Neutron Scattering Reveals the Dynamic Basis of Protein Adaptation to Extreme Temperature

Inward rectifier K+ (Kir) channels are activated by phosphatidylinositol-(4,5)-bisphosphate (PIP2), but G protein-gated Kir (KG) channels further require either G protein βγ subunits (Gβγ) or intracellular Na+ for their activation. To reveal the mechanism(s) underlying this regulation, we compared the crystal structures of the cytoplasmic domain of KG channel subunit Kir3.2 obtained in the presence and the absence of Na+. The Na+-free Kir3.2, but not the Na+-plus Kir3.2, possessed an ionic bond connecting the N terminus and the CD loop of the C terminus. Functional analyses revealed that the ionic bond between His-69 on the N terminus and Asp-228 on the CD loop, which are known to be critically involved in Gβγ- and Na+-dependent activation, lowered PIP2 sensitivity. The conservation of these residues within the KG channel family indicates that the ionic bond is a character that maintains the channels in a closed state by controlling the PIP2 sensitivity.

Changes in chromatin architecture induced by epigenetic mechanisms are essential for normal cellular processes such as gene expression, DNA repair, and cellular division. Compact chromatin presents a barrier to these processes and is highly regulated by epigenetic markers binding to components of the nucleosome. Histone modifications directly influence chromatin dynamics and facilitate recruitment of additional factors such as chromatin remodelers and histone chaperones. One member of this last class of factors, FACT (facilitates chromatin transcription), is categorized as a histone chaperone critical for nucleosome reorganization during replication, transcription, and DNA repair. Significant discoveries regarding the role of histone chaperones and specifically FACT have come over the past dozen years from a number of independent laboratories. Here, we review the structural and biophysical basis for FACT-mediated nucleosome reorganization and discuss up-to-date models for FACT function.

The Eph family of receptor tyrosine kinases has been implicated in tumorigenesis as well as pathological forms of angiogenesis. Understanding how to modulate the interaction of Eph receptors with their ephrin ligands is therefore of critical interest for the development of therapeutics to treat cancer. Previous work identified a set of 12-mer peptides that displayed moderate binding affinity but high selectivity for the EphB2 receptor. The SNEW antagonistic peptide inhibited the interaction of EphB2 with ephrinB2, with an IC50 of approximately 15 microm. To gain a better molecular understanding of how to inhibit Eph/ephrin binding, we determined the crystal structure of the EphB2 receptor in complex with the SNEW peptide to 2.3-A resolution. The peptide binds in the hydrophobic ligand-binding cleft of the EphB2 receptor, thus competing with the ephrin ligand for receptor binding. However, the binding interactions of the SNEW peptide are markedly different from those described for the TNYL-RAW peptide, which binds to the ligand-binding cleft of EphB4, indicating a novel mode of antagonism. Nevertheless, we identified a conserved structural motif present in all known receptor/ligand interfaces, which may serve as a scaffold for the development of therapeutic leads. The EphB2-SNEW complex crystallized as a homodimer, and the residues involved in the dimerization interface are similar to those implicated in mediating tetramerization of EphB2-ephrinB2 complexes. The structure of EphB2 in complex with the SNEW peptide reveals novel binding determinants that could serve as starting points in the development of compounds that modulate Eph receptor/ephrin interactions and biological activities.

The exchange protein directly activated by cAMP (EPAC) is a key receptor of cAMP in eukaryotes and controls critical signaling pathways. Currently, no residue resolution information is available on the full-length EPAC dynamics, which are known to be pivotal determinants of allostery. In addition, no information is presently available on the intermediates for the classical induced fit and conformational selection activation pathways. Here these questions are addressed through molecular dynamics simulations on five key states along the thermodynamic cycle for the cAMP-dependent activation of a fully functional construct of EPAC2, which includes the cAMP-binding domain and the integral catalytic region. The simulations are not only validated by the agreement with the experimental trends in cAMP-binding domain dynamics determined by NMR, but they also reveal unanticipated dynamic attributes, rationalizing previously unexplained aspects of EPAC activation and autoinhibition. Specifically, the simulations show that cAMP binding causes an extensive perturbation of dynamics in the distal catalytic region, assisting the recognition of the Rap1b substrate. In addition, analysis of the activation intermediates points to a possible hybrid mechanism of EPAC allostery incorporating elements of both the induced fit and conformational selection models. In this mechanism an entropy compensation strategy results in a low free-energy pathway of activation. Furthermore, the simulations indicate that the autoinhibitory interactions of EPAC are more dynamic than previously anticipated, leading to a revised model of autoinhibition in which dynamics fine tune the stability of the autoinhibited state, optimally sensitizing it to cAMP while avoiding constitutive activation.

Prodiginines are a class of red-pigmented natural products with immunosuppressant, anticancer, and antimalarial activities. Recent studies on prodiginine biosynthesis in Streptomyces coelicolor have elucidated the function of many enzymes within the pathway. However, the function of RedJ, which was predicted to be an editing thioesterase based on sequence similarity, is unknown. We report here the genetic, biochemical, and structural characterization of the redJ gene product. Deletion of redJ in S. coelicolor leads to a 75% decrease in prodiginine production, demonstrating its importance for prodiginine biosynthesis. RedJ exhibits thioesterase activity with selectivity for substrates having long acyl chains and lacking a β-carboxyl substituent. The thioesterase has 1000-fold greater catalytic efficiency with substrates linked to an acyl carrier protein (ACP) than with the corresponding CoA thioester substrates. Also, RedJ strongly discriminates against the streptomycete ACP of fatty acid biosynthesis in preference to RedQ, an ACP of the prodiginine pathway. The 2.12 Å resolution crystal structure of RedJ provides insights into the molecular basis for the observed substrate selectivity. A hydrophobic pocket in the active site chamber is positioned to bind long acyl chains, as suggested by a long-chain ligand from the crystallization solution bound in this pocket. The accessibility of the active site is controlled by the position of a highly flexible entrance flap. These data combined with previous studies of prodiginine biosynthesis in S. coelicolor support a novel role for RedJ in facilitating transfer of a dodecanoyl chain from one acyl carrier protein to another en route to the key biosynthetic intermediate 2-undecylpyrrole.

Sorting nexin 9 (SNX9) functions in a complex with the GTPase dynamin-2 at clathrin-coated pits, where it provokes fission of vesicles to complete endocytosis. Here the SNX9.dynamin-2 complex binds to clathrin and adapter protein complex 2 (AP-2) that line these pits, and this occurs through interactions of the low complexity domain (LC4) of SNX9 with AP-2. Intriguingly, localization of the SNX9.dynamin-2 complex to clathrin-coated pits is blocked by interactions with the abundant glycolytic enzyme aldolase, which also binds to the LC4 domain of SNX9. The crystal structure of the LC4 motif of human SNX9 in complex with aldolase explains the biochemistry and biology of this interaction, where SNX9 binds near the active site of aldolase via residues 165-171 that are also required for the interactions of SNX9 with AP-2. Accordingly, SNX9 binding to aldolase is structurally precluded by the binding of substrate to the active site. Interactions of SNX9 with aldolase are far more extensive and differ from those of the actin-nucleating factor WASP with aldolase, indicating considerable plasticity in mechanisms that direct the functions of the aldolase as a scaffold protein.

PICK1 (protein interacting with C kinase 1) contains a single PDZ domain known to mediate interaction with the C termini of several receptors, transporters, ion channels, and kinases. In contrast to most PDZ domains, the PICK1 PDZ domain interacts with binding sequences classifiable as type I (terminating in (S/T)XPhi; X, any residue) as well as type II (PhiXPhi; Phi, any hydrophobic residue). To enable direct assessment of the affinity of the PICK1 PDZ domain for its binding partners we developed a purification scheme for PICK1 and a novel quantitative binding assay based on fluorescence polarization. Our results showed that the PICK1 PDZ domain binds the type II sequence presented by the human dopamine transporter (-WLKV) with an almost 15-fold and >100-fold higher affinity than the type I sequences presented by protein kinase Calpha (-QSAV) and the beta(2)-adrenergic receptor (-DSLL), respectively. Mutational analysis of Lys(83) in the alphaB1 position of the PDZ domain suggested that this residue mimics the function of hydrophobic residues present in this position in regular type II PDZ domains. The PICK1 PDZ domain was moreover found to prefer small hydrophobic residues in the C-terminal P(0) position of the ligand. Molecular modeling predicted a rank order of (Val > Ile > Leu) that was verified experimentally with up to a approximately 16-fold difference in binding affinity between a valine and a leucine in P(0). The results define the structural basis for the unusual binding pattern of the PICK1 PDZ domain by substantiating the critical role of the alphaB1 position (Lys(83)) and of discrete side chain differences in position P(0) of the ligands.

Variants in Autophagy Genes Affect Susceptibility to Both Crohn's Disease and Helicobacter pylori Infection

During apoptosis, Smac (second mitochondria-derived activator of caspases)/DIABLO, an IAP (inhibitor of apoptosis protein)-binding protein, is released from mitochondria and potentiates apoptosis by relieving IAP inhibition of caspases. We demonstrate that exposure of MCF-7 cells to the death-inducing ligand, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), results in rapid Smac release from mitochondria, which occurs before or in parallel with loss of cytochrome c. Smac release is inhibited by Bcl-2/Bcl-xL or by a pan-caspase inhibitor demonstrating that this event is caspase-dependent and modulated by Bcl-2 family members. Following release, Smac is rapidly degraded by the proteasome, an effect suppressed by co-treatment with a proteasome inhibitor. As the RING finger domain of XIAP possesses ubiquitin-protein ligase activity and XIAP binds tightly to mature Smac, an in vitro ubiquitination assay was performed which revealed that XIAP functions as a ubiquitin-protein ligase (E3) in the ubiquitination of Smac. Both the association of XIAP with Smac and the RING finger domain of XIAP are essential for ubiquitination, suggesting that the ubiquitin-protein ligase activity of XIAP may promote the rapid degradation of mitochondrial-released Smac. Thus, in addition to its well characterized role in inhibiting caspase activity, XIAP may also protect cells from inadvertent mitochondrial damage by targeting pro-apoptotic molecules for proteasomal degradation.

We have reconstituted human mitochondrial transcription in vitro on DNA oligonucleotide templates representing the light strand and heavy strand-1 promoters using protein components (RNA polymerase and transcription factors A and B2) isolated from Escherichia coli. We show that 1 eq of each transcription factor and polymerase relative to the promoter is required to assemble a functional initiation complex. The light strand promoter is at least 2-fold more efficient than the heavy strand-1 promoter, but this difference cannot be explained solely by the differences in the interaction of the transcription machinery with the different promoters. In both cases, the rate-limiting step for production of the first phosphodiester bond is open complex formation. Open complex formation requires both transcription factors; however, steps immediately thereafter only require transcription factor B2. The concentration of nucleotide required for production of the first dinucleotide product is substantially higher than that required for subsequent cycles of nucleotide addition. In vitro, promoter-specific differences in post-initiation control of transcription exist, as well as a second rate-limiting step that controls conversion of the transcription initiation complex into a transcription elongation complex. Rate-limiting steps of the biochemical pathways are often those that are targeted for regulation. Like the more complex multisubunit transcription systems, multiple steps may exist for control of transcription in human mitochondria. The tools and mechanistic framework presented here will facilitate not only the discovery of mechanisms regulating human mitochondrial transcription but also interrogation of the structure, function, and mechanism of the complexes that are regulated during human mitochondrial transcription.

Pyrin domain (PYD)-containing proteins are key components of pathways that regulate inflammation, apoptosis, and cytokine processing. Their importance is further evidenced by the consequences of mutations in these proteins that give rise to autoimmune and hyperinflammatory syndromes. PYDs, like other members of the death domain (DD) superfamily, are postulated to mediate homotypic interactions that assemble and regulate the activity of signaling complexes. However, PYDs are presently the least well characterized of all four DD subfamilies. Here we report the three-dimensional structure and dynamic properties of ASC2, a PYD-only protein that functions as a modulator of multidomain PYD-containing proteins involved in NF-κB and caspase-1 activation. ASC2 adopts a six-helix bundle structure with a prominent loop, comprising 13 amino acid residues, between helices two and three. This loop represents a divergent feature of PYDs from other domains with the DD fold. Detailed analysis of backbone 15N NMR relaxation data using both the Lipari-Szabo model-free and reduced spectral density function formalisms revealed no evidence of contiguous stretches of polypeptide chain with dramatically increased internal motion, except at the extreme N and C termini. Some mobility in the fast, picosecond to nanosecond timescale, was seen in helix 3 and the preceding α2-α3 loop, in stark contrast to the complete disorder seen in the corresponding region of the NALP1 PYD. Our results suggest that extensive conformational flexibility in helix 3 and the α2-α3 loop is not a general feature of pyrin domains. Further, a transition from complete disorder to order of the α2-α3 loop upon binding, as suggested for NALP1, is unlikely to be a common attribute of pyrin domain interactions. Pyrin domain (PYD)-containing proteins are key components of pathways that regulate inflammation, apoptosis, and cytokine processing. Their importance is further evidenced by the consequences of mutations in these proteins that give rise to autoimmune and hyperinflammatory syndromes. PYDs, like other members of the death domain (DD) superfamily, are postulated to mediate homotypic interactions that assemble and regulate the activity of signaling complexes. However, PYDs are presently the least well characterized of all four DD subfamilies. Here we report the three-dimensional structure and dynamic properties of ASC2, a PYD-only protein that functions as a modulator of multidomain PYD-containing proteins involved in NF-κB and caspase-1 activation. ASC2 adopts a six-helix bundle structure with a prominent loop, comprising 13 amino acid residues, between helices two and three. This loop represents a divergent feature of PYDs from other domains with the DD fold. Detailed analysis of backbone 15N NMR relaxation data using both the Lipari-Szabo model-free and reduced spectral density function formalisms revealed no evidence of contiguous stretches of polypeptide chain with dramatically increased internal motion, except at the extreme N and C termini. Some mobility in the fast, picosecond to nanosecond timescale, was seen in helix 3 and the preceding α2-α3 loop, in stark contrast to the complete disorder seen in the corresponding region of the NALP1 PYD. Our results suggest that extensive conformational flexibility in helix 3 and the α2-α3 loop is not a general feature of pyrin domains. Further, a transition from complete disorder to order of the α2-α3 loop upon binding, as suggested for NALP1, is unlikely to be a common attribute of pyrin domain interactions. Inflammatory caspases such as caspase-1 play an essential role in innate immune responses to infection by regulating the processing of pro-inflammatory cytokines interleukin-1β and interleukin-18 into their mature, secreted forms (1Burns K. Martinon F. Tschopp J. Curr. Opin. Immunol. 2003; 15: 26-30Crossref PubMed Scopus (120) Google Scholar, 2Martinon F. Tschopp J. Cell. 2004; 117: 561-574Abstract Full Text Full Text PDF PubMed Scopus (795) Google Scholar). Tight regulation of the production of these cytokines is required to maintain the homeostasis of host tissues. Excessive interleukin-1β production and chronic inflammation are hallmarks of many autoimmune diseases that present both systemically and within the central nervous system, including rheumatoid arthritis and multiple sclerosis (3Christodoulou C. Choy E.H. Clin. Exp. Med. 2006; 6: 13-19Crossref PubMed Scopus (87) Google Scholar, 4Lucas S.-M. Rothwell N.J. Gibson R.M. Br. J. Pharmacol. 2006; 147: S232-S240Crossref PubMed Scopus (1035) Google Scholar). Similar to initiator caspases involved in apoptosis, the activation of inflammatory caspases requires their recruitment into a multiprotein signaling complex, which promotes dimerization and cleavage to produce the active enzyme via an induced proximity mechanism (2Martinon F. Tschopp J. Cell. 2004; 117: 561-574Abstract Full Text Full Text PDF PubMed Scopus (795) Google Scholar, 5Riedl S.J. Shi Y. Nat. Rev. Mol. Cell. Biol. 2004; 5: 897-907Crossref PubMed Scopus (1571) Google Scholar). Recently, models for caspase-1 activation have been proposed whereby inflammatory stimuli promote the formation of molecular platforms referred to as inflammasomes (6Petrilli V. Papin S. Tschopp J. Curr. Biol. 2005; 15: R581Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The NALP1 inflammasome induces the activation of both caspase-1 and caspase-5 through the formation of a complex that also contains the proteins NALP1 and ASC (7Martinon F. Burns K. Tschopp J. Mol. Cell. 2002; 10: 417-426Abstract Full Text Full Text PDF PubMed Scopus (4207) Google Scholar). Similarly, the NALP2/NALP3 inflammasome is involved in the activation of caspase-1 through the recruitment of NALP2 or NALP3, ASC, Cardinal, and caspase-1 (8Agostini L. Martinon F. Burns K. McDermott M.F. Hawkins P.N. Tschopp J. Immunity. 2004; 20: 319-325Abstract Full Text Full Text PDF PubMed Scopus (1382) Google Scholar). The components of the inflammasome encode multiple protein-protein interaction domains, including the pyrin domain (PYD), 2The abbreviations used are: PYD, pyrin domain; CARD, caspase recruitment domain; DED, death effector domain; DD, death domain; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy; RDC, residual dipolar coupling; r.m.s.d., root mean square deviation. 2The abbreviations used are: PYD, pyrin domain; CARD, caspase recruitment domain; DED, death effector domain; DD, death domain; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy; RDC, residual dipolar coupling; r.m.s.d., root mean square deviation. caspase recruitment domain (CARD), and a nucleotide binding and oligomerization domain, that promote homotypic interactions between molecules to facilitate assembly. For instance, the adaptor protein ASC has a dualdomain structure consisting of an N-terminal PYD and a C-terminal CARD that enables it to function as a bridge between the PYD of various NALPs and the CARD of caspase-1 (2Martinon F. Tschopp J. Cell. 2004; 117: 561-574Abstract Full Text Full Text PDF PubMed Scopus (795) Google Scholar, 7Martinon F. Burns K. Tschopp J. Mol. Cell. 2002; 10: 417-426Abstract Full Text Full Text PDF PubMed Scopus (4207) Google Scholar). Inflammasome assembly is modulated by other PYD family members, including pyrin and ASC2, which interact with the inflammasome via their PYD domains to promote or inhibit activity (9Richards N. Schaner P. Diaz A. Stuckey J. Shelden E. Wadhwa A. Gumucio D.L. J. Biol. Chem. 2001; 276: 39320-39329Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 10Stehlik C. Krajewska M. Welsh K. Krajewski S. Godzik A. Reed J.C. Biochem. J. 2003; 373: 101-113Crossref PubMed Scopus (137) Google Scholar, 11Yu J.W. Wu J. Zhang Z. Datta P. Ibrahimi I. Taniguchi S. Sagara J. Fernandes-Alnemri T. Alnemri E.S. Cell Death Differ. 2006; 13: 236-249Crossref PubMed Scopus (283) Google Scholar). Furthermore, hereditary mutations in key components of the inflammasome are thought to contribute to several types of autoinflammatory disease. For example, an increased incidence of familial Mediterranean fever has been associated with mutations in pyrin, whereas mutations in NALP3 have been linked to familial cold autoinflammatory syndrome, Muckle-Wells syndrome, and neonatal-onset multiple-system inflammatory disease (12Hull K.M. Shoham N. Chae J.J. Aksentijevich I. Kastner D.L. Curr. Opin. Rheumatol. 2003; 15: 61-69Crossref PubMed Scopus (204) Google Scholar, 13Ting J.P. Kastner D.L. Hoffman H.M. Nat. Rev. Immunol. 2006; 6: 183-195Crossref PubMed Scopus (277) Google Scholar). The molecular mechanisms by which PYD-containing molecules regulate inflammatory responses are under intense investigation. Recent studies have identified a small protein, ASC2 (also known as POP1 and ASCI), as a regulator of multidomain PYD-containing proteins involved in NF-κB and caspase-1 activation (10Stehlik C. Krajewska M. Welsh K. Krajewski S. Godzik A. Reed J.C. Biochem. J. 2003; 373: 101-113Crossref PubMed Scopus (137) Google Scholar, 14Pawlowski K. Pio F. Chu Z. Reed J.C. Godzik A. Trends Biochem. Sci. 2001; 26: 85-87Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). ASC2 consists solely of a PYD and appears to function as a dominant-negative inhibitor, similar to the CARD-only proteins ICEBERG and pseudo-ICE, which suppress caspase-1 activation (15Humke E.W. Shriver S.K. Starovasnik M.A. Fairbrother W.J. Dixit V.M. Cell. 2000; 103: 99-111Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 16Lee S.H. Stehlik C. Reed J.C. J. Biol. Chem. 2001; 276: 34495-34500Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 17Druilhe A. Srinivasula S.M. Razmara M. Ahmad M. Alnemri E.S. Cell Death Differ. 2001; 8: 649-657Crossref PubMed Scopus (146) Google Scholar), or the death effector domain (DED)-only FLIP proteins in regulating apoptosis by affecting the recruitment of caspase-8 to tumor necrosis factor family death receptors (18Thome M. Tschopp J. Nat. Rev. Immunol. 2001; 1: 50-58Crossref PubMed Scopus (352) Google Scholar). Consistent with this notion, ASC2 associates with the adaptor protein ASC to inhibit its ability to collaborate with pyrin and NALP3 in NF-κB and caspase-1 activation (10Stehlik C. Krajewska M. Welsh K. Krajewski S. Godzik A. Reed J.C. Biochem. J. 2003; 373: 101-113Crossref PubMed Scopus (137) Google Scholar). Interestingly, ASC2 closely resembles the pyrin domain of ASC (64% sequence identity), and the genes encoding these proteins are located on nearby regions of chromosome 16, suggesting that they arose by gene duplication (10Stehlik C. Krajewska M. Welsh K. Krajewski S. Godzik A. Reed J.C. Biochem. J. 2003; 373: 101-113Crossref PubMed Scopus (137) Google Scholar). The predominant expression of ASC2 in monocytes, macrophages, and granulocytes (10Stehlik C. Krajewska M. Welsh K. Krajewski S. Godzik A. Reed J.C. Biochem. J. 2003; 373: 101-113Crossref PubMed Scopus (137) Google Scholar) further supports a role for ASC2 in the regulation of inflammatory responses. Despite the importance of PYD-containing proteins in the regulation of inflammation and apoptosis, the underlying mechanisms remain largely unknown. At present, there is only limited structural information on the isolated PYDs of ASC and NALP1 (19Liepinsh E. Barbals R. Dahl E. Sharipo A. Staub E. Otting G. J. Mol. Biol. 2003; 332: 1155-1163Crossref PubMed Scopus (121) Google Scholar, 20Hiller S. Kohl A. Fiorito F. Herrmann T. Wider G. Tschopp J. Gru¨tter M.G. Wu¨thrich K. Structure. 2003; 11: 1199-1205Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The structure of ASC PYD was found to conform to the canonical fold of the death domain (DD) superfamily comprising six antiparallel α-helices, which is also shared by the DD, DED, and CARD subfamilies (21Fesik S.W. Cell. 2000; 103: 273-282Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). In contrast, the region that normally corresponds to the third helix in the DD fold was found to be completely disordered in NALP1 PYD (20Hiller S. Kohl A. Fiorito F. Herrmann T. Wider G. Tschopp J. Gru¨tter M.G. Wu¨thrich K. Structure. 2003; 11: 1199-1205Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). This has fueled speculation that PYD interactions may involve a conformational change upon binding, and that the folding/unfolding transition of helix 3 may be an important determinant of the function and disease-related dysfunction of this domain (20Hiller S. Kohl A. Fiorito F. Herrmann T. Wider G. Tschopp J. Gru¨tter M.G. Wu¨thrich K. Structure. 2003; 11: 1199-1205Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 22Eliezer D. Structure. 2003; 11: 1190-1191Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 23Liu T. Rojas A. Ye Y. Godzik A. Protein Sci. 2003; 12: 1872-1881Crossref PubMed Scopus (31) Google Scholar). In an effort to further characterize the molecular basis of PYD interactions and the contribution of dynamics, we have determined the three-dimensional solution structure and dynamic properties of ASC2 using NMR spectroscopy. ASC2 adopts a core six-helix bundle structure characteristic of the DD superfamily. As in other members of the PYD subfamily, including ASC and NALP1, ASC2 is characterized by the presence of an insertion between helices 2 and 3 that forms a prominent, exposed loop of non-regular secondary structure. This feature is divergent from other members of the DD superfamily. 15N relaxation data indicate that ASC2 is a globular protein with an approximately isotropic diffusion tensor and a rotational correlation time of 6.2 ns. A detailed analysis of backbone 15N relaxation data using the Lipari-Szabo model-free formalism (24Lipari G. Szabo A. J. Am. Chem. Soc. 1982; 104: 4546-4559Crossref Scopus (3397) Google Scholar, 25Clore G.M. Szabo A. Bax A. Kay L.E. Driscoll P.C. Gronenborn A.M. J. Am. Chem. Soc. 1990; 112: 4989-4991Crossref Scopus (970) Google Scholar, 26Fushman D. Cahill S. Cowburn D. J. Mol. Biol. 1997; 266: 173-194Crossref PubMed Scopus (202) Google Scholar) as well as the reduced spectral density function approach (27Farrow N.A. Zhang O. Szabo A. Torchia D.A. Kay L.E. J. Biomol. NMR. 1995; 6: 153-162Crossref PubMed Scopus (465) Google Scholar) revealed that the α2-α3 loop and the third α-helix (α3) displayed a marginally greater degree of conformational disorder than the other five α-helices. This was in stark contrast to the complete disorder observed in the α3 region of NALP1 PYD (20Hiller S. Kohl A. Fiorito F. Herrmann T. Wider G. Tschopp J. Gru¨tter M.G. Wu¨thrich K. Structure. 2003; 11: 1199-1205Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). This observation indicates that the mechanism of PYD interactions, namely a local disorder/order transition upon binding as proposed for NALP1 PYD (20Hiller S. Kohl A. Fiorito F. Herrmann T. Wider G. Tschopp J. Gru¨tter M.G. Wu¨thrich K. Structure. 2003; 11: 1199-1205Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 22Eliezer D. Structure. 2003; 11: 1190-1191Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar), may be a unique mode of interaction in that case and not a general feature of interactions involving PYDs. Protein Expression and Purification—Full-length ASC2 (residues 1-89) was subcloned into the bacterial expression vector pQE-30 (Qiagen), which produces the recombinant protein with an N-terminal His6 tag. Uniformly 15N- and 15N/13C-labeled proteins were overexpressed in Escherichia coli BL21 cells in minimal media containing 15NH4Cl (1 g/liter) with or without [13C]glucose (2 g/liter) as the sole nitrogen and carbon sources, respectively. Cells were grown at 37 °C to an optical density (A600 nm) of ∼1.0 and then induced with 1 mm isopropyl-β-d-thiogalactopyranoside for 5 h. After harvesting, the cells were suspended in 50 mm Tris buffer (pH 8.0), 1 m NaCl, 30 mm imidazole, and 10 mm benzamidine hydrochloride, lysed by sonication, and centrifuged. The protein was purified using Ni2+ affinity chromatography and judged to be >95% pure by SDS-PAGE analysis. Samples for NMR contained 1 mm protein in 10 mm sodium phosphate buffer (pH 7.3) and 140 mm NaCl in H2O/D2O (9:1) or D2O. NMR Spectroscopy—All NMR experiments were acquired at 25 °C on a Bruker Avance 600-MHz spectrometer equipped with a z-shielded gradient triple resonance probe. The backbone and side-chain 1H, 13C, and 15N resonances of the protein were assigned using CBCA(CO)NH, HNCA, HNCACB, HNCO, HN(CA)CO, HBHA(CO)NH, C(CO)NH, H(CCO)NH and three-dimensional HCCH-TOCSY experiments (28Sattler M. Schleucher J. Griesinger C. Prog. NMR Spectrosc. 1999; 34: 93-158Abstract Full Text Full Text PDF Scopus (1389) Google Scholar). NOE-derived distance restraints were obtained from 15N- or 13C-separated three-dimensional NOESY spectra (mixing times of 110 and 120 ms, respectively). 3JNHα and 3JNHβ coupling constants were measured by quantitative J correlation spectroscopy (29Vuister G.W. Tessari M. Karini-Nejad Y. Whitehead B. Biological Magnetic Resonance 16. Kluwer Press, New York, NY1999: 195-259Google Scholar). 1DNH residual dipolar couplings were measured on a 15N-labeled sample partially aligned in 5% polyethylene glycol (C12E5)/hexanol media with a surfactant to alcohol ratio of 0.96 (30Ru¨ckert M. Otting G. J. Am. Chem. Soc. 2000; 122: 7793-7797Crossref Scopus (540) Google Scholar). 15N-HN splittings were measured on the isotropic and partially aligned samples using two-dimensional 1H-15N HSQC-IPAP experiments (31Cordier F. Dingley A.J. Grzesiek S. J. Biomol. NMR. 1999; 13: 175-180Crossref PubMed Scopus (101) Google Scholar). Slowly exchanging amide protons were identified from a series of two-dimensional 1H-15N HSQC spectra recorded after the buffer was changed to D2O. NMR spectra were processed with NMRPipe/NMRDraw (32Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11549) Google Scholar) and analyzed using PIPP and STAPP (33Garrett D.S. Powers R. Gronenborn A.M. Clore G.M. J. Magn. Reson. 1991; 95: 214-220Crossref Scopus (802) Google Scholar). Structure Calculations—NOEs within the protein were grouped into four distance ranges, 1.8-2.7, 1.8-3.3, 1.8-5.0, and 1.8-6.0 Å, corresponding to strong, medium, weak, and very weak intensities. Distances involving methyl groups, aromatic ring protons, and non-stereospecifically assigned methylene protons were represented as a (∑r−6)−1/6 sum (34Nilges M. Proteins. 1993; 17: 297-309Crossref PubMed Scopus (308) Google Scholar). 80 φ and 48 χ1 angle restraints were derived from an analysis of 3JNHα and 3JNHβ coupling constants (29Vuister G.W. Tessari M. Karini-Nejad Y. Whitehead B. Biological Magnetic Resonance 16. Kluwer Press, New York, NY1999: 195-259Google Scholar), and 69 ψ angle restraints were determined by chemical shift data base analysis using the program TALOS (35Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2738) Google Scholar). The minimum range employed for φ, ψ, and χ1 torsion angle restraints was ± 30°. Hydrogen bond distance restraints (rNH-O = 1.5-2.8 Å and rN-O = 2.4-3.5 Å) were added during the final stages of refinement for residues within α-helices as derived from an analysis of amide proton exchange, 13Cα chemical shifts, and characteristic NOE patterns. The structures were calculated with the program XPLOR-NIH 2.11.2 (36Schwieters C.D. Kuszewski J.J. Tjandra N. 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The neuronal adaptor protein Fe65 is involved in brain development, Alzheimer disease amyloid precursor protein (APP) signaling, and proteolytic processing of APP. It contains three protein-protein interaction domains, one WW domain, and a unique tandem array of phosphotyrosine-binding (PTB) domains. The N-terminal PTB domain (Fe65-PTB1) was shown to interact with a variety of proteins, including the low density lipoprotein receptor-related protein (LRP-1), the ApoEr2 receptor, and the histone acetyltransferase Tip60. We have determined the crystal structures of human Fe65-PTB1 in its apo- and in a phosphate-bound form at 2.2 and 2.7A resolution, respectively. The overall fold shows a PTB-typical pleckstrin homology domain superfold. Although Fe65-PTB1 has been classified on an evolutionary basis as a Dab-like PTB domain, it contains attributes of other PTB domain subfamilies. The phosphotyrosine-binding pocket resembles IRS-like PTB domains, and the bound phosphate occupies the binding site of the phosphotyrosine (Tyr(P)) within the canonical NPXpY recognition motif. In addition Fe65-PTB1 contains a loop insertion between helix alpha2 and strand beta2(alpha2/beta2 loop) similar to members of the Shc-like PTB domain subfamily. The structural comparison with the Dab1-PTB domain reveals a putative phospholipid-binding site opposite the peptide binding pocket. We suggest Fe65-PTB1 to interact with its target proteins involved in translocation and signaling of APP in a phosphorylation-dependent manner.

hormone response element peroxisome proliferator-activated receptor thyroid hormone receptor estrogen receptor ligand binding domain nuclear receptor corepressor silencing mediator of retinoic acid and thyroid hormone receptor imitation SWI cAMP response element-binding protein CREB-binding protein histone acetyltransferase mitogen-activated protein histone deacetylase steroid receptor coactivator RAR interacting protein glucocorticoid receptor interacting protein T3R receptor associated protein vitamin receptor D interacting protein Members of the nuclear receptor superfamily directly activate or repress target genes by binding to hormone response elements (HREs)1 in promoter or enhancer regions, and by binding to other DNA sequence-specific activators and can inhibit the transcriptional activities of other classes of transcription factors by transrepression. Hormone response elements provide specificity to receptor homodimer heterodimer binding (reviewed in Ref. 2Bourguet W. Germain P. Gronemeyer H. Trends Pharm. Sci. 2000; 21: 381-388Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). Nuclear receptor functions are directed by specific activation domains, referred to as activation function 1 (AF-1), which resides in the N terminus, and activation function 2 (AF-2), which resides in the C-terminal ligand binding domain (LBD) (reviewed in Ref. 1Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar). Regulation of gene transcription by nuclear receptors requires the recruitment of proteins characterized as coregulators, with ligand-dependent exchange of corepressors for coactivators serving as the basic mechanism for switching gene repression to activation. In this review, we discuss biochemical and genetic studies suggesting that coregulatory complexes are differentially utilized in both a cell- and promoter-specific fashion to activate or repress gene transcription. These coregulatory components, themselves targets of diverse intracellular signaling pathways, provide a combinatorial code for tissue- and gene-specific responses, utilizing both enzymatic and platform assembly functions to mediate the actions of nuclear receptor genetic programs critical for developmental and homeostatic processes in metazoan organisms. A diverse group of proteins have emerged as potential coactivators for nuclear receptors. Ligand-dependent recruitment of coactivators is dependent on AF-2, which consists of a short conserved helical sequence within the C terminus of the LBD (2Bourguet W. Germain P. Gronemeyer H. Trends Pharm. Sci. 2000; 21: 381-388Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). Biochemical and expression cloning approaches have been used to identify a large number of factors that interact with nuclear receptors in either a ligand-independent or a ligand-dependent manner and are often components of large multiprotein complexes. Many of these factors are capable of potentiating nuclear receptor activity in transient cotransfection assays. In addition, a distinct set of coactivators is associated with the AF-1 domain. As the number of potential coregulators clearly exceeds the capacity for direct interaction by a single receptor, the most plausible hypothesis is that transcriptional activation by nuclear receptors involves the actions of multiple factors. These factors act in a sequential and/or combinatorial manner to reorganize chromatin templates and to modify and recruit basal factors and RNA polymerase II (3Wu C. J. Biol. Chem. 1997; 272: 28171-28174Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 4Wade P.A. Wollfe A.P. Curr. Biol. 1999; 9: R221-R224Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). As chromatinized transcription units are “repressed” compared with naked DNA, a critical aspect of gene activation involves nucleosomal remodeling (reviewed in Refs. 3Wu C. J. Biol. Chem. 1997; 272: 28171-28174Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 4Wade P.A. Wollfe A.P. Curr. Biol. 1999; 9: R221-R224Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 5Struhl K. Cell. 1999; 98: 1-4Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). Two general classes of chromatin remodeling factors that appear to play critical roles in transcriptional activation by nuclear receptors have been identified. These are ATP-dependent nucleosome remodeling complexes and factors that contain histone acetyltransferase activity. The yeast SWI·SNF complex facilitates the binding of sequence-specific transcription factors to nucleosomal DNA and can cause local changes in chromatin structure in an ATP-dependent manner (3Wu C. J. Biol. 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Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1549) Google Scholar, 11Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2448) Google Scholar, 12Grant P.A. Duggan L. Cote J. Roberts S.M. Brownell J.E. Candau R. Ohba R. Owen-Hughes T. Allis C.D. Winston F. Berger S.L. Workman J.L. Genes Dev. 1997; 11: 1640-1650Crossref PubMed Scopus (897) Google Scholar). Mammalian homologues of Drosophila SWI2/SNF2 such as BRG1/hBrm function as components of large multiprotein complexes. Transfection of ATPase-defective alleles of either Brg1 orhBrm into several mammalian cell lines leads to a significant decrease in the ability of several nuclear receptors to activate transcription (3Wu C. J. Biol. Chem. 1997; 272: 28171-28174Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 4Wade P.A. Wollfe A.P. Curr. Biol. 1999; 9: R221-R224Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 5Struhl K. Cell. 1999; 98: 1-4Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 6Pazin M.J. Kadonaga J.T. Cell. 1997; 88: 737-740Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Remodeling complexes containing ISWI (imitation SWI) may also be involved in nuclear receptor function (7Pazin M.J. Kadonaga J.T. Cell. 1997; 89: 325-328Abstract Full Text Full Text PDF PubMed Scopus (773) Google Scholar, 8Mizzen C.A. Yang X.-J. Kokubo T. Brownell J.E. Bannister A.J. Owen-Hughes T. Workman J. Wang L. Berger S.L. Kouzarides T. Nakatani Y. Allis C.D. Cell. 1996; 87: 1261-1270Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar, 9Ogryzko V.V. Kotani T. Zhang R.L. Howard S.T. Yang X.J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, 10Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1549) Google Scholar, 11Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2448) Google Scholar). Rates of gene transcription roughly correlate with the degree of histone acetylation, with hyperacetylated regions of the genome appearing to be more actively transcribed than hypoacetylated regions (reviewed in Ref. 7Pazin M.J. Kadonaga J.T. Cell. 1997; 89: 325-328Abstract Full Text Full Text PDF PubMed Scopus (773) Google Scholar). The specific recruitment of a complex with histone acetyltransferase activity to a promoter may play a critical role in overcoming repressive effects of chromatin structure on transcription (4Wade P.A. Wollfe A.P. Curr. Biol. 1999; 9: R221-R224Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 5Struhl K. Cell. 1999; 98: 1-4Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 6Pazin M.J. 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The central conserved domain mediates ligand-dependent interactions with the nuclear receptor LBD, whereas the conserved C-terminal transcriptional activation domains mediate interactions with either CBP/p300 or protein-arginine methyltransferase (16Chen D. Ma H. Hong H. Koh S.S. Huang S.-M. Schurter B.T. Aswad D.W. Stallcup M.R. Science. 1999; 284: 2174-2176Crossref PubMed Scopus (1019) Google Scholar, 17Koh S. Chen D. Lee Y. Stallcup M. J. Biol. Chem. 2001; 276: 1089-1098Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). Based on the presence of three regulatory domains, members of the p160 family have been suggested to function as coactivators, at least in part, by serving as adapter molecules that recruit CBP and/or p300 complexes to promoter-bound nuclear receptors in a ligand-dependent manner (18Kurokawa R. Kalafus D. Ogliastro M.-H. Kioussi C. Xu L. Torchia J. Rosenfeld M.G. Glass C.K. 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Mutations and polymorphisms in the regulator of complement activation, factor H, have been linked to atypical hemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis, and age-related macular degeneration. Many aHUS patients carry mutations in the two C-terminal modules of factor H, which normally confer upon this abundant 155-kDa plasma glycoprotein its ability to selectively bind self-surfaces and prevent them from inappropriately triggering the complement cascade via the alternative pathway. In the current study, the three-dimensional solution structure of the C-terminal module pair of factor H has been determined. A binding site for a fully sulfated heparin-derived tetrasaccharide has been delineated using chemical shift mapping and the C3d/C3b-binding site inferred from sequence comparisons and computational docking. The resultant information allows assessment of the likely consequences of aHUS-associated amino acid substitutions in this critical region of factor H. It is striking that, excepting those likely to perturb the three-dimensional structure, aHUS-associated missense mutations congregate in the polyanion-binding site delineated in this study, thus potentially disrupting a vital mechanism for control of complement on self-surfaces in the microvasculature of the kidney. It is intriguing that a single nucleotide polymorphism predisposing to age-related macular degeneration occupies another region of factor H that harbors a polyanion-binding site.

The editing domain of valyl-tRNA synthetase (ValRS) is known to deacylate, or edit, misformed Thr-tRNA(Val) (post-transfer editing). Here, we determined the 1.7-Angstroms resolution crystal structure of the Thermus thermophilus ValRS editing domain. A comparison of the structure with the previously reported tRNA complex structure revealed conformational changes of the editing domain upon accommodation of the terminal A76; the "GTG loop" moves to expand the pocket, and the side chain of Phe-264 on the GTG loop rotates to interact with the A76 adenine ring. If these conformational changes did not occur, then C75 and A76 of the tRNA would clash with Phe-264. To elucidate the mechanism of the threonine side-chain recognition, we determined the crystal structure of the editing domain bound with [N-(L-threonyl)-sulfamoyl]adenosine at 1.7-Angstroms resolution. The gamma-OH of the threonyl moiety is recognized by the Lys-270, Thr-272, and Asp-279 side chains, which may reject the cognate valyl moiety. Accordingly, ValRS mutants with an Ala substitution for Lys-270 or Asp-279 synthesized significant amounts of Thr-tRNA(Val). The misproduced Thr-tRNA(Val) was hydrolyzed efficiently by the wild-type ValRS, but this post-transfer editing activity was drastically impaired by the Ala substitutions for Lys-270 and Asp-279 and was also decreased by those for Arg-216, Phe-264, and Thr-272. These results indicate that the threonyl moiety and A76 of Thr-tRNA(Val) are recognized by the Lys-270, Thr-272, and Asp-279 side chains and by the Phe-264 side chain, respectively, of the ValRS editing domain.