liposomes (Fig. 1) self-assembled from phospholipids are the first mimics of single-bilayer biological membranes (13–16), but they are polydisperse, unstable, and permeable (14). Stealth liposomes coassembled from phospholipids, cholesterol, and phospholipids conjugated with poly(ethylene glycol) exhibit improved stability, permeability, and mechanical properties (17–20). Polymersomes (21–24) assembled from amphiphilic block copolymers exhibit better mechanical properties and permeability, but are not always biocompatible and are polydisperse. Dendrimersomes (25–28) self-assembled from amphiphilic Janus dendrimers and minidendrimers (26–28) have also been elaborated to mimic single-bilayer biological membranes. Amphiphilic Janus dendrimers take advantage of multivalency both in their hydrophobic and hydrophilic parts (23, 29–32). Dendrimersomes are assembled by simple injection (33) of a solution of an amphiphilic Janus dendrimer (26) in a water-soluble solvent into water or buffer and produce uniform (34), impermeable, and stable vesicles with excellent mechanical properties. In addition, their size and properties can be predicted by their primary structure (27). Amphiphilic Janus glycodendrimers self-assemble into glycodendrimersomes that mimic the glycan ligands of biological membranes (35). They have been demonstrated to be bioactive toward biomedically relevant bacterial, plant, and human lectins, and could have numerous applications in nanomedicine (20).Open in a separate windowFig. 1.Strategies for the preparation of single-bilayer vesicles and multibilayer onion-like vesicles.More complex and functional cell mimics such as multivesicular vesicles (36, 37) and multibilayer onion-like vesicles (38–40) have also been discovered. Multivesicular vesicles compartmentalize a larger vesicle (37) whereas multibilayer onion-like vesicles consist of concentric alternating bilayers (40). Currently multibilayer vesicles are obtained by very complex and time-consuming methods that do not control their size (39) and size distribution (40) in a precise way. Here we report the discovery of “single–single” (28) amphiphilic Janus dendrimer primary structures that self-assemble into uniform multibilayer onion-like dendrimersomes (Fig. 1) with predictable size and number of bilayers by simple injection of their solution into water or buffer. 相似文献
19.
Ashley J. Pratt David S. Shin Gregory E. Merz Robert P. Rambo W. Andrew Lancaster Kevin N. Dyer Peter P. Borbat Farris L. Poole II Michael W. W. Adams Jack H. Freed Brian R. Crane John A. Tainer Elizabeth D. Getzoff 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(43):E4568-E4576
Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misassembly and aggregation in cells affected by the motor neuron disease ALS. However, the mechanistic relationship between superoxide dismutase 1 (SOD1) mutations and human disease is controversial, with many hypotheses postulated for the propensity of specific SOD mutants to cause ALS. Here, we experimentally identify distinguishing attributes of ALS mutant SOD proteins that correlate with clinical severity by applying solution biophysical techniques to six ALS mutants at human SOD hotspot glycine 93. A small-angle X-ray scattering (SAXS) assay and other structural methods assessed aggregation propensity by defining the size and shape of fibrillar SOD aggregates after mild biochemical perturbations. Inductively coupled plasma MS quantified metal ion binding stoichiometry, and pulsed dipolar ESR spectroscopy evaluated the Cu2+ binding site and defined cross-dimer copper–copper distance distributions. Importantly, we find that copper deficiency in these mutants promotes aggregation in a manner strikingly consistent with their clinical severities. G93 mutants seem to properly incorporate metal ions under physiological conditions when assisted by the copper chaperone but release copper under destabilizing conditions more readily than the WT enzyme. Altered intradimer flexibility in ALS mutants may cause differential metal retention and promote distinct aggregation trends observed for mutant proteins in vitro and in ALS patients. Combined biophysical and structural results test and link copper retention to the framework destabilization hypothesis as a unifying general mechanism for both SOD aggregation and ALS disease progression, with implications for disease severity and therapeutic intervention strategies.ALS is a lethal degenerative disease of the human motor system (1). Opportunities for improved understanding and clinical intervention arose from the discovery that up to 23.5% of familial ALS cases and 7% of spontaneous cases are caused by mutations in the superoxide dismutase 1 (SOD1) gene encoding human Cu, Zn SOD (2–4). SOD is a highly conserved (5), dimeric, antioxidant metalloenzyme that detoxifies superoxide radicals (6, 7), but overexpression of SOD1 ALS mutants is sufficient to cause disease in mice (8). Misfolded and/or aggregated SOD species are deposited within mouse neuronal and glial inclusions (9, 10), even before symptoms appear (11, 12). Although human familial ALS has a symptomatic phenotype indistinguishable from sporadic cases (13), individual SOD1 mutations can result in highly variable disease progression and penetrance (14, 15).Many nongeneral mechanisms, including loss of activity or gain of function, were postulated to explain the roles of SOD mutants in ALS (3, 16–19). Recently, however, an initial hypothesis proposing that SOD manifests disease symptoms by framework destabilization (protein instability caused by structural defects) and consequent protein misassembly and aggregation has gained renewed support (2, 10, 14, 20–23). Ironically, WT SOD is an unusually stable protein (7, 24–26), and precisely how SOD mutations cause disease remains unclear. For instance, human SOD free cysteine residues C6 and C111 have been implicated in protein aggregation by promoting cross-linking (27, 28) and/or stability changes associated with oxidative modifications (29–33). Mutation of the chemically reactive thiols significantly decreases the irreversible denaturation rate for human and bovine SOD (24, 34). However, ALS mutants in a C6A/C111S SOD (AS-SOD) background (35, 36) maintain the native C57–C146 disulfide bond but can still undergo aggregation, and mutations of the free cysteines can cause ALS (37, 38). These results imply that free cysteines are not strictly required but rather, may alter aggregation kinetics (20). SOD also contains two metal ion cofactors in each subunit: a catalytic copper ion (6) and a structurally stabilizing zinc ion (34, 39, 40) (Fig. 1A). In higher eukaryotes, a copper chaperone for SOD (CCS) plays an important role in catalyzing both the copper incorporation and native disulfide bond formation (41). Structural analyses of apo WT SOD point to greater flexibility or increased solvent accessibility of C6 otherwise buried in the stable dimer interface (42, 43), and molecular dynamics simulations also suggest a critical role for metal ions in protein structure, because SOD’s β-sheet propensity decreases in the absence of metals (44). As a result, apo SOD readily forms protein aggregates (45, 46), but the molecular structures of SOD aggregates are likely polymorphic and represent a controversial topic (23, 47–51). The intertwined effects of the aggregation-enhancing free cysteines, dimer-stabilizing metal ions, and CCS maturation of SOD complicate the study of the ALS-causing SOD mutations themselves, and therefore, a clear cause-and-effect relationship remains obscure and requires deconvolution.Open in a separate windowFig. 1.Comparison of crystallographic and solution structures of WT and G93A SOD. (A) Overall architecture of the WT SOD dimer is displayed in 90° rotated views. G93 (small red spheres) resides on a surface-exposed interstrand loop between the fifth and sixth sequential β-strands of SOD and is expected to be innocuous in facilitating protein stability; however, this site harbors the most substitutions observed to result in ALS. G93 is also distant from both (Upper) the dimer interface and (Lower Left) the SOD active site (gold and silver spheres), which are generally implicated as the major determinants for SOD stability. Small blue spheres denote free cysteines. (Lower Right) The close-up view of the mutation site (boxed region in Lower Left tilted forward) shows high similarity between WT (purple) and G93A (red) SOD crystal structures [Protein Data Bank ID codes 1PU0 (WT) and 2ZKY (G93A)]. Hydrogen bonds characteristic of a β-bulge motif are indicated, whereby G93 (or A93) represents position 1. The main chain carbonyl group of β-barrel cork residue L38 is adjacent to the G93 site. (B) SAXS-derived electron pair P(r) distributions from WT (purple) and G93A (red) SOD samples in solution are compared with the theoretical curve for 1PU0. P(r) plots are normalized to peak height. Ab initio models of WT SOD derived from P(r) data are depicted in purple, with crystal structure docked into mesh envelope. Contributions to major and minor peaks from subunit and dimer dimensions are indicated.To better understand the structural effects of ALS mutations on SOD architecture, we coupled the wealth of crystallographic knowledge on SOD structure (7, 52, 53) with small-angle X-ray scattering (SAXS) experiments to characterize misassembly aggregates of ALS mutant SODs in solution. Over 20 y ago, we solved the first atomic structure of the human WT SOD protein (Fig. 1A) (20, 34) and proposed the framework destabilization hypothesis to explain how diverse mutations located throughout the 153-residue β-barrel enzyme might produce a similar disease phenotype (2), albeit with distinctions in the progression trajectory. Since that time, a staggering number of ALS mutations has been documented in patients [178 (mostly missense) (54)], with a similar phenotype in dogs (55, 56). Solution-based techniques are increasingly being applied to connect structure to biological outcome, for instance, through examination of intermolecular interactions within stress-activated pathways, for instance (57, 58). SAXS, which can probe structures for a wide size range of species, also provides higher resolution insights (59), for instance, over visible light-scattering techniques, readily distinguishing unfolded from folded proteins (60).Here, we monitor the initial events of protein aggregation in a subset of ALS mutants localized to a mutational hotspot site at glycine 93. Specifically, we wished to test a possible structural basis for how G93 mutations (to A, C, D, R, S, or V) modulate age of onset and clinical severity in ALS patients (14, 15). The G93 substitution occurs in a β-bulge region (61) between sequential β-strands of the protein (Fig. 1A) on a protruding loop roughly ∼20 Å from T54, the nearest residue of the opposing subunit, and the metal-containing active site (Fig. S1). A priori, mutation of this outer loop position would not be expected to interfere with active site chemistry or buried molecular interfaces. However, we discovered correlations of aggregation nucleation kinetics of SOD proteins with ALS mutations at this site, the stabilizing effects of metal ion retention, and available data for clinical phenotypes in patients with the same mutation. Furthermore, by measuring and exploiting the dimer geometry to observe intrinsic SOD conformers, we show that G93 mutant proteins natively reveal increased intradimer conformational flexibility in the absence of aggregation, which may reflect an increased tendency for ALS mutants to become metal-deficient and misfolding-prone and further explain the correlation to disease severity. Collective results on G93 mutants, thus, support and extend the framework destabilization hypothesis. 相似文献
20.
Wei Ding Wan-Qiu Liu Youli Jia Yongzhen Li Wilfred A. van der Donk Qi Zhang 《Proceedings of the National Academy of Sciences of the United States of America》2016,113(13):3521-3526
Production of ribosomally synthesized and posttranslationally modified peptides (RiPPs) has rarely been reported in fungi, even though organisms of this kingdom have a long history as a prolific source of natural products. Here we report an investigation of the phomopsins, antimitotic mycotoxins. We show that phomopsin is a fungal RiPP and demonstrate the widespread presence of a pathway for the biosynthesis of a family of fungal cyclic RiPPs, which we term dikaritins. We characterize PhomM as an S-adenosylmethionine–dependent α-N-methyltransferase that converts phomopsin A to an N,N-dimethylated congener (phomopsin E), and show that the methyltransferases involved in dikaritin biosynthesis have evolved differently and likely have broad substrate specificities. Genome mining studies identified eight previously unknown dikaritins in different strains, highlighting the untapped capacity of RiPP biosynthesis in fungi and setting the stage for investigating the biological activities and unknown biosynthetic transformations of this family of fungal natural products.Ascomycetes constitute the largest phylum of the fungal kingdom and are a prolific source of natural products that are of great significance to humankind (1–4). Beneficial ascomycetous natural products include many clinically important drugs and various food and industrial chemicals. Many other compounds produced by ascomycetes are toxic and are known as mycotoxins, which pose substantial threats to human food supplies and health (5). Major classes of natural products from Ascomycetes include alkaloids (6, 7), terpenoids (8), polyketides (PKs) (9–12), nonribosomal peptides (NRPs) (12–14), and PK-NRP hybrids (15, 16). However, ribosomally synthesized and posttranslationally modified peptides (RiPPs), a rapidly growing class of natural products (17), have rarely been found in Ascomycetes. It has been known since 2007 that amatoxins and phallotoxins are RiPPs produced by strains of another fungal phylum, Basidiomycetes (which, together with Ascomycetes, compose the subkingdom Dikarya) (17–19). It also was recently reported that the ustiloxins produced by Aspergillus flavus and Ustilaginoidea virens belong to the RiPP class (20, 21).Phomopsins are a group of hexapeptide mycotoxins produced by the pathogenic Ascomycetes Phomopsis leptostromiformis (class Sordariomycetes, order Diaporthales) that infects lupins (Lupinus). Lupin-based animal food contaminated with phomopsins causes lupinosis, a liver disease of sheep and cattle that is particularly problematic in Australia and South Africa but affects other countries as well (22). By targeting the vinca domain of tubulin (23), phomopsins exhibit potent antimitotic activity and thus also represent a potential lead for antitumor drug development. Phomopsins have a 13-member macrocyclic ring formed by an ether bridge linking an Ile residue to the phenyl ring of a Tyr (Fig. 1A). A similar Tyr-containing macrocycle is also present in ustiloxins (Fig. 1B). Unlike ustiloxins, however, phomopsins contain no proteinogenic amino acids and feature a set of dehydroamino acids found only in this group of compounds, including 2,3-dehydroisoleucine, 2,3-dehydroaspartic acid, 3,4-dehydroproline, and 3,4-dehydrovaline (Fig. 1A).Open in a separate windowFig. 1.Chemical structures of phomopsins (A) and ustiloxins (B). The compounds labeled in red are previously unknown N,N-dimethylated dikaritins identified in this study.Early isotopic labeling studies showed that Ile, Pro, and Phe were all incorporated into the phomopsin scaffold (24). Because a Phe hydroxylase that can convert Phe to Tyr has not been found in Ascomycetes, the labeling study seems incongruent with a ribosomal route to phomopsins, in which Tyr, not Phe, would need to be incorporated into the phomopsin structure via a tRNA-dependent pathway. Here we report our investigation of the biosynthetic pathway of phomopsins, which explains the paradoxical observations in the early labeling studies and unequivocally demonstrates that phomopsins belong to the family of RiPP natural products. Moreover, our study shows that RiPPs are widespread in fungi, paving the way for a study of their biological activities and biosynthesis. 相似文献