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Mutation of C9orf72 is the most prevalent defect associated with amyotrophic lateral sclerosis and frontotemporal degeneration.sup.1. Together with hexanucleotide-repeat expansion.sup.2,3, haploinsufficiency of C9orf72 contributes to neuronal dysfunction.sup.4-6. Here we determine the structure of the C9orf72-SMCR8-WDR41 complex by cryo-electron microscopy. C9orf72 and SMCR8 both contain longin and DENN (differentially expressed in normal and neoplastic cells) domains.sup.7, and WDR41 is a [beta]-propeller protein that binds to SMCR8 such that the whole structure resembles an eye slip hook. Contacts between WDR41 and the DENN domain of SMCR8 drive the lysosomal localization of the complex in conditions of amino acid starvation. The structure suggested that C9orf72-SMCR8 is a GTPase-activating protein (GAP), and we found that C9orf72-SMCR8-WDR41 acts as a GAP for the ARF family of small GTPases. These data shed light on the function of C9orf72 in normal physiology, and in amyotrophic lateral sclerosis and frontotemporal degeneration. The cryo-electron microscopy structure of C9orf72-SMCR8-WDR41 suggests that this complex is a GTPase-activating protein for ARF-family small GTPases, which sheds light on the role of C9orf72 mutations in neuronal dysfunction., Author(s): Ming-Yuan Su [sup.1] [sup.2] , Simon A. Fromm [sup.1] [sup.2] , Roberto Zoncu [sup.1] [sup.2] , James H. Hurley [sup.1] [sup.2] [sup.3] Author Affiliations: (1) Department of Molecular and [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2010.11.030 Byline: James H. Hurley (1), Evzen Boura (1), Lars-Anders Carlson (1), Bartosz RoA1/4ycki (2) Abstract: Membrane budding is a key step in vesicular transport, multivesicular body biogenesis, and enveloped virus release. These events range from those that are primarily protein driven, such as the formation of coated vesicles, to those that are primarily lipid driven, such as microdomain-dependent biogenesis of multivesicular bodies. Other types of budding reside in the middle of this spectrum, including caveolae biogenesis, HIV-1 budding, and ESCRT-catalyzed multivesicular body formation. Some of these latter events involve budding away from cytosol, and this unusual topology involves unique mechanisms. This Review discusses progress toward understanding the structural and energetic bases of these different membrane-budding paradigms. Author Affiliation: (1) Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0580, USA (2) Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0580, USA

When internalized receptors and other cargo are destined for lysosomal degradation, they are ubiquitinated and sorted by the endosomal sorting complex required for transport (ESCRT) complexes 0, I, II and III into multivesicular bodies. Multivesicular bodies are formed when cargo-rich patches of the limiting membrane of endosomes bud inwards by an unknown mechanism and are then cleaved to yield cargo-bearing intralumenal vesicles. The biogenesis of multivesicular bodies was reconstituted and visualized using giant unilamellar vesicles, fluorescent ESCRT-0, -I, -II and -III complexes, and a membrane-tethered fluorescent ubiquitin fusion as a model cargo. Here we show that ESCRT-0 forms domains of clustered cargo but does not deform membranes. ESCRT-I and ESCRT-II in combination deform the membrane into buds, in which cargo is confined. ESCRT-I and ESCRT-II localize to the bud necks, and recruit ESCRT-0-ubiquitin domains to the buds. ESCRT-III subunits localize to the bud neck and efficiently cleave the buds to form intralumenal vesicles. Intralumenal vesicles produced in this reaction contain the model cargo but are devoid of ESCRTs. The observations explain how the ESCRTs direct membrane budding and scission from the cytoplasmic side of the bud without being consumed in the reaction., It was shown 30 years ago that after stimulation, internalized EGF receptor localized to multivesicular bodies (MVBs) of human cells within minutes (1). MVBs subsequently fuse with lysosomes where their [...]

The endosomal sorting complex required for transport (ESCRT) system is essential for multivesicular body biogenesis, in which cargo sorting is coupled to the invagination and scission of intralumenal vesicles. The ESCRTs are also needed for budding of enveloped viruses including human immunodeficiency virus 1, and for membrane abscission in cytokinesis. In Saccharomyces cerevisiae, ESCRT-III consists of Vps20, Snf7, Vps24 and Vps2 (also known as Did4), which assemble in that order and require the ATPase Vps4 for their disassembly. In this study, the ESCRT-III-dependent budding and scission of intralumenal vesicles into giant unilamellar vesicles was reconstituted and visualized by fluorescence microscopy. Here we show that three subunits of ESCRT-III, Vps20, Snf7 and Vps24, are sufficient to detach intralumenal vesicles. Vps2, the ESCRT-III subunit responsible for recruiting Vps4, and the ATPase activity of Vps4 were required for ESCRT-III recycling and supported additional rounds of budding. The minimum set of ESCRT-III and Vps4 proteins capable of multiple cycles of vesicle detachment corresponds to the ancient set of ESCRT proteins conserved from archaea to animals., The ESCRT machinery is essential for several fundamental cellular pathways (1-7). The biogenesis of lysosomes involves the maturation of early endosomes into multivesicular bodies (MVBs) (1,2,5). In this pathway, portions [...]

The retromer complex (1,2) is required for the sorting of acid hydrolases to lysosomes (3-7), transcytosis of the polymeric immunoglobulin receptor (8), Wnt gradient formation (9,10), iron transporter recycling (11) [...]

Sterols are moved between cellular membranes by nonvesicular pathways whose functions are poorly understood. In yeast, one such pathway transfers sterols from the plasma membrane (PM) to the endoplasmic reticulum (ER). We show that this transport requires oxysterol-binding protein (OSBP)-related proteins (ORPs), which are a large family of conserved lipid-binding proteins. We demonstrate that a representative member of this family, Osh4p/Kes1p, specifically facilitates the nonvesicular transfer of cholesterol and ergosterol between membranes in vitro. In addition, Osh4p transfers sterols more rapidly between membranes containing phosphoinositides (PIPs), suggesting that PIPs regulate sterol transport by ORPs. We confirmed this by showing that PM to ER sterol transport slows dramatically in mutants with conditional defects in PIP biosynthesis. Our findings argue that ORPs move sterols among cellular compartments and that sterol transport and intracellular distribution are regulated by PIPs.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.01.049 Byline: Michael S. Kostelansky (1), Ji Sun (2), Sangho Lee (1), Jaewon Kim (1), Rodolfo Ghirlando (1), Aitor Hierro (1), Scott D. Emr (2), James H. Hurley (1) Abstract: The endosomal sorting complex required for transport (ESCRT) complexes are central to receptor downregulation, lysosome biogenesis, and budding of HIV. The yeast ESCRT-I complex contains the Vps23, Vps28, and Vps37 proteins, and its assembly is directed by the C-terminal steadiness box of Vps23, the N-terminal half of Vps28, and the C-terminal half of Vps37. The crystal structures of a Vps23:Vps28 core subcomplex and the Vps23:Vps28:Vps37 core were solved at 2.1 and 2.8 A resolution. Each subunit contains a structurally similar pair of helices that form the core. The N-terminal domain of Vps28 has a hydrophobic binding site on its surface that is conformationally dynamic. The C-terminal domain of Vps28 binds the ESCRT-II complex. The structure shows how ESCRT-I is assembled by a compact core from which the Vps23 UEV domain, the Vps28 C domain, and other domains project to bind their partners. Author Affiliation: (1) Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, US Department of Health and Human Services, Bethesda, MD 20892, USA (2) Department of Cellular and Molecular Medicine, Department of Chemistry and Biochemistry, and Howard Hughes Medical Institute, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA Article History: Received 27 September 2005; Revised 6 December 2005; Accepted 12 January 2006 Article Note: (miscellaneous) Published: April 6, 2006

Author(s): Young Jun Im [1, 3]; Sumana Raychaudhuri [2]; William A. Prinz [2]; James H. Hurley (corresponding author) [1] The oxysterol-binding-protein (OSBP)-related proteins (ORPs) are conserved from yeast to humans [...]

The Golgi-localized, [gamma]-ear-containing, Arf (ADP-ribosylation factor)-binding (GGA) proteins are clathrin adaptors that mediate the sorting of transmembrane-cargo molecules at the trans-Golgi network and endosomes. Cargo proteins can be directed into the GGA pathway by at least two different types of sorting signals: acidic cluster-dileucine motifs and covalent modification by ubiquitin. The latter modification is recognized by the GGAs through binding to their GAT [GGA and TOM (target of Myb)] domain. Here we report the crystal structure of the GAT domain of human GGA3 in a 1:1 complex with ubiquitin at 2.8-A resolution. Ubiquitin binds to a hydrophobic and acidic patch on helices [alpha]1 and [alpha]2 of the GAT three-helix bundle that includes Asn-223, Leu-227, Glu-230, Met231, Asp-244, Glu-246, Leu-247, Glu-250, and Leu-251. The GAT-binding surface on ubiquitin is a hydrophobic patch centered on Ile-44 that is also responsible for binding most other ubiquitin effectors. The ubiquitin-binding site observed in the crystal is distinct from the Rabaptin-5-binding site on helices [alpha]2 and [alpha]3 of the GAT domain. Mutational analysis and modeling of the ubiquitin-Rabaptin-5-GAT ternary complex indicates that ubiquitin and Rabaptin-5 can bind to the GAT domain at two different sites without any steric conflict. This ability highlights the GAT domain as a hub for interactions with multiple partners in trafficking. protein-protein interactions | structural biology | trafficking

The crystal structure at 3.2 angstron unit resolution of one such protein, beta 2-chimaerin, a GTPase-activating protein for the small GTPase Rac, in its inactive conformation is reported. The structure shows that in the inactive state, the N terminus of beta2-chimaerin protrudes into the inactive site of the RacGAP domain, sterically blocking Rac binding.

UDP-GalNAc:polypeptide [alpha]-N-acetylgalactosaminyltransferases (ppGaNTases) initiate the formation of mucin-type, O-linked glycans by catalyzing the transfer of [alpha]-N-acetylgalactosamine from UDP-GalNAc to Ser or Thr residues of core proteins to form the Tn antigen (GalNAc-[alpha]-1-O-Ser/Thr). ppGaNTases are unique among glycosyltransferases in containing a C-terminal lectin domain. We present the x-ray crystal structure of a ppGaNTase, murine ppGaNTase-T1, and show that it folds to form distinct catalytic and lectin domains. The association of the two domains forms a large cleft in the surface of the enzyme that contains a [Mn.sup.2+] ion complexed by invariant D209 and H211 of the 'DXH' motif and by invariant H344. Each of the three potential lectin domain carbohydrate-binding sites ([alpha],[beta], and [gamma],) is located on the active-site face of the enzyme, suggesting a mechanism by which the transferase may accommodate multiple conformations of glycosylated acceptor substrates. A model of a mucin 1 glycopeptide substrate bound to the enzyme shows that the spatial separation between the lectin [alpha] site and a modeled active site UDP-GalNAc is consistent with the in vitro pattern of glycosylation observed for this peptide catalyzed by ppGaNTase-T1. The structure also provides a template for the larger ppGaNTase family, and homology models of several ppGaNTase isoforms predict dramatically different surface chemistries consistent with isoform-selective acceptor substrate recognition. glycosyltransferase | mucin

Author(s): Aitor Hierro [1]; Ji Sun [2]; Alexander S. Rusnak [2]; Jaewon Kim [1]; Gali Prag [1]; Scott D. Emr [2]; James H. Hurley (corresponding author) [1] The multivesicular-body (MVB) [...]

The Golgi-associated, [gamma]-adaptin homologous, ADP-ribosylation factor (ARF)-interacting proteins (GGAs) are adaptors that sort receptors from the trans-Golgi network into the endosomal/lysosomal pathway. The GGAs and TOM1 (GAT) domains of the GGAs are responsible for their ARF-dependent localization. The 2.4-[Angstrom] crystal structure of the GAT domain of human GGA1 reveals a three-helix bundle, with a long N-terminal helical extension that is not conserved in GAT domains that do not bind ARF. The ARF binding site is located in the N-terminal extension and is separate from the core three-helix bundle. An unanticipated structural similarity to the N-terminal domain of syntaxin 1a was discovered, comprising the entire three-helix bundle. A conserved binding site on helices 2 and 3 of the GAT domain three-helix bundle is predicted to interact with coiled-coil-containing proteins. We propose that the GAT domain is descended from the same ancestor as the syntaxin 1a N-terminal domain, and that both protein families share a common function in binding coiled-coil domain proteins.

The authors review the publications on membrane curvature. The topics of interest include the curvature creation and stabilization mechanisms and the structural analysis of the epsin N-terminal homology domain of the endocytic protein epsin.

Author(s): Saurav Misra [1]; Rosa Puertollano [2]; Yukio Kato [2]; Juan S. Bonifacino [2]; James H. Hurley (corresponding author) [1] Specific sorting signals direct transmembrane proteins to the compartments of [...]

Many novel signal transduction domains are being identified in the wake of genome sequencing projects and improved sensitivity in homology-detection techniques. The functions of these domains are being discovered by hypothesis-driven experiments and structural genomics approaches. This article reviews the recent highlights of research on modular signaling domains, and the relative contributions and limitations of the various approaches being used.

The role of lipids in signal transduction with reference to phosphatidylinositol 3-phosphate directing endosomal localization of regulatory proteins is discused. Specific phosphate and hydroxyl group interactions distinguish phosphatidylinositol 3-phosphate from other lipids. The lipid binds to FYVE and PX domains found in intracellular membrane transport/signal transduction proteins and phagocyte proteins, respectively.

Research describes the crystal structure of inositol polyphosphate 5-phosphatase domain and its product complex together with structure/function analysis of dephosphorylation site specific 5-phosphatases, substrate specificity, and membrane binding determinants.

Results show that VHS domain containing human target of myb 1 proteins are localized to membranes. Crystallographic analysis of VHS domains indicates that helicies 2 and 4 are involved in the protein-protein interaction.

The flattened face adjacent to the active site of type IIbeta phosphatidylinositol phosphate kinase is essential for catalysis. The face binds acidic phospholipids via electrostatic interactions.

The structure of type II-beta phosphatidylinositol phosphate kinase (PIPK) shows a protein kinase ATP-binding core. It indicates that all phosphoinositide kinases are part of one superfamily. The flatness of the membrane-binding site, along with the shallowness of the substrate-binding pocket and its closeness to the membrane indicate that PIPKs phosphyorylate their substrates in situ in membranes. This allows the enzyme to avoid an energetic penalty for removing the substrate from its low-energy conformation within the membrane.

Guanylyl cyclases (GCs) and adenylyl cyclases (ACs) have fundamental roles in a wide range of cellular processes. Whereas GCs use GTP as a substrate to form cGMP, ACs catalyze the analogous conversion of ATP to cAMP. Previously, a model based on the structure of adenylate cyclase was used to predict the structure of the nucleotide-binding pocket of a membrane guanylyl cyclase, RetGC-1. based on this model, we replaced specific amino acids in the guanine-binding pocket of GC with their counterparts from AC. A change of two amino acids, E925K together with C995D, is sufficient to completely alter the nucleotide specificity from GTP to ATP. These experiments strongly validate the AC-derived RetGC-1 structural model and functionally confirm the role of these residues in nucleotide discrimination.

Phosphoinositide-specific phospholipase C has four binding sites for calcium. One at the catalytic domain, another at the C2 domain and two at the EF-hands. To determine which calcium binding site is responsible for the dependence of the catalytic reaction on calcium, C2 binding site deletion mutants were synthesized. The activities of the native phospholipase C and the deletion mutants are comparable relative to the calcium ion concentration indicating that the calcium binding site at the catalytic domain is responsible for the calcium dependence of the process.

The adenylyl and guanylyl cyclases catalyze the formation of 3[prime],5[prime]-cyclic adenosine or guanosine monophosphate from the corresponding nucleoside 5[prime]-triphosphate. The guanylyl cyclases, the mammalian adenylyl cyclases, and their microbial homologues function as pairs of homologous catalytic domains. The crystal structure of the rat type II adenylyl cyclase [C.sub.2] catalytic domain was used to model by homology a mammalian adenylyl cyclase [C.sub.1]-[C.sub.2] domain pair, a homodimeric adenylyl cyclase of Dictyostelium discoideum, a heterodimeric soluble guanylyl cyclase, and a homodimeric membrane guanylyl cyclase. [Mg.sup.2+]ATP or [Mg.sup.2+]GTP were docked into the active sites based on known stereochemical constraints on their conformation. The models are consistent with the activities of seven active-site mutants. Asp-310 and Glu-432 of type I adenylyl cyclase coordinate a [Mg.sup.2+] ion. The D310S and D310A mutants have 10-fold reduced [V.sub.max] and altered [[Mg.sup.2+]] dependence. The NTP purine moieties bind in mostly hydrophobic pockets. Specificity is conferred by a Lys and an Asp in adenylyl cyclase, and a Glu, an Arg, and a Cys in guanylyl cyclase. The models predict that an Asp from one domain is a general base in the reaction, and that the transition state is stabilized by a conserved Asn-Arg pair on the other domain.

The mammalian adenylyl cyclase has two conserved regions, C(sub 1) and C(sub 2), which are responsible for forskolin- and G-protein-stimulated catalysis. The C(sub 2) catalytic region of type II rat adenylyl cyclase contains an alpha/beta class fold that has a central cleft. At the end of the cleft are two forskolin molecules that bind in hydrophobic pockets while at the center are charged residues implicated in adenosine triphosphate binding. Forskolin seems to activate adenylyl cyclase by promoting the assembly of the active dimer and by direct interaction within the catalytic cleft. Other adenylyl cyclase regulators work at the dimer interface or on a flexible C-terminal region.

Isocitrate dehydrogenase (IDH) is a decarboxylating dehydrogenase that is known to be involved with the Krebs Cycle, glutamic acid production and the provision of NADPH for biosyntheses. IDH, like many decarboxylating dehydrogenases, has been the subject of much research because its strong preference for either NAD or NADP has made it a target of enzyme redesign efforts. Determinants of IDH's cofactor specificity are examined using the structure of an engineered NADP - NAD specificity-reversal mutant as a reference point.

The binding domain for phorbol 13-acetate on the cys2 domain of protein kinase C-delta (PKC-delta) is formed by pulling apart the two beta-sheets and five hydrogen bonds are formed between the phorbol and PKC-delta. The binding of phorbol to the protein forms a hydrophobic surface which extends over 1/3 of the domain and the formation of the hydrogen bonds and the free energy released helps in the insertion of the protein into the membrane. The insertion of the unliganded protein into the membrane is unfavorable because hydrogen bonds at the membrane-protein interface are not formed.

The phosphocarrier protein [III.sup.Glc] is an integral component of the bacterial phosphotransferase (PTS) system. Unphosphorylated [III.sup.Glc] inhibits non-PTS carbohydrate transport systems by binding to diverse target proteins. The crystal structure at 2.6 [angstrom] resolution of one of the targets, glycerol kinase (GK), in complex with unphosphorylated [III.sup.Glc], glycerol, and adenosine diphosphate was determined. GK contains a region that is topologically identical to the adenosine triphosphate binding domains of hexokinase, the 70-kD heat shock cognate, and actin. [III.sup.Glc] binds far from the catalytic site of GK, indicating that long-range conformational changes mediate the inhibition of GK by [III.sup.Glc.] GK and [III.sup.Glc] are bound by hydrophobic and electrostatic interactions, with only one hydrogen bond involving an uncharged group. The phosphorylation site of [III.sup.Glc], [His.sup.90], is buried in a hydrophobic environment formed by the active site region [III.sup.Glc] and a [3.sup.10] helix of GK, suggesting that phosphorylation prevents [III.sup.Glc] binding to GK by directly disrupting protein-protein interactions., In Escherichia coli, the phosphoenolpyruvate:glycose phosphotransferase system (PTS) functions both as a transport pathway for its sugar substrates and as a negative regulator of the transport of several energy sources [...]

Classical electrostatic calculations are applied to the study of the origin of enzymatic rate enhancement of one class of biochemical carbon-carbon bond cleavage reactions. A model based on the three-dimensional structure of magnesium(II) isocitrate joined to isocitrate dehydrogenase is used for the charge distribution in the ground and transition states in the decarboxylation reaction.

The endosomal sorting complex required for transport (ESCRT) complexes sort ubiquitinated membrane proteins into multivesicular bodies, which is a key step in the lysosomal degradation pathway. Shields et al. (Shields, S.B., A.J. Oestreich, S. Winistorfer, D. Nguyen, J.A. Payne, D.J. Katzmann, and R. Piper. 2009. J. Cell Biol. 185:213-224) identify a new ubiquitin-binding site in ESCRT-I and provide evidence that the upstream ESCRT-I and -II complexes sort cargo in parallel rather than in series.

The Kvebaek Family Sculpture Technique (KFST) is a symbolic figure placement procedure for family assessment. Family members arrange blocks representing themselves on a standard grid to depict the emotional distance or closenss they feel toward each other. However, published evidence for covergent validity of the technique is scant. This study compared KFST emotional distance with cohesion measured by the Family Adaptability and Cohesion Evaluation Scale III (FACES III). Families (N = 31) having at least one adolescent and two parents participated. KFST distance was significantly related to FACES III cohesion, suggesting some covergent validity for the KFST. Potential uses of the KFST for family therapy are considered.

The structural and functional results presented provide a picture of the organization of the ESCRT-1 complex, which is the central to receptor downregulation, lysosome biogenesis, and budding of HIV. It is found that the fundamental unit of the ESCRT-1 complex is a 1:1:1 heterotrimer, which appears to be capable of forming weakly interacting dimers in vitro and higher order oligomers in vivo.

The CUE domain of the Vps9p sorting protein binds to monoubiquitin. The two binding surfaces of the CUE monomer are opposite to each other and cannot bind a ubiquitin molecule simultaneously. However, dimerization facilitates both sides to bind a monoubiquitin molecule.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2007.06.001 Byline: Thomas A. Leonard (1), James H. Hurley (1) Abstract: In this issue, report the structure of focal adhesion kinase (FAK) and reveal how FAK maintains an autoinhibited state. Together with the structure of another tyrosine kinase, ZAP-70 (), this work highlights the diversity of mechanisms that nature has evolved within the kinase superfamily to regulate their activity through autoinhibition. Author Affiliation: (1) Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0580, USA

The FYVE domains' interaction with the phosphatidylinositol three-phosphate membranes controls membrane trafficking and signalling pathways. Study of the FYVE domain structure revealed that this amino acid region is composed of two antiparallel beta sheets and an L helix stabilized by two Zn2+-binding clusters. It was also observed that the tip of the FYVE domain has basic and hydrophobic surfaces that promote nonspecific interactions with the phospholipid bilayer.