CAFASP-1: Critical assessment of fully automated structure prediction methods

Fischer, Daniel; Barret, Christian; Bryson, Kevin; Elofsson, Arne; Godzik, Adam; Jones, David T.; Karplus, Kevin; Kelley, Lawrence A.; MacCallum, Robert M.; Pawowski, Krzysztof; Rost, Burkhard; Rychlewski, Leszek; Sternberg, Michael J.E.

doi:10.1002/(sici)1097-0134(1999)37:3+<209::aid-prot27>3.0.co;2-y

Cited by 123 publications

(43 citation statements)

References 18 publications

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“…The structure of mGluR1 was chosen as a template for the model building because running the sequences of GluR3-NTD and GluR6-NTD on several threading servers (such as SAM_T02 (14 -16), 3DPSSM (17,18), and mGenTHREADER (19,20)) indicated the mGluR1 structures as good candidates for serving as a template for GluR3-NTD and GluR6-NTD model building. The alignment was done using the procedure Align 2D of Modeler, giving identity values of 8.3 and 10% and similarity values of 21.8 and 26.8% for mGluR1 with GluR3-NTD and mGluR1 with GluR6-NTD, respectively.…”

Section: Methodsmentioning

confidence: 99%

Two Regions in the N-terminal Domain of Ionotropic Glutamate Receptor 3 Form the Subunit Oligomerization Interfaces That Control Subtype-specific Receptor Assembly

Ayalon¹,

Segev²,

Elgavish

et al. 2005

Journal of Biological Chemistry

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The N-terminal domain (NTD) of ␣-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) and kainate glutamate receptors plays an important role in controlling subtype specific receptor assembly. To identify NTD subdomains involved in this process we generated AMPA glutamate receptor 3 (GluR3) mutants having intra-NTD substitutions with the corresponding regions of the kainate receptor GluR6 and tested their ability to form functional heteromers with wild-type subunits. The chimeric design was based on the homology of the NTD to the NTD of the metabotropic GluR1, shown to form two globular lobes and to assemble in dimers. Accordingly, the NTD was divided into four regions, termed here N1-N4, of which N1 and N3 correspond to the regions forming lobe-1 and N2 and N4 to those forming lobe-2. Substituting N1 or N3 impaired functional heteromerization but allowed protein-protein interactions. Conversely, exchanging N2 or N4 preserved functional heteromerization, although it significantly decreased homomeric activity, indicating a role in subunit folding. Moreover, a deletion in GluR3 corresponding to the hotfoot mouse mutation of the glutamate receptor ␦2, covering part of N2, N3, and N4, impaired both homomeric and heteromeric oligomerization, thus explaining the null-like mouse phenotype. Finally, computer modeling suggested that the dimer interface, largely formed by N1, is highly hydrophobic in GluR3, whereas in GluR6 it contains electrostatic interactions, hence offering an explanation for the subtype assembly specificity conferred by this region. N3, however, is positioned perpendicular to the dimer interface and therefore may be involved in secondary interactions between dimers in the assembled tetrameric receptor.Glutamate, the major excitatory neurotransmitter, activates two receptor families: metabotropic glutamate receptors (mGluRs), 1 which are coupled to G-proteins, and ionotropic glutamate receptors (iGluRs), which form cation selective ion channels. The iGluRs are further divided into three subtypes: AMPA, kainate, and NMDA receptors, originally named after their most selective agonist (1). Each subtype, in turn, consists of several subunits that assemble as tetramers in various combinations to form channels with distinct properties (2). Of the cloned subunits, GluR1-4 form the AMPA receptors, GluR5-7 and KA1-2 form the kainate receptors, and NR1, NR2A-D, and NR3A-B form the NMDA receptors. The ␦1-2 subunits constitute an orphan group of iGluRs, and their relation to the three principal subtypes is unclear.Overall, all iGluR subunits have a common domain organization (3). Each subunit contains four membrane regions (M1-M4), of which M1, M3, and M4 traverse the plasma membrane and M2 is a reentrant loop aligning the channel pore. The N terminus is extracellular and comprises an ϳ400-residue Nterminal domain (NTD; also termed "X domain"), which is homologous to bacterial periplasmic amino acid-binding proteins and to the N-terminal domain of mGluRs (4). Following the NTD is S1, an ϳ150-residue segment, which ...

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Section: Methodsmentioning

confidence: 99%

Two Regions in the N-terminal Domain of Ionotropic Glutamate Receptor 3 Form the Subunit Oligomerization Interfaces That Control Subtype-specific Receptor Assembly

Ayalon¹,

Segev²,

Elgavish

et al. 2005

Journal of Biological Chemistry

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“…More than 80% sequence similarities are shown in dark gray; Ͼ60% sequence similarities are shown in light gray. Sequences showing local similarities (motifs I, II/Y, IV, and V) to methyltransferases are boxed, using the 3D-PSSM web-based method (27,28).…”

Section: Methodsmentioning

confidence: 99%

Mutational Analysis of the N-Methyltransferase Domain of the Multifunctional Enzyme Enniatin Synthetase

Hacker

Glinski

Hornbogen

et al. 2000

Journal of Biological Chemistry

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N-Methylated peptides like cyclosporins and enniatins constitute a class of pharmacologically interesting compounds. They are synthesized by a special class of enzymes representing hybrid systems of peptide synthetases and integrated Nmethyltransferase domains (1).Like other peptide synthetases, N-methylcyclopeptide synthetases follow a so-called thiol template mechanism, in which the substrate amino acids are activated as thioesters mediated by enzyme-bound 4Ј-phosphopantetheine residues (1-3). Enniatin synthetase was the first N-methylcyclopeptide synthetase to characterized (4). Sequencing of the enniatin synthetase-corresponding gene (esyn1) from Fusarium scirpi revealed that the enzyme is one single polypeptide chain of 347 kDa (5). It consists of the two modules EA and EB containing the two catalytic binding sites for the substrates D-hydroxyisovaleric acid and the branched-chain L-amino acid, respectively (5). The 55-kDa N-methyltransferase portion M of the enzyme is located within the EB module. N-Methylation takes place after covalent binding of the amino acid on the surface of the corresponding peptide synthetase prior to peptide bond formation (4, 6). S-Adenosyl-L-methionine (AdoMet) 1 serves as the methyl donor (4, 7). The mechanism of formation of N-methylated peptides has been elucidated in the case of the cyclodepsipeptides enniatin (8), beauvericin (9), and cyclosporin (10) and in actinomycin biosynthesis (11).Biochemical investigations of the N-methyltransferase function of enniatin synthetase (4) revealed that, similar to other methyltransferases, S-adenosyl-L-homocysteine (AdoHcy) and sinefungin are potent inhibitors of the AdoMet-dependent reaction. Sinefungin acted as a competitive inhibitor with respect to AdoMet, whereas AdoHcy exhibited an inhibition pattern characteristic for a partial competitive inhibitor, suggesting a discrete binding site for this inhibitor. Like other methyltransferases, the N-methyltransferase domain of enniatin synthetase can be affinity-labeled by UV irradiation in the presence of AdoMet labeled at the methyl group (4). The photoreaction was shown to be site-specific, and a binding stoichiometry of one methyl group/enzyme molecule was observed (4). It could be shown that AdoHcy diminished photolabeling of enniatin synthetase with [methyl-14 C]AdoMet or [methyl-3 H]AdoMet; but even in the presence of excess AdoHcy (100 M), it was not able to totally prevent the photoreaction, as did sinefungin; in contrast, the enzyme-bound radioactivity reached a reduced but constant level of 40% of the uninhibited control (4). This indicates that AdoHcy does not directly compete with AdoMet, but binds to a discrete inhibitory site. AdoHcy reduces the affinity of AdoMet for the enzyme, but, even at infinite high inhibitor concentrations, allows formation of an enzyme⅐AdoMet⅐AdoHcy complex, which still yields product. Furthermore, neither sinefungin nor AdoHcy affected substrate activation (adenylation and subsequent thioacylation). Interestingly, AdoHcy inhibited the formation of...

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“…The 3D-PSSM web-based homology server (16,17) identified the Der p 2 dust mite allergen as an excellent template for modeling the NPC2 protein structure (E value 7.14e-13) based on multiple sequence alignment, secondary structure prediction, and solvation potential. The Der p 2 structure is a ␤ sandwich, and all six cysteine residues involved in disulfides are conserved between the two proteins.…”

Section: Purification Of Npc2mentioning

confidence: 99%

The integrity of a cholesterol-binding pocket in Niemann–Pick C2 protein is necessary to control lysosome cholesterol levels

Binkley

Sidow

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

182

196

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The neurodegenerative disease Niemann-Pick Type C2 (NPC2) results from mutations in the NPC2 (HE1) gene that cause abnormally high cholesterol accumulation in cells. We find that purified NPC2, a secreted soluble protein, binds cholesterol specifically with a much higher affinity (K d ‫؍‬ 30 -50 nM) than previously reported. Genetic and biochemical studies identified single amino acid changes that prevent both cholesterol binding and the restoration of normal cholesterol levels in mutant cells. The amino acids that affect cholesterol binding surround a hydrophobic pocket in the NPC2 protein structure, identifying a candidate sterol-binding location. On the basis of evolutionary analysis and mutagenesis, three other regions of the NPC2 protein emerged as important, including one required for efficient secretion.point mutants ͉ secretion ͉ protein evolution N iemann-Pick Type C (NPC) is an autosomal-recessive disorder characterized by progressive ataxia, dystonia, and dementia (1). Substantial cell death, particularly in the cerebellum, accounts for some of the symptoms. At the cellular level, mutant cells accumulate cholesterol and other lipids in aberrant compartments with features of late endosomes and lysosomes, and the normal homeostatic response to this excess cholesterol is abolished (2-4).Our understanding of the molecular basis of this disease has progressed substantially over the last several years, because the two genes damaged by NPC mutations, NPC1 and NPC2, have been identified (5-7). NPC1 encodes a multiple-membranespanning protein containing sequences similar to the sterol cleavage activating protein (SCAP) regulator of cholesterol metabolism and the receptor Patched, a transducer of the Hedgehog family of protein signals. Mutations in NPC1 are responsible for Ϸ95% of the NPC cases (8). The exact role that NPC1 plays in maintaining normal levels of cholesterol in late endosomes and lysosomes remains mysterious. Even less is known about the NPC2 [human epididymis (HE1)] protein, the loss of which is responsible for 5% of NPC cases.NPC2 is a small secreted glycoprotein originally identified as a transcript enriched in the HE1 (9). The porcine HE1 homolog was reported to bind cholesterol with micromolar affinity (K d ϭ 2.3 M; ref. 10), information that was crucial in identifying HE1 as a candidate npc2 gene product (7). However, the similarity between the reported K d and the solubility of cholesterol in aqueous solution (5 M, Merck Index) warrants reexamination of the strength and specificity of this interaction. If the binding is meaningful, NPC2 activity could be altered when cholesterol is bound, thus serving as a sensor of cholesterol levels, accessibility, or location. Alternatively, NPC2 could process or transport cholesterol. Resolving these questions is crucial for determining why cholesterol accumulates in people lacking functional NPC2 protein and for designing rational treatment strategies.In this report, we combine in vitro binding experiments, genetic analysis, a quantitative cell-based ...

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CAFASP-1: Critical assessment of fully automated structure prediction methods

Cited by 123 publications

References 18 publications

Two Regions in the N-terminal Domain of Ionotropic Glutamate Receptor 3 Form the Subunit Oligomerization Interfaces That Control Subtype-specific Receptor Assembly

Two Regions in the N-terminal Domain of Ionotropic Glutamate Receptor 3 Form the Subunit Oligomerization Interfaces That Control Subtype-specific Receptor Assembly

Mutational Analysis of the N-Methyltransferase Domain of the Multifunctional Enzyme Enniatin Synthetase

The integrity of a cholesterol-binding pocket in Niemann–Pick C2 protein is necessary to control lysosome cholesterol levels

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