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At least 667 records · Page 37Linked to original sources

PubMed2005

A structural model for the membrane-bound form of the juxtamembrane domain of the epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family involved in the regulation of cellular proliferation and differentiation. Its juxtamembrane domain (JX), the region located between the transmembrane and kinase domains, plays important roles in receptor trafficking. Two sorting signals, a PXXP motif and a 658LL659 motif, are responsible for basolateral sorting in polarized epithelial cells, and a 679LL680 motif targets the ligand-activated receptor for lysosomal degradation. To understand the regulation of these signals, we characterized the structural properties of recombinant JX domain in aqueous solution and in dodecylphosphocholine (DPC) detergent. JX is inherently unstructured in aqueous solution, albeit a nascent helix encompasses the lysosomal sorting signal. In DPC micelles, structures derived from NMR data showed three amphipathic, helical segments. A large, internally inconsistent group of long range nuclear Overhauser effects suggest a close proximity of the helices, and the presence of significant conformational averaging. Models were determined for the average JX conformation using restraints representing the translational restriction due to micelle-surface adsorption, and the helix orientations were determined from residual dipolar couplings. Two equivalent average structural models were obtained that differ only in the relative orientation between first and second helices. In these models, the 658LL659 and 679LL680 motifs are located in the first and second helices and face the micelle surface, whereas the PXXP motif is located in a flexible helix-connecting region. The data suggest that the activity of these signals may be regulated by their membrane association and restricted accessibility in the intact receptor.

Amino Acid Sequence↗

PubMed2005

Characterization of disulfide bonds in human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3): implications for NTPDase structural modeling.

Cell-surface nucleotidases (NTPDases) contain 10 invariant cysteine residues in their extracellular regions. To investigate disulfide structure in human NTPDase3, we made single and double mutants of these 10 cysteines, and analyzed their enzymatic activity, glycosylation pattern, trafficking to the cell membrane, and sensitivity to reduction. The mutants constituted five distinct phenotypes, thus, strongly suggesting disulfide bonds between C92-C116 (first bond), C261-C308 (second bond), C289-C334 (third bond), C347-C353 (fourth bond), and C399-C422 (fifth bond). Due to conservation of the 10 cysteines, the identified five disulfide bonds are likely to exist in all cell-surface NTPDases. The third and fifth bonds are also present in the soluble NTPDases and are critical for processing, trafficking, and enzymatic activity. The fourth bond has minimal effect on processing and function, while the first and second bonds are of intermediate importance. Most of the N-linked glycosylation sites in the wild-type enzyme are processed to complex oligosaccharides, but at least one site is high-mannose or hybrid in structure. Interestingly, disruption of the first disulfide bond resulted in some enzyme that lost sensitivity to endoglycosidase H, suggesting that the first disulfide bond in the wild-type enzyme shields some high-mannose glycans from terminal glycosylation. Comparative modeling by threading and homology modeling of the NTPDase3 sequence revealed a high degree of structural fold similarity with a bacterial exopolyphosphatase (PDB ). The resultant theoretical 3-D model of the extracellular portion of NTPDase3, based on homology with this exopolyphosphatase, is consistent with the assignment of the disulfide bonds occurring in regions of good fold similarity between NTPDase3 and the exopolyphosphatase. The 3-D model obtained for NTPDase3 also suggests the structural basis for the importance of several apyrase conserved regions for the nucleotidase activities of the NTPDases.

Amino Acid Sequence↗

PubMed1992

Activity, disulphide mapping and structural modelling of the fifth domain of human beta 2-glycoprotein I.

Complexes formed by the interaction of negatively charged phospholipids and beta 2-glycoprotein I (beta 2-I) are the target of autoantibodies in systemic lupus erythematosus. The highly positively charged fifth (C-terminal) domain of human beta 2-I was produced as a fusion protein in an Escherichia coli expression system and was shown to bind to the negatively charged phospholipid, cardiolipin, almost as well as the intact protein. In an attempt to define the 3D structure of this domain, the disulphide linkage pattern was determined and shown to be Cys 1-4, Cys 2-5 and Cys 3-6 in contradiction to an earlier report. In the light of this information, the sequence of the fifth domain of beta 2 I (beta 2-I-5) is readily aligned with that of the 16th repeat of factor H, of which the 3D structure is known, and a model of beta 2I-5 has been built by homology. On the basis of the model we suggest residues that might be the target of profitable site-directed mutagenesis in structure-function studies.

Amino Acid Sequence↗

PubMed2006

Putting Wilson and Cleary to the test: analysis of a HRQOL conceptual model using structural equation modeling.

Wilson and Cleary (1995) proposed a conceptual model of health-related quality of life (HRQOL) that integrates both biological and psychological aspects of health outcomes. There are five different levels in their model, namely, physiological factors, symptom status, functional health, general health perceptions, and overall quality of life. Their model has been widely applied to different populations, including patients living with cancer, Parkinson's disease, arthritis, and HIV+/AIDS. However, their conceptual model has only been partially examined. That is, the five major concepts have not been examined simultaneously. Using structural equation modeling (SEM), the Wilson and Cleary HRQOL model was validated in patients living with HIV from the AIDS Time-Oriented Health Outcomes Study. The results showed that the HRQOL model fit the data adequately, and the relationships between the constructs were all significant (at p<0.05 level). Based on the modification indexes, an alternative model linking symptom status directly with general health perceptions and overall quality of life was specified. Implication and limitation of the findings are discussed.

Adult↗

PubMed2006

Semiparametric maximum likelihood variance component estimation using mixture moment structure models.

Nonnormal phenotypic distributions introduce significant problems in the estimation and selection of genetic models. Here, a semiparametric maximum likelihood approach to analyzing nonnormal phenotypes is described. In this approach, distributions are explicitly modeled together with genetic and environmental effects. Distributional parameters are introduced through mixture constraints, where the distribution of effects are discretized and freely estimated rather than assumed to be normal. Semiparametric maximum likelihood estimation can be used with a variety of genetic models, can be extended to a variety of pedigree structures, and has various advantages over other approaches to modeling nonnormal data.

Humans↗

PubMed1994

A new kind of immobilized lipase in organic solvent and its structure model.

In this paper, we used Ca-alginate gel beads coated with polyetheneimine and glutaraldehyde to adsorb Expansum penicillium lipase. The immobilized lipase catalyzed esterification of 1-dodecanol with dodecanoic acid in benzene. The results show that when the concentration of Ca-alginate, polyetheneimine (PEI) and glutaraldehyde is 1%, 6% and 1%, respectively, the activity of the immobilized lipase and the amount of adsorbed protein are the highest. The immobilized lipase is better than the SDS-immobilized lipase. The activity of the immobilized lipase connected by glutaraldehyde is higher than the activity of that without glutaraldehyde. The initial rate of the immobilized lipase and lyophilized lipase powder is 5.9 x 10(2) nmol/min.mgpr and 2.8 x 10(1) nmol/min.mgpr, respectively. After the immobilized lipase catalyzed the esterification reaction at 37 degrees C for about 12 hours, 93.3% of 1-dodecanol was converted to ester, but for lyophilized lipase powder, only 17.5% converted. Based on all above results, we have presumed and explained the structure of this kind of immobilized lipase.

Alginates↗

PubMed2006

A structural model reveals energy transduction in dynein.

Intracellular active transport is driven by ATP-hydrolyzing motor proteins that move along cytoskeletal filaments. In particular, the microtubule-associated dynein motor is involved in the transport of organelles and vesicles, the maintenance of the Golgi, and mitosis. However, unlike kinesin and myosin, the mechanism by which dynein converts chemical energy into mechanical force remains largely a mystery, due primarily to the lack of a high-resolution molecular structure. Using homology modeling and normal mode analysis, we propose a complete atomic structure and a mechanism for force generation by the motor protein dynein. In agreement with very recent electron microscopy (EM) reconstructions showing dynein as a ring-shaped heptamer, our model consists of six ATPases of the AAA (ATPases associated with various cellular activities) superfamily and a C-terminal domain, which is experimentally known to control motor function. Our model shows a coiled coil spanning the diameter of the motor that accounts for previously unidentified structures in EM studies and provides a potential mechanism for long-range communication between the AAA domains. Furthermore, normal mode analysis reveals that the subunits of the motor that contain the nucleotide binding sites exhibit minimal movement, whereas the rest of the motor is very mobile. Our analysis suggests the likely domain rearrangements of the motor unit that generate its power stroke. This study provides insights into the structure and function of dynein that can guide further experimental investigations into energy transduction in dynein.

Amino Acid Sequence↗

PubMed2003

Chromosome location, genomic organization of the porcine COL10A1 gene and model structure of the NC1 domain.

The porcine COL10A1 gene, encoding the alpha1(X) chain of type X collagen, has been sequenced. The gene structure is evolutionarily conserved, consisting of three exons and two introns spanning 7100 bp. Linkage mapping localized the gene to chromosome 1, which is in agreement with human-pig homology maps. Furthermore, protein structure comparison of the functionally important carboxyl domain between species revealed that amino acid changes were few and mainly situated in loop regions.

Animals↗

PubMed1996

Secondary structure model of the coat protein gene of turnip yellow mosaic virus RNA: long, C-rich, single-stranded regions.

The RNA of all tymoviruses, a group of ssRNA plant viruses, has a base composition that is different from that of most other viruses. The excess of cytosines (35-42%) and the low number of guanosines (15-17%) must impel an RNA structure with a relatively low amount of base pairing and a high incidence of unpaired cytosines. These unpaired cytosines probably function in RNA-protein interactions. To gain Insight into the way the RNA is positioned inside the virion, the secondary structure has been determined of a part of TYMV RNA, including the so-called tymobox, the coat protein gene, and the 3' untranslated region, by structure probing, sequence comparison, and computer predictions. Conservation of secondary structure elements in tymoviruses is not high and does not parallel the conservation of the primary structure. A combination of structure prediction and probing experiments, however, results in a model consisting of structured domains of 100-200 nucleotides interspersed by long unpaired cytosine-rich regions. The latter may interact with the coat protein inside the virion. The structure of some functionally interesting regions of the 3' part of TYMV RNA is also discussed.

Base Sequence↗

PubMed2005

Structural model of human GAD65: prediction and interpretation of biochemical and immunogenic features.

The 65 kDa human isoform of glutamate decarboxylase, GAD65, plays a central role in neurotransmission in higher vertebrates and is a typical autoantigen in several human autoimmune diseases, such as insulin-dependent diabetes mellitus (IDDM), Stiff-man syndrome and autoimmune polyendocrine syndrome type I. In autoimmune diabetes, an attack of inflammatory cells to endocrine pancreatic beta-cells leads to their complete destruction, eventually resulting in the inability to produce sufficient insulin for the body's requirements. Even though the etiology of beta-cell destruction is still a matter of debate, the role and antigenic potency of GAD65 are widely recognized. Herein a model of GAD65 is presented, which is based on the recently solved crystal structures of mammalian DOPA decarboxylase and of bacterial glutamate decarboxylase. The model provides for the first time a detailed and accurate structure of the GAD65 subunit (all three domains) and of its dimeric quaternary assembly. It reveals the structural basis for specific antibody recognition to GAD65 as opposed to GAD67, the other human isoform, which shares 81% sequence similarity with GAD65 and is much less antigenic. Literature data on monoclonal antibody binding are perfectly consistent with the detailed features of the model, which allows explanation of several findings on GAD65 immunogenicity. Importantly, by analyzing the active site, we identified the residues most likely involved in catalysis and substrate recognition, paving the way for rational mutagenesis studies of the GAD65 reaction mechanism, specificity and inhibition.

Animals↗

PubMed1994

The effect of residual strain on the diastolic function of the left ventricle as predicted by a structural model.

The unloaded heart is not stress-free. It is subjected to residual stress and strain. Their extent and influence on the global performance of the left ventricle and on local phenomena in the ventricular wall are studied by model simulation. The analysis focuses on the equatorial region of the ventricle, with an approximate thick-walled cylindrical geometry. The in vivo myocardium is considered to be incompressible, consisting of fibers embedded in a fluid matrix, with transmurally varying anisotropic microstructure in accordance with morphological characteristics. The results show that residual strain is transmurally distributed with a pattern and magnitude which agree well with measurements. The calculated residual strains are within mean +/- one standard deviation of the measured ones. Their magnitude was found to increase with increasing opening angle and with increasing wall thickness. The residual strain was found to have several effects on ventricular function: At volumes higher than the reference one it gives rise to more uniform transmural distributions of stress and intramyocardial pressure; it causes about 50% increase in the ventricular compliance at high volumes and doubles the suction of atrial blood at low volumes, thus facilitating the diastolic filling. In addition, residual strains cause bias of in vivo measured strains from their true values. This may significantly affect physiological interpretation of measured ventricular deformations. In conclusion, the present structural analysis predicts that residual strain has favorable effect on left-ventricular diastolic performance, and gives rise to more uniform ventricular stress distribution.

Atrial Function, Left↗

PubMed1993

Three-dimensional structural model of the serine receptor ligand-binding domain.

Computer-based homology modeling techniques were used to construct a three-dimensional model of the Escherichia coli serine receptor ligand-binding domain based on the crystal structure of the Salmonella typhimurium aspartate receptor and the sequence homology between the two receptors. Residues that have been found in mutagenesis studies to be necessary for serine binding are located in a proposed serine-binding site. Several other mutations that affect swimming behavior require relatively small shifts in alpha-carbon positions in the model to give a minimized structure, suggesting that small changes in receptor conformation can affect the signaling state of the receptor.

Amino Acid Sequence↗

PubMed1999

A preliminary comparison of structural models for catalytic intermediates of acetylcholinesterase.

Determination of the three dimensional structure of Torpedo Californica acetylcholinesterase (TcAChE) provided an experimental tool for directly visualizing interaction of AChE with cholinesterase inhibitors of fundamental, pharmacological and toxicological interest. The structure revealed that the active site is located near the bottom of a deep and narrow gorge lined with 14 conserved aromatic amino acids. The structure of a complex of TcAChE with the powerful 'transition state analog' inhibitor, TMTFA, suggested that its orientation in the experimentally determined structure was very similar to that proposed for the natural substrate, acetylcholine, by manual docking. The array of enzyme-ligand interactions visualized in the TMFTA complex also are expected to envelope the unstable TI that forms with acetylcholine during acylation, and to sequester it from solvent. In our most recent studies, the crystal structures of several 'aged' conjugates of TcAChE obtained with OP nerve agents have been solved and compared with that of the native enzyme. The methylphosphonylated-enzyme obtained by reaction with soman provides a useful structural analog for the TI that forms during deacylation after the reaction of TcAChE with acetylcholine. By comparing these structures, we conclude that the same 'oxyanion hole' residues, as well as the aromatic side chains constituting the 'acyl pocket', participate in acylation (TMTFA-AChE) and deacylation (OP-AChE), and that AChE can accommodate both TIs at the bottom of the gorge without major conformational movements.

Acetylcholinesterase↗

PubMed1997

RNA recognition by the joint action of two nucleolin RNA-binding domains: genetic analysis and structural modeling.

The interaction of nucleolin with a short stem-loop structure (NRE) requires two contiguous RNA-binding domains (RBD 1+2). The structural basis for RNA recognition by these RBDs was studied using a genetic system in Escherichia coli. Within each of the two domains, we identified several mutations that severely impair interaction with the RNA target. Mutations that alter RNA-binding specificity were also isolated, suggesting the identity of specific contacts between RBD 1+2 amino acids and nucleotides within the NRE stem-loop. Our data indicate that both RBDs participate in a joint interaction with the NRE and that each domain uses a different surface to contact the RNA. The constraints provided by these genetic data and previous mutational studies have enabled us to propose a three-dimensional model of nucleolin RBD 1+2 bound to the NRE stem-loop.

Amino Acid Sequence↗

PubMed2002

Structured model to predict intracellular amino acid shortages during recombinant protein overexpression in E. coli.

Recombinant protein overexpression and the classical stringent response have been shown to induce the same proteases. Since the stringent response was the result of an intracellular amino acid shortage, it was hypothesized that the overexpression of the recombinant protein also caused an intracellular amino acid shortage. A structured non-segregated kinetic mathematical model of recombinant Escherichia coli was developed to predict intracellular amino acid shortages during recombinant protein overexpression, and thus the induction of the stringent response. Two model recombinant proteins were examined, chloramphenicol acetyl-transferase (CAT) and an 'average protein'. The model predicted an aromatic amino acid shortage during CAT overexpression, as predicted based on the CAT's amino acid content. The model also predicted a shortage of the intracellular alanine family amino acid pool during CAT overexpression. This was unexpected due to the relatively low content of alanine family amino acids in CAT compared to the average E. coli protein. The model predicted alanine, glutarate, and aspartate family amino acid shortages during recombinant 'average protein' overexpression. Additionally, the model predicted a decrease in the ribosome pool at induction for both recombinant proteins, which agrees with published experimental results. Thus, the structured kinetic model was able to predict amino acid shortages, that could potentially cause a stringent response and elevated protease activity.

Amino Acids↗

PubMed2001

Evaluation of a structural model of Pseudomonas aeruginosa outer membrane protein OprM, an efflux component involved in intrinsic antibiotic resistance.

The outer membrane protein OprM of Pseudomonas aeruginosa is involved in intrinsic and mutational multiple-antibiotic resistance as part of two resistance-nodulation-division efflux systems. The crystal structure of TolC, a homologous protein in Escherichia coli, was recently published (V. Koronakis, A. Sharff, E. Koronakis, B. Luisl, and C. Hughes, Nature 405:914-919, 2000), demonstrating a distinctive architecture comprising outer membrane beta-barrel and periplasmic helical-barrel structures, which assemble differently from the common beta-barrel-only conformation of porins. Based on their sequence similarity, a similar content of alpha-helical and beta-sheet structure determined by circular dichroism spectroscopy, and our observation that OprM, like TolC, reconstitutes channels in planar bilayer membranes, OprM and TolC were considered to be structurally homologous, and a model of OprM was constructed by threading its sequence to the TolC crystal structure. Residues thought to be important for the TolC structure were conserved in space in this OprM model. Analyses of deletion mutants and previously isolated insertion mutants of OprM in the context of this model allowed us to propose roles for different protein domains. Our data indicate that the helical barrel of the protein is critical for both the function and the integrity of the protein, while a C-terminal domain localized around the equatorial plane of this helical barrel is dispensable. Extracellular loops appear to play a lesser role in substrate specificity for this efflux protein compared to classical porins, and there appears to be a correlation between the change in antimicrobial activity for OprM mutants and the pore size. Our model and channel formation studies support the "iris" mechanism of action for TolC and permit us now to form more focused hypotheses about the functional domains of OprM and its related family of efflux proteins.

Amino Acid Sequence↗

PubMed2002

High-spin Ni(II), a surprisingly good structural model for [NiFe] hydrogenase.

The first density functional calculations on high-spin (HS) Ni(II) models for the active site of the [NiFe] hydrogenases predict a ligand arrangement about Ni that is in better agreement with the crystal structures than previous predictions for low-spin (LS) Ni(II) models. With the crystal structures' geometry, the HS form is approximately 20 kcal/mol lower in energy than the LS one.

Binding Sites↗

PubMed1994

The de novo design of an antibody combining site. Crystallographic analysis of the VL domain confirms the structural model.

In a protein design approach the molecular model of an artificial antibody Fv fragment was generated with predicted complementarity to part of the known crystal structure of chicken egg-white cystatin. The model of the Fv fragment was based on the three-dimensional structure of the anti-lysozyme antibody HyHEL-10, which was modified by substituting amino acid side-chains in the complementarity-determining regions (CDRs) as well as the framework without altering the backbone. In the course of crystallization experiments with the bacterially produced Fv fragment crystals of the VL domain alone were obtained. These crystals diffracted X-rays to a resolution of 2.17 A and were shown to belong to the space group P2(1)2(1)2(1) with unit cell dimensions a = 46.89 A, b = 58.05 A, c = 83.22 A containing two VL monomers in the asymmetric unit. The crystal structure was solved by molecular replacement and refined to a crystallographic R-factor of 17.5%. The two VL monomers exhibit an asymmetric mode of association, which is different from other crystallized VL domains described before and shows the peculiar feature of an isopropanol precipitant molecule buried at the interface. Both VL structures reveal a high level of similarity to the predicted three-dimensional model. With the exception of two loop segments in the framework region that are involved in crystal packing contacts, the backbone structures of the two VL monomers in the crystal and the molecular model of the VL domain are practically identical. Although six amino acid residues had been replaced in the hypervariable regions, the CDR conformations remained conserved and only minor deviations in the orientation of some side-chains and peptide planes were detected. The crystallographic analysis of the VL domain modelled as part of a complex between an artificial Fv fragment and the small protein cystatin, deliberately chosen as antigen target, confirms the concept of distinct structural classes for CDR backbones and supports our strategy for the de novo design of an antibody combining site.

Amino Acid Sequence↗