Conserved structural domains among species and tissues-specific differences in the mitochondrial phosphate-transport protein and the ADP/ATP carrier

Peptide maps were generated of the CNBr-digested mitochondrial phosphate-transport protein and ADP/ATP carrier from bovine and rat heart, rat liver and blowfly flight muscle. Total mitochondrial proteins from the same sources plus pig heart were separated by SDS-polyacrylamide gel electrophoresis. The peptide maps and the total mitochondrial proteins were electroblotted onto nitrocellulose membranes and reacted with rabbit antisera raised against the purified bovine heart phosphate-transport protein and the ADP/ATP carrier. On the basis of antibody specificity, mobility in SDS-polyacrylamide gel electrophoresis, and peptide maps the following was concluded. (1) Phosphate-transport protein and phosphate-transport protein β (pig and bovine heart) react equally with the first and also with the second of two independent phosphate-transport protein-antisera. (2) Tissue-specific structural domains exist for both the phosphate-transport protein and the ADP/ATP carrier, i.e., one phosphate-transport protein-antiserum reacts with the phosphate-transport protein from all assayed sources, the other only with the cardiac phosphate-transport protein. These differences may reflect tissue-specific regulation of phosphate and adenine nucleotide transport. (3) Homologies among the different species are found for the phosphate transport protein and the ADP/ATP carrier, except for the flight muscle ADP/ATP carrier. These conserved structural domains of the phosphate-transport protein may relate directly to catalytic activity. (4) Alkylation of the purified phosphate-transport proteins and the ADP/ATP carriers by the transport inhibitor N-ethylmaleimide affects electrophoretic mobilities but not the antibody binding. (5) Neither of the two phosphate-transport protein-antisera nor the ADP/ATP-carrier antiserum react with both phosphate transport protein and ADP/ATP carrier, even though these two proteins possess similarities in primary structure and function. Possible mechanisms for generating tissue-specific structural differences in the proteins are discussed.     

Conserved structural domains among species and tissues-specific differences in the mitochondrial phosphate-transport protein and the ADP/ATP carrier

Peptide maps were generated of the CNBr-digested mitochondrial phosphate-transport protein and ADP/ATP carrier from bovine and rat heart, rat liver and blowfly flight muscle. Total mitochondrial proteins from the same sources plus pig heart were separated by SDS-polyacrylamide gel electrophoresis. The peptide maps and the total mitochondrial proteins were electroblotted onto nitrocellulose membranes and reacted with rabbit antisera raised against the purified bovine heart phosphate-transport protein and the ADP/ATP carrier. On the basis of antibody specificity, mobility in SDS-polyacrylamide gel electrophoresis, and peptide maps the following was concluded. Phosphate-transport protein alpha and phosphate-transport protein beta (pig and bovine heart) react equally with the first and also with the second of two independent phosphate-transport protein-antisera. Tissue-specific structural domains exist for both the phosphate-transport protein and the ADP/ATP carrier, i.e., one phosphate-transport protein-antiserum reacts with the phosphate-transport protein from all assayed sources, the other only with the cardiac phosphate-transport protein. These differences may reflect tissue-specific regulation of phosphate and adenine nucleotide transport. Homologies among the different species are found for the phosphate transport protein and the ADP/ATP carrier, except for the flight muscle ADP/ATP carrier. These conserved structural domains of the phosphate-transport protein may relate directly to catalytic activity. Alkylation of the purified phosphate-transport proteins and the ADP/ATP carriers by the transport inhibitor N-ethylmaleimide affects electrophoretic mobilities but not the antibody binding. Neither of the two phosphate-transport protein-antisera nor the ADP/ATP-carrier antiserum react with both phosphate transport protein and ADP/ATP carrier, even though these two proteins possess similarities in primary structure and function. Possible mechanisms for generating tissue-specific structural differences in the proteins are discussed.     

Conserved protein domains are maintained in an average ratio to proteome size

Background  

Conserved domains (CD) in proteins play a crucial role in protein interactions, DNA binding, enzyme activity, and other important cellular processes. We proposed to study ratios of genes containing these domains to ratios of proteome size of different eukaryotes.     

Conserved domains of glycosyltransferases.

Glycosyltransferases catalyze the synthesis of glycoconjugates by transferring a properly activated sugar residue to an appropriate acceptor molecule or aglycone for chain initiation and elongation. The acceptor can be a lipid, a protein, a heterocyclic compound, or another carbohydrate residue. A catalytic reaction is believed to involve the recognition of both the donor and acceptor by suitable domains, as well as the catalytic site of the enzyme. To elucidate the structural requirements for substrate recognition and catalytic reactions of glycosyltransferases, we have searched the databases for homologous sequences, identified conserved amino acid residues, and proposed potential domain motifs for these enzymes. Depending on the configuration of the anomeric functional group of the glycosyl donor molecule and of the resulting glycoconjugate, all known glycosyltransferases can be divided into two major types: retaining glycosyltransferases, which transfer sugar residue with the retention of anomeric configuration, and inverting glycosyltransferases, which transfer sugar residue with the inversion of anomeric configuration. One conserved domain of the inverting glycosyltransferases identified in the database is responsible for the recognition of a pyrimidine nucleotide, which is either the UDP or the TDP portion of a donor sugar-nucleotide molecule. This domain is termed "Nucleotide Recognition Domain 1 beta," or NRD1 beta, since the type of nucleotide is the only common structure among the sugar donors and acceptors. NRD1 beta is present in 140 glycosyltransferases. The central portion of the NRD1 beta domain is very similar to the domain that is present in one family of retaining glycosyltransferases. This family is termed NRD1 alpha to designate the similarity and stereochemistry of sugar transfer, and it consists of 77 glycosyltransferases identified thus far. In the central portion there is a homologous region for these two families and this region probably has a catalytic function. A third conserved domain is found exclusively in membrane-bound glycosyltransferases and is termed NRD2; this domain is present in 98 glycosyltransferases. All three identified NRDs are present in archaebacterial, eubacterial, viral, and eukaryotic glycosyltransferases. The present article presents the alignment of conserved NRD domains and also presents a brief overview of the analyzed glycosyltransferases which comprise about 65% of all known sugar-nucleotide dependent (Leloir-type) and putative glycosyltransferases in different databases. A potential mechanism for the catalytic reaction is also proposed. This proposed mechanism should facilitate the design of experiments to elucidate the regulatory mechanisms of glycosylation reactions. Amino acid sequence information within the conserved domain may be utilized to design degenerate primers for identifying DNA encoding new glycosyltransferases.     

Conserved DNA binding and self-association domains of the Drosophila zeste protein.

The zeste gene product is involved in two types of genetic effects dependent on chromosome pairing: transvection and the zeste-white interaction. Comparison of the predicted amino acid sequence with that of the Drosophila virilis gene shows that several blocks of amino acid sequence have been very highly conserved. One of these regions corresponds to the DNA binding domain. Site-directed mutations in this region indicate that a sequence resembling that of the homeodomain DNA recognition helix is essential for DNA binding activity. The integrity of an amphipathic helical region is also essential for binding activity and is likely to be responsible for dimerization of the DNA binding domain. Another very strongly conserved domain of zeste is the C-terminal region, predicted to form a long helical structure with two sets of heptad repeats that constitute two long hydrophobic ridges at opposite ends and on opposite faces of the helix. We show that this domain is responsible for the extensive aggregation properties of zeste that are required for its role in transvection phenomena. A model is proposed according to which the hydrophobic ridges induce the formation of open-ended coiled-coil structures holding together many hundreds of zeste molecules and possibly anchoring these complexes to other nuclear structures.     

Conserved domains in polynucleotide phosphorylase among eubacteria

Polynucleotide phosphorylase (PNPase) is a polynucleotide nucleotidyl transferase (E. C. 2.7.7.8) that is involved in mRNA degradation in prokaryotes. PNPase structure analysis has been performed in Streptomyces antibioticus; this revealed the presence of five domains: two ribonuclease PH (RPH)-like (pnp1 and pnp2), one alpha helical, one KH, and one S1 domains. The trimeric nature of this enzyme was also confirmed. In this work, we have investigated conserved domains or subdomains in bacterial PNPases (55), for this structure-based sequence homology analysis between predicted amino acid sequences from bacterial PNPases and that of S. antibioticus was performed. Our findings indicate that while pnp2 (% similarity average S = 84/% identity average I = 22), KH (S = 74.3%/I = 5.3%), S1 (S = 71.3%/I = 1.2%); and pnp1 (S = 52.8%/I = 0.3%) domain; structure and sequence are well conserved among different bacteria, alpha helical domain (S = 39.5%/I = 0) although conservation of the structure is somewhat maintained, the sequence is not conserved at all. Implications of such findings in PNPase activity will be discussed.     

Sarcoplasmic reticulum: structural determinants and protein dynamics

The sarcoplasmic reticulum is a unique organelle found in muscle cells that is dedicated to the regulation of Ca(2+) homeostasis and activation of myofilament contraction. The functional requirement for an efficient and synchronous activation of Ca(2+) release from the SR, following the depolarization of the plasma membrane, accounts for the complex three-dimensional organization of internal membranes observed in muscle cells and for the localization of proteins at specific sites of the SR. Recent advancements in understanding the molecular basis of SR structure and function have greatly increased our understanding of muscle cellular physiology and biology. Parallel work has revealed that several human diseases affecting skeletal and cardiac tissues are linked to either mutations or altered post-translational modifications of SR proteins.     

Towards an automatic classification of protein structural domains based on structural similarity

Background  

Formal classification of a large collection of protein structures aids the understanding of evolutionary relationships among them. Classifications involving manual steps, such as SCOP and CATH, face the challenge of increasing volume of available structures. Automatic methods such as FSSP or Dali Domain Dictionary, yield divergent classifications, for reasons not yet fully investigated. One possible reason is that the pairwise similarity scores used in automatic classification do not adequately reflect the judgments made in manual classification. Another possibility is the difference between manual and automatic classification procedures. We explore the degree to which these two factors might affect the final classification.     

Scaffold-associated regions: cis-acting determinants of chromatin structural loops and functional domains.

It has been proposed that scaffold-associated regions are DNA elements that form the bases of chromatin loops in eukaryotic cells. Recent evidence supports a role for these elements as cis-acting 'handlers' of both structural and functional chromatin domains.     

3Dee: a database of protein structural domains

The 3Dee database is a repository of protein structural domains. It stores alternative domain definitions for the same protein, organises domains into sequence and structural hierarchies, contains non-redundant set(s) of sequences and structures, multiple structure alignments for families of domains, and allows previous versions of the database to be regenerated. AVAILABILITY: 3Dee is accessible on the World Wide Web at the URL http://barton.ebi.ac.uk/servers/3Dee.html.     

Conserved features of imprinted differentially methylated domains

Genomic imprinting is a conserved epigenetic phenomenon in eutherian mammals, with regards both to the genes that are imprinted and the mechanism underlying the expression of just one of the parental alleles. Epigenetic modifications of alleles of imprinted genes are established during oogenesis and spermatogenesis, and these modifications are then inherited. Differentially methylated domains (DMDs) of imprinted genes are the genomic sites of these inherited epigenetic imprints. We previously showed that CpG-rich imperfect tandem direct repeats within three different mouse DMDs (Snurf/Snrpn, Kcnq1 and Igf2r), each with a unique sequence, play a central role in maintaining the differential methylation. This finding implicates repeat-related DNA structure, not sequence, in the imprinting mechanism. To better define the important features of this signal, we compared sequences of these three DMD tandem repeats among mammalian species. All DMD repeats contain short indirect repeats, many of which are organized into larger unit repeats. Even though the larger repeat units undergo deletion and addition during evolution (most likely through unequal crossovers during meiosis), the size of DMD tandem repeated regions has remained remarkably stable during mammalian evolution. Moreover, all three DMD tandem repeats have a high-CpG content, an ordered arrangement of CpG dinucleotides, and similar predicted secondary structures. These observations suggest that a structural feature or features of these DMD tandem repeats is the conserved DMD imprinting signal.     

Enhanced recognition of protein transmembrane domains with prediction-based structural profiles

MOTIVATION: Membrane domain prediction has recently been re-evaluated by several groups, suggesting that the accuracy of existing methods is still rather limited. In this work, we revisit this problem and propose novel methods for prediction of alpha-helical as well as beta-sheet transmembrane (TM) domains. The new approach is based on a compact representation of an amino acid residue and its environment, which consists of predicted solvent accessibility and secondary structure of each amino acid. A recently introduced method for solvent accessibility prediction trained on a set of soluble proteins is used here to indicate segments of residues that are predicted not to be accessible to water and, therefore, may be 'buried' in the membrane. While evolutionary profiles in the form of a multiple alignment are used to derive these simple 'structural profiles', they are not used explicitly for the membrane domain prediction and the overall number of parameters in the model is significantly reduced. This offers the possibility of a more reliable estimation of the free parameters in the model with a limited number of experimentally resolved membrane protein structures. RESULTS: Using cross-validated training on available sets of structurally resolved and non-redundant alpha and beta membrane proteins, we demonstrate that membrane domain prediction methods based on such a compact representation outperform approaches that utilize explicitly evolutionary profiles and multiple alignments. Moreover, using an external evaluation by the TMH Benchmark server we show that our final prediction protocol for the TM helix prediction is competitive with the state-of-the-art methods, achieving per-residue accuracy of approximately 89% and per-segment accuracy of approximately 80% on the set of high resolution structures used by the TMH Benchmark server. At the same time the observed rates of confusion with signal peptides and globular proteins are the lowest among the tested methods. The new method is available online at http://minnou.cchmc.org.     

Conformational domains and structural transitions of human von Willebrand protein

The conformational states of human von Willebrand protein (vWF) were studied by using ultraviolet (UV) difference, circular dichroism, and fluorescence spectrophotometric techniques in order to gain insight into the forces that maintain its asymmetric, flexible shape. vWF has 24% alpha-helix and 18% beta-pleated sheet structure in the native state. Disulfide bond reduction and carboxamidation reduced the beta-pleated sheet content by 50% without affecting the content of alpha-helix. In addition, the quantum yield of intrinsic (tryptophan/tyrosine) fluorescence decreased by 33% after reduction and alkylation, and the affinity of the hydrophobic fluorescent probes 8-anilino-1-naphthalenesulfonate and 6-(p-toluidino)-2-naphthalenesulfonate for vWF was reduced 2.5-fold. In contrast, intrinsic fluorescence quenching by acrylamide and the UV difference spectrum did not change following reduction. An analysis of changes in the intrinsic fluorescence polarization and the emission maximum shift induced by thermal and guanidine hydrochloride denaturation revealed single, smooth transitions for both native and reduced vWF, suggesting the existence of an ordered structure in both species. This study shows that (1) disulfide reduction and carboxamidation cause significant conformational changes in vWF, (2) vWF may contain discrete, ordered, conformational domains linked by regions of random polypeptide chain, and (3) specific tertiary structural domains within vWF are not affected by disulfide reduction and carboxamidation. This structural model would explain both the asymmetry and flexibility of the molecule.     

Conserved structural and functional properties of D-domain containing redox-active and -inactive protein disulfide isomerase-related protein chaperones

The structure and mode of binding of the endoplasmic reticulum protein disulfide isomerase-related proteins to their substrates is currently a focus of intensive research. We have recently determined the crystal structure of the Drosophila melanogaster protein disulfide isomerase-related protein Wind and have described two essential substrate binding sites within the protein, one within the thioredoxin b-domain and another within the C-terminal D-domain. Although a mammalian ortholog of Wind (ERp29/28) is known, conflicting interpretations of its structure and putative function have been postulated. Here, we have provided evidence indicating that ERp29 is indeed similar in both structure and function to its Drosophila ortholog. Using a site-directed mutagenesis approach, we have demonstrated that homodimerization of the b-domains is significantly reduced in vitro upon replacement of key residues at the predicted dimerization interface. Investigation of Wind-ERp29 fusion constructs showed that mutants of the D-domain of ERp29 prevent transport of a substrate protein (Pipe) in a manner consistent with the presence of a discrete, conserved peptide binding site in the D-domain. Finally, we have highlighted the general applicability of these findings by showing that the D-domain of a redox-active disulfide isomerase, from the slime mold Dictyostelium discoideum, can also functionally replace the Wind D-domain in vivo.     

Conserved domains subserve novel mechanisms and functions in DKF-1, a Caenorhabditis elegans protein kinase D

Protein kinase D (PKD) isoforms are effectors in signaling pathways controlled by diacylglycerol. PKDs contain conserved diacylglycerol binding (C1a, C1b), pleckstrin homology (PH), and Ser/Thr kinase domains. However, the properties of conserved domains may vary within the context of distinct PKD polypeptides. Such functional/structural malleability (plasticity) was explored by studying Caenorhabditis elegans D kinase family-1 (DKF-1), a PKD that governs locomotion in vivo. Phorbol ester binding with C1b alone activates classical PKDs by relieving C1-mediated inhibition. In contrast, C1a avidly ligated phorbol 12-myristate 13-acetate (PMA) and anchored DKF-1 at the plasma membrane. C1b bound PMA (moderate affinity) and cooperated with C1a in targeting DKF-1 to membranes. Mutations at a "Pro(11)" position in C1 domains were inactivating; kinase activity was minimal at PMA concentrations that stimulated wild type DKF-1 approximately 10-fold. DKF-1 mutants exhibited unchanged, maximum kinase activity after cells were incubated with high PMA concentrations. Titration in situ revealed that translocation and activation of wild type and mutant DKF-1 were tightly and quantitatively linked at all PMA concentrations. Thus, C1 domains positively regulated phosphotransferase activity by docking DKF-1 with pools of activating lipid. A PH domain inhibits kinase activity in classical PKDs. The DKF-1 PH module neither inhibited catalytic activity nor bound phosphoinositides. Consequently, the PH module is an obligatory, positive regulator of DKF-1 activity that is compromised by mutation of Lys(298) or Trp(396). Phosphorylation of Thr(588) switched on DKF-1 kinase activity. Persistent phosphorylation of Thr(588) (activation loop) promoted ubiquitinylation and proteasome-mediated degradation of DKF-1. Each DKF-1 domain displayed novel properties indicative of functional malleability (plasticity).