The evolutionary origins of eukaryotic protein disulfide isomerase domains: new evidence from the Amitochondriate protist Giardia lamblia

A phylogenetic analysis of protein disulfide isomerase (PDI) domain evolution was performed with the inclusion of recently reported PDIs from the amitochondriate protist Giardia lamblia, yeast PDIs that contain a single thioredoxin-like domain, and PDIs from a diverse selection of protists. We additionally report and include two new giardial PDIs, each with a single thioredoxin-like domain. Inclusion of protist PDIs in our analyses revealed that the evolutionary history of the endoplasmic reticulum may not be simple. Phylogenetic analyses support common ancestry of all eukaryotic PDIs from a thioredoxin ancestor and independent duplications of thioredoxin-like domains within PDIs throughout eukaryote evolution. This was particularly evident for Acanthamoeba PDI, Dictyostelium PDI, and mammalian erp5 domains. In contrast, gene duplication, instead of domain duplication, produces PDI diversity in G. lamblia. Based on our results and the known diversity of PDIs, we present a new hypothesis that the five single-domain PDIs of G. lamblia may reflect an ancestral mechanism of protein folding in the eukaryotic endoplasmic reticulum. The PDI complement of G. lamblia and yeast suggests that a combination of PDIs may be used as a redox chain analogous to that known for bacterial Dsb proteins.     

Structural classification of thioredoxin-like fold proteins

Protein structure classification is necessary to comprehend the rapidly growing structural data for better understanding of protein evolution and sequence-structure-function relationships. Thioredoxins are important proteins that ubiquitously regulate cellular redox status and various other crucial functions. We define the thioredoxin-like fold using the structure consensus of thioredoxin homologs and consider all circular permutations of the fold. The search for thioredoxin-like fold proteins in the PDB database identified 723 protein domains. These domains are grouped into eleven evolutionary families based on combined sequence, structural, and functional evidence. Analysis of the protein-ligand structure complexes reveals two major active site locations for the thioredoxin-like proteins. Comparison to existing structure classifications reveals that our thioredoxin-like fold group is broader and more inclusive, unifying proteins from five SCOP folds, five CATH topologies and seven DALI domain dictionary globular folding topologies. Considering these structurally similar domains together sheds new light on the relationships between sequence, structure, function and evolution of thioredoxins.     

Nucleotide sequence of phospholipase A(2) gene expressed in snake pancreas reveals the molecular evolution of toxic phospholipase A(2) genes

A protein disulfide isomerase (PDI) coding sequence was cloned from a cDNA library derived from carrot (Daucus carota L.) somatic embryos. The cDNA is 2060 bp in length and encodes for a protein of 581 amino acids and molecular weight of 64.4 kDa. Primary structure analysis of the deduced protein revealed two thioredoxin-like active sites and an endoplasmic reticulum-retention signal at its C-terminus, which is also found in PDIs in plants and animals. Although between the carrot protein and other plant PDIs there is only about 30% identity, the active site regions are almost identical. The corresponding mRNA was found in varying amounts, in all tissues investigated. A recombinant protein expressed from the carrot cDNA clone effectively catalyzed both glutathione-insulin transhydrogenation and the oxidative renaturation of denatured RNase A. These results suggest that the protein coded for by the carrot gene is a novel member of the PDI family in plants. We therefore designated this novel carrot gene PDIL1. The protein expressed by the PDIL1 cDNA sequence had a highly acidic stretch at its N-terminal region (no such domain exists in known plant PDIs), and was located far from known plant PDIs on a maximum likelihood tree. The PDIL1 gene, together with closely-related genes identified in Arabidopsis and tomato, was suggested to belong to a novel subfamily of PDIs.     

TrbB from conjugative plasmid F is a structurally distinct disulfide isomerase that requires DsbD for redox state maintenance

TrbB, a periplasmic protein encoded by the conjugative plasmid F, has a predicted thioredoxin-like fold and possesses a C-X-X-C redox active site motif. TrbB may function in the conjugative process by serving as a disulfide bond isomerase, facilitating proper folding of a subset of F-plasmid-encoded proteins in the periplasm. Previous studies have demonstrated that a ΔtrbB F plasmid in Escherichia coli lacking DsbC(E.coli), its native disulfide bond isomerase, experiences a 10-fold decrease in mating efficiency but have not provided direct evidence for disulfide bond isomerase activity. Here we demonstrate that trbB can partially restore transfer of a variant of the distantly related R27 plasmid when both chromosomal and plasmid genes encoding disulfide bond isomerases have been disrupted. In addition, we show that TrbB displays both disulfide bond isomerase and reductase activities on substrates not involved in the conjugative process. Unlike canonical members of the disulfide bond isomerase family, secondary structure predictions suggest that TrbB lacks both an N-terminal dimerization domain and an α-helical domain found in other disulfide bond isomerases. Phylogenetic analyses support the conclusion that TrbB belongs to a unique family of plasmid-based disulfide isomerases. Interestingly, although TrbB diverges structurally from other disulfide bond isomerases, we show that like those isomerases, TrbB relies on DsbD from E. coli for maintenance of its C-X-X-C redox active site motif.     

Thioredoxin fold as homodimerization module in the putative chaperone ERp29: NMR structures of the domains and experimental model of the 51 kDa dimer.

BACKGROUND: ERp29 is a ubiquitously expressed rat endoplasmic reticulum (ER) protein conserved in mammalian species. Fold predictions suggest the presence of a thioredoxin-like domain homologous to the a domain of human protein disulfide isomerase (PDI) and a helical domain similar to the C-terminal domain of P5-like PDIs. As ERp29 lacks the double-cysteine motif essential for PDI redox activity, it is suggested to play a role in protein maturation and/or secretion related to the chaperone function of PDI. ERp29 self-associates into 51 kDa dimers and also higher oligomers. RESULTS: 3D structures of the N- and C-terminal domains determined by NMR spectroscopy confirmed the thioredoxin fold for the N-terminal domain and yielded a novel all-helical fold for the C-terminal domain. Studies of the full-length protein revealed a short, flexible linker between the two domains, homodimerization by the N-terminal domain, and the presence of interaction sites for the formation of higher molecular weight oligomers. A gadolinium-based relaxation agent is shown to present a sensitive tool for the identification of macromolecular interfaces by NMR. CONCLUSIONS: ERp29 is the first eukaryotic PDI-related protein for which the structures of all domains have been determined. Furthermore, an experimental model of the full-length protein and its association states was established. It is the first example of a protein where the thioredoxin fold was found to act as a specific homodimerization module, without covalent linkages or supporting interactions by further domains. A homodimerization module similar as in ERp29 may also be present in homodimeric human PDI.     

The Protein Disulfide Isomerase Family: from proteostasis to pathogenesis

In mammalian cells, nearly one-third of proteins are inserted into the endoplasmic reticulum (ER), where they undergo oxidative folding and chaperoning assisted by approximately 20 members of the protein disulfide isomerase family (PDIs). PDIs consist of multiple thioredoxin-like domains and recognize a wide variety of proteins via highly conserved interdomain flexibility. Although PDIs have been studied intensely for almost 50 years, exactly how they maintain protein homeostasis in the ER remains unknown, and is important not only for fundamental biological understanding but also for protein misfolding- and aggregation-related pathophysiology. Herein, we review recent advances in structural biology and biophysical approaches that explore the underlying mechanism by which PDIs fulfil their distinct functions to promote productive protein folding and scavenge misfolded proteins in the ER, the primary factory for efficient production of the secretome.     

Domain a' of Bombyx mori protein disulfide isomerase has chaperone activity

Protein disulfide isomerase (PDI) is an endoplasmic reticulum (ER)-localized multifunctional enzyme that can function as a disulfide oxidase, a reductase, an isomerase, and a chaperone. The domain organization of PDI is abb'xa'c, with two catalytic (CxxC) motifs and a KDEL ER retention motif. The members of the PDI family exhibit differences in tissue distribution, specificity, and intracellular localization. We previously identified and characterized the PDI of Bombyx mori (bPDI) as a thioredoxin-like protein that shares primary sequence homology with other PDIs. Here we compare the reactivation of inactivated rRNase and sRNase by bPDI and three bPDI mutants, and show that bPDI has mammalian PDI-like activity. On its own, the N-terminal a domain does not retain this activity, but the a' domain does. This is the first report of chaperone activity only in the a' domain, but not in the a domain.     

Structure of the Catalytic aa Fragment of the Protein Disulfide Isomerase ERp72

Protein disulfide isomerases (PDIs) are responsible for catalyzing the proper oxidation and isomerization of disulfide bonds of newly synthesized proteins in the endoplasmic reticulum (ER). The ER contains many different PDI-like proteins. Some, such as PDI, are general enzymes that directly recognize misfolded proteins while others, such as ERp57 and ERp72, have more specialized roles. Here, we report the high-resolution X-ray crystal structure of the N-terminal portion of ERp72 (also known as CaBP2 or PDI A4), which contains two a⁰a catalytic thioredoxin-like domains. The structure shows that the a⁰ domain contains an additional N-terminal β-strand and a different conformation of the β5-α4 loop relative to other thioredoxin-like domains. The structure of the a domain reveals that a conserved arginine residue inserts into the hydrophobic core and makes a salt bridge with a conserved glutamate residue in the vicinity of the catalytic site. A structural model of full-length ERp72 shows that all three catalytic sites roughly face each other and positions the adjacent hydrophobic patches that are likely involved in protein substrate binding.     

Structure-function analysis of the endoplasmic reticulum oxidoreductase TMX3 reveals interdomain stabilization of the N-terminal redox-active domain

Disulfide bond formation in the endoplasmic reticulum is catalyzed by enzymes of the protein disulfide-isomerase family that harbor one or more thioredoxin-like domains. We recently discovered the transmembrane protein TMX3, a thiol-disulfide oxidoreductase of the protein disulfide-isomerase family. Here, we show that the endoplasmic reticulum-luminal region of TMX3 contains three thioredoxin-like domains, an N-terminal redox-active domain (named a) followed by two enzymatically inactive domains (b and b'). Using the recombinantly expressed TMX3 domain constructs a, ab, and abb', we compared structural stability and enzymatic properties. By structural and biophysical methods, we demonstrate that the reduced a domain has features typical of a globular folded domain that is, however, greatly destabilized upon oxidization. Importantly, interdomain stabilization by the b domain renders the a domain more resistant toward chemical denaturation and proteolysis in both the oxidized and reduced form. In combination with molecular modeling studies of TMX3 abb', the experimental results provide a new understanding of the relationship between the multidomain structure of TMX3 and its function as a redox enzyme. Overall, the data indicate that in addition to their role as substrate and co-factor binding domains, redox-inactive thioredoxin-like domains also function in stabilizing neighboring redox-active domains.     

Solution structure and dynamics of the reduced and oxidized forms of the N-terminal domain of PilB from Neisseria meningitidis

The secreted form of the PilB protein was proposed to be involved in pathogen survival fighting against the defensive host's oxidative burst. PilB protein is composed of three domains. The central and the C-terminal domains display methionine sulfoxide reductase A and B activities, respectively. The N-terminal domain, which possesses a CXXC motif, was recently shown to regenerate in vitro the reduced forms of the methionine sulfoxide reductase domains of PilB from their oxidized forms, as does the thioredoxin 1 from E. coli, via a disulfide bond exchange. The thioredoxin-like N-terminal domain belongs to the cytochrome maturation protein structural family, but it possesses a unique additional segment (99)FLHE (102) localized in a loop. This segment covers one edge of the active site in the crystal structure of the reduced form of the N-terminal domain of PilB. We have determined the solution structure and the dynamics of the N-terminal domain from Neisseria meningitidis, in its reduced and oxidized forms. The FLHE loop adopts, in both redox states, a well-defined conformation. Subtle conformational and dynamic changes upon oxidation are highlighted around the active site, as well as in the FLHE loop. The functional consequences of the cytochrome maturation protein topology and those of the presence of FLHE loop are discussed in relation to the enzymatic properties of the N-terminal domain.     

模式匹配分析方法检索免疫球蛋白样功能区

运用计算机进行核酸和蛋白质的序列分析是分子生物学研究的一个较新发展,这项技术已越来越多地用于研究大量积累的序列数据。蛋白质功能区是蛋白质分子中能独立折叠成具有一定结构并执行特定功能的结构域,所有具有同一类功能区的分子统称为一个蛋白质的超族(protein superfamily)。本文通过对免疫球蛋白(Ig)超族及其功能区序列所进行的分析,建立了一种根据功能区之保守片段残基组成的模式匹配分析检索蛋白质功能区的方法,它先根据多序列的对准比较确定某一类功能区之保守片段,再对已知的保守片段各位置上氨基酸残基组成进行统计分析,然后根据与统计数值相匹配的方法,计算待检序列残基组成的统计学意义,由此确定功能区的存在。该方法的优点在于它不仅可以检出已知的具有某一类功能区的分子,而且还可能发现新的具有该功能区的分子,从而推测后者的功能。     

Structure of intact AhpF reveals a mirrored thioredoxin-like active site and implies large domain rotations during catalysis

AhpF, a homodimer of 57 kDa subunits, is a flavoenzyme which catalyzes the NADH-dependent reduction of redox-active disulfide bonds in the peroxidase AhpC, a member of the recently identified peroxiredoxin class of antioxidant enzymes. The structure of AhpF from Salmonella typhimurium at 2.0 A resolution, determined using multiwavelength anomalous dispersion, shows that the C-terminal portion of AhpF (residues 210-521) is structurally like Escherichia coli thioredoxin reductase. In addition, AhpF has an N-terminal domain (residues 1-196) formed from two contiguous thioredoxin folds, but containing just a single redox-active disulfide (Cys129-Cys132). A flexible linker (residues 197-209) connects the domains, consistent with experiments showing that the N-terminal domain acts as an appended substrate, first being reduced by the C-terminal portion of AhpF, and subsequently reducing AhpC. Modeling studies imply that an intrasubunit electron transfer accounts for the reduction of the N-terminal domain in dimeric AhpF. Furthermore, comparing the N-terminal domain with protein disulfide oxidoreductase from Pyrococcus furiosis, we describe a new class of protein disulfide oxidoreductases based on a novel mirror-image active site arrangement, with a distinct carboxylate (Glu86) being functionally equivalent to the key acid (Asp26) of E. coli thioredoxin. A final fortuitous result is that the N-terminal redox center is reduced and provides a high-resolution view of the thiol-thiolate hydrogen bond that has been predicted to stabilize the attacking thiolate in thioredoxin-like proteins.     

Molecular Evolution of the Domain Structures of Protein Disulfide Isomerases

Protein disulfide isomerase (PDI) is an enzyme that promotes protein folding by catalyzing disulfide bridge isomerization. PDI and its relatives form a diverse protein family whose members are characterized by thioredoxin-like (TX) domains in the primary structures. The family was classified into four classes by the number and the relative positions of the TX domains. To investigate the evolution of the domain structures, we aligned the amino acid sequences of the TX domains, and the molecular phylogeny was examined by the NJ and ML methods. We found that all of the current members of the PDI family have evolved from an ancestral enzyme, which has two TX domains in the primary structure. The diverse domain structures of the members have been generated through domain duplications and deletions.     

Residue-residue contact substitution probabilities derived from aligned three-dimensional structures and the identification of common folds.

We report the derivation of scores that are based on the analysis of residue-residue contact matrices from 443 3-dimensional structures aligned structurally as 96 families, which can be used to evaluate sequence-structure matches. Residue-residue contacts and the more than 3 x 10(6) amino acid substitutions that take place between pairs of these contacts at aligned positions within each family of structures have been tabulated and segregated according to the solvent accessibility of the residues involved. Contact maps within a family of structures are shown to be highly conserved (approximately 75%) even when the sequence identity is approaching 10%. In a comparison involving a globin structure and the search of a sequence databank (> 21,000 sequences), the contact probability scores are shown to provide a very powerful secondary screen for the top scoring sequence-structure matches, where between 69% and 84% of the unrelated matches are eliminated. The search of an aligned set of 2 globins against a sequence databank and the subsequent residue contact-based evaluation of matches locates all 618 globin sequences before the first non-globin match. From a single bacterial serine proteinase structure, the structural template approach coupled with residue-residue contact substitution data lead to the detection of the mammalian serine proteinase family among the top matches in the search of a sequence databank.     

Matching nucleotide sequences of human antibodies with other known sequences

From an evolutionary point of view, the complementarity-determining regions of antibodies are distinct from other proteins including the framework regions of antibodies. A search for identical nucleotide sequences of eighty-four 15 consecutive bp in the complementary-determining regions of human antibody heavy chains with other known sequences yielded four matches: two sequential 15-bp matches, or one 16-bp match, with the coding region of a sea-urchin testis histone H2b-2, one 15-bp match with the promotor region of a cauliflower mosaic virus inclusion body protein, and a 15-bp match with an intron between exons 1 and 2 of human factor IX. As a control, an identical search of eighty-four 15 consecutive bp in the framework regions of human antibody heavy chains yielded no matches with other sequences except those from other antibody framework regions. Since the currently available nucleotide sequence database used in the search consisted of about 1 x 10(7) bp, finding such matches in the complementarity-determining regions might not be random.     

The sequence determinants of cadherin molecules

The sequence and structural analysis of cadherins allow us to find sequence determinants-a few positions in sequences whose residues are characteristic and specific for the structures of a given family. Comparison of the five extracellular domains of classic cadherins showed that they share the same sequence determinants despite only a nonsignificant sequence similarity between the N-terminal domain and other extracellular domains. This allowed us to predict secondary structures and propose three-dimensional structures for these domains that have not been structurally analyzed previously. A new method of assigning a sequence to its proper protein family is suggested: analysis of sequence determinants. The main advantage of this method is that it is not necessary to know all or almost all residues in a sequence as required for other traditional classification tools such as BLAST, FASTA, and HMM. Using the key positions only, that is, residues that serve as the sequence determinants, we found that all members of the classic cadherin family were unequivocally selected from among 80,000 examined proteins. In addition, we proposed a model for the secondary structure of the cytoplasmic domain of cadherins based on the principal relations between sequences and secondary structure multialignments. The patterns of the secondary structure of this domain can serve as the distinguishing characteristics of cadherins.     

Interdomain Contacts and the Stability of Serralysin Protease from Serratia marcescens

The serralysin family of bacterial metalloproteases is associated with virulence in multiple modes of infection. These extracellular proteases are members of the Repeats-in-ToXin (RTX) family of toxins and virulence factors, which mediated virulence in E. coli, B. pertussis, and P. aeruginosa, as well as other animal and plant pathogens. The serralysin proteases are structurally dynamic and their folding is regulated by calcium binding to a C-terminal domain that defines the RTX family of proteins. Previous studies have suggested that interactions between N-terminal sequences and this C-terminal domain are important for the high thermal and chemical stabilities of the RTX proteases. Extending from this, stabilization of these interactions in the native structure may lead to hyperstabilization of the folded protein. To test this hypothesis, cysteine pairs were introduced into the N-terminal helix and the RTX domain and protease folding and activity were assessed. Under stringent pH and temperature conditions, the disulfide-bonded mutant showed increased protease activity and stability. This activity was dependent on the redox environment of the refolding reaction and could be blocked by selective modification of the cysteine residues before protease refolding. These data demonstrate that the thermal and chemical stability of these proteases is, in part, mediated by binding between the RTX domain and the N-terminal helix and demonstrate that stabilization of this interaction can further stabilize the active protease, leading to additional pH and thermal tolerance.     

The spvB gene-product of the Salmonella enterica virulence plasmid is a mono(ADP-ribosyl)transferase

A number of well-known bacterial toxins ADP-ribosylate and thereby inactivate target proteins in their animal hosts. Recently, several vertebrate ecto-enzymes (ART1-ART7) with activities similar to bacterial toxins have also been cloned. We show here that PSIBLAST, a position-specific-iterative database search program, faithfully connects all known vertebrate ecto-mono(ADP-ribosyl)transferases (mADPRTs) with most of the known bacterial mADPRTs. Intriguingly, no matches were found in the available public genome sequences of archaeabacteria, the yeast Saccharomyces cerevisiae or the nematode Caenorhabditis elegans. Significant new matches detected by PSIBLAST from the public sequence data bases included only one open reading frame (ORF) of previously unknown function: the spvB gene contained in the virulence plasmids of Salmonella enterica. Structure predictions of SpvB indicated that it is composed of a C-terminal ADP-ribosyltransferase domain fused via a poly proline stretch to a N-domain resembling the N-domain of the secretory toxin TcaC from nematode-infecting enterobacteria. We produced the predicted catalytic domain of SpvB as a recombinant fusion protein and demonstrate that it, indeed, acts as an ADP-ribosyltransferase. Our findings underscore the power of the PSIBLAST program for the discovery of new family members in genome databases. Moreover, they open a new avenue of investigation regarding salmonella pathogenesis.     

Conservation of intrinsic disorder in protein domains and families: I. A database of conserved predicted disordered regions

Many protein regions have been shown to be intrinsically disordered, lacking unique structure under physiological conditions. These intrinsically disordered regions are not only very common in proteomes, but also crucial to the function of many proteins, especially those involved in signaling, recognition, and regulation. The goal of this work was to identify the prevalence, characteristics, and functions of conserved disordered regions within protein domains and families. A database was created to store the amino acid sequences of nearly one million proteins and their domain matches from the InterPro database, a resource integrating eight different protein family and domain databases. Disorder prediction was performed on these protein sequences. Regions of sequence corresponding to domains were aligned using a multiple sequence alignment tool. From this initial information, regions of conserved predicted disorder were found within the domains. The methodology for this search consisted of finding regions of consecutive positions in the multiple sequence alignments in which a 90% or more of the sequences were predicted to be disordered. This procedure was constrained to find such regions of conserved disorder prediction that were at least 20 amino acids in length. The results of this work included 3,653 regions of conserved disorder prediction, found within 2,898 distinct InterPro entries. Most regions of conserved predicted disorder detected were short, with less than 10% of those found exceeding 30 residues in length.