Sequence-related Proteins Research Articles

Understanding the assembly mechanism of the twin arginine transport system.

The twin-arginine protein translocation (Tat) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of chloroplasts. The Escherichia coli Tat system comprises TatC and two additional sequence-related proteins, TatA and TatB. Tat-directed proteins are distinguished by a conserved twin-arginine (RR-) motif in their signal sequences. Substrate binding promotes a conformation rearrangement of the Tat complex. TatA is then recruited to the substrate activated TatBC complex, where it polymerizes to form the substrate translocation pathway. The central challenge in understanding Tat transport is to determine how the Tat components are assembled to form the active translocon allowing folded substrates to be transferred across the membrane bilayer. In this work, we used molecular dynamics simulations to describe the TatA oligomerization mechanism and how the translocation pathway is formed. Our results show that the Tat complex becomes more dynamic after substrate binding. The presence of the substrate induces a “breathing” movement, increasing the separation between two adjacent TatC subunits. Moreover, our results also suggest that substrate binding induces an asymmetry in the Tat Complex, favouring TatA aggregation around the interface opposite to the TatC subunit where the signal peptide is bound. The TatA oligomer is anchored to the Tat complex through contacts with the TM1 and the TM6 of the adjacent TatC subunits, which promotes a local weakening of the membrane bilayer. Our extensive coarse-grained MD simulations provide a molecular description of the Tat translocon assembly and new insights into the Tat transport cycle.

VTR: A Web Tool for Identifying Analogous Contacts on Protein Structures and Their Complexes.

Evolutionarily related proteins can present similar structures but very dissimilar sequences. Hence, understanding the role of the inter-residues contacts for the protein structure has been the target of many studies. Contacts comprise non-covalent interactions, which are essential to stabilize macromolecular structures such as proteins. Here we show VTR, a new method for the detection of analogous contacts in protein pairs. The VTR web tool performs structural alignment between proteins and detects interactions that occur in similar regions. To evaluate our tool, we proposed three case studies: we 1) compared vertebrate myoglobin and truncated invertebrate hemoglobin; 2) analyzed interactions between the spike protein RBD of SARS-CoV-2 and the cell receptor ACE2; and 3) compared a glucose-tolerant and a non-tolerant β-glucosidase enzyme used for biofuel production. The case studies demonstrate the potential of VTR for the understanding of functional similarities between distantly sequence-related proteins, as well as the exploration of important drug targets and rational design of enzymes for industrial applications. We envision VTR as a promising tool for understanding differences and similarities between homologous proteins with similar 3D structures but different sequences. VTR is available at http://bioinfo.dcc.ufmg.br/vtr.

Substrate-triggered position switching of TatA and TatB during Tat transport in Escherichia coli.

The twin-arginine protein transport (Tat) machinery mediates the translocation of folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. The Escherichia coli Tat system comprises TatC and two additional sequence-related proteins, TatA and TatB. The active translocase is assembled on demand, with substrate-binding at a TatABC receptor complex triggering recruitment and assembly of multiple additional copies of TatA; however, the molecular interactions mediating translocase assembly are poorly understood. A ‘polar cluster’ site on TatC transmembrane (TM) helix 5 was previously identified as binding to TatB. Here, we use disulfide cross-linking and molecular modelling to identify a new binding site on TatC TM helix 6, adjacent to the polar cluster site. We demonstrate that TatA and TatB each have the capacity to bind at both TatC sites, however in vivo this is regulated according to the activation state of the complex. In the resting-state system, TatB binds the polar cluster site, with TatA occupying the TM helix 6 site. However when the system is activated by overproduction of a substrate, TatA and TatB switch binding sites. We propose that this substrate-triggered positional exchange is a key step in the assembly of an active Tat translocase.

Analysis of distinct molecular assembly complexes of keratin K8 and K18 by hydrogen-deuterium exchange.

Keratins are intermediate filament (IF) proteins that form complex filament systems in epithelial cells, thus serving as scaffolding elements and mechanical stress absorbers. The building blocks of keratin IFs are parallel coiled-coil dimers of two distinct sequence-related proteins distinguished as type I and type II keratins. To gain more insight into their structural dynamics, we resorted to hydrogen-deuterium exchange mass spectrometry of keratins K8 and K18, which are characteristic for simple epithelial cells. Using this powerful technique not employed with IFs before, we mapped patterns of protected versus unprotected regions in keratin complexes at various assembly levels. In particular, we localized protein segments exhibiting different hydrogen exchange patterns in tetramers versus filaments. We observed a general pattern of precisely positioned regions of stability intertwining with flexible regions, mostly represented by the non-α-helical segments. Notably, some regions within the coiled-coil domains are significantly more dynamic than others, while the IF-consensus motifs at the end domains of the central α-helical "rod" segment, which mediate the "head-to-tail" dimer-dimer interaction in the filament elongation process, become distinctly more protected upon formation of filaments. Moreover, to gain more insight into the dynamics of the individual keratins, we investigated the properties of homomeric preparations of K8 and K18. The physiological importance of keratins without a partner is encountered in both pathological and experimental situations when one of the two species is present in robust excess or completely absent, such as in gene-targeted mice.

Characterization, mutagenesis and mechanistic analysis of an ancient algal sterol C24-methyltransferase: Implications for understanding sterol evolution in the green lineage

Sterol C24-methyltransferases (SMTs) constitute a group of sequence-related proteins that catalyze the pattern of sterol diversity across eukaryotic kingdoms. The only gene for sterol alkylation in green algae was identified and the corresponding catalyst from Chlamydomonas reinhardtii (Cr) was characterized kinetically and for product distributions. The properties of CrSMT were similar to those predicted for an ancient SMT expected to possess broad C3-anchoring requirements for substrate binding and formation of 24β-methyl/ethyl Δ25(27)-olefin products typical of primitive organisms. Unnatural Δ24(25)-sterol substrates, missing a C4β-angular methyl group involved with binding orientation, convert to product ratios in favor of Δ24(28)-products. Remodeling the active site to alter the electronics of Try110 (to Leu) results in delayed timing of the hydride migration from methyl attack of the Δ24-bond, that thereby produces metabolic switching of product ratios in favor of Δ25(27)-olefins or impairs the second C1-transfer activity. Incubation of [27-13C]lanosterol or [methyl-2H3]SAM as co-substrates established the CrSMT catalyzes a sterol methylation pathway by the “algal” Δ25(27)-olefin route, where methylation proceeds by a conserved SN2 reaction and de-protonation proceeds from the pro-Z methyl group on lanosterol corresponding to C27. This previously unrecognized catalytic competence for an enzyme of sterol biosynthesis, together with phylogenomic analyses, suggest that mutational divergence of a promiscuous SMT produced substrate- and phyla-specific SMT1 (catalyzes first biomethylation) and SMT2 (catalyzes second biomethylation) isoforms in red and green algae, respectively, and in the case of SMT2 selection afforded modification in reaction channeling necessary for the switch in ergosterol (24β-methyl) biosynthesis to stigmasterol (24α-ethyl) biosynthesis during the course of land plant evolution.

Increased Expression of hypoxia-glycolysis-acidosis sequence related proteins: An important role in keloid pathogenesis

Keloids represent a prolonged inflammatory fibrotic state with areas that display distinctive histological features characterized by an abundant extracellular matrix stroma, a local infiltration of inflammatory cells, and a milieu of enriched cytokines. Previous studies demonstrated that hypoxia regulates extracellular matrix remodeling via hypoxia-inducible factor-1α (HIF-1α). Stabilization of HIF-1α upregulates the expression of downstream genes such as carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF).

Expanding the biocatalytic toolbox of flavoprotein monooxygenases from Rhodococcus jostii RHA1

With the aim to enlarge the set of available flavoprotein monooxygenases, we have cloned 8 unexplored genes from Rhodococcus jostii RHA1 that were predicted to encode class B flavoprotein monooxygenases. Each monooxygenase can be expressed as soluble protein and has been tested for conversion of sulfides and ketones. Not only enantioselective sulfoxidations, but also enantioselective Baeyer–Villiger oxidations could be performed with this set of monooxygenases. Interestingly, in contrast to known class B flavoprotein monooxygenases, all studied biocatalysts showed no nicotinamide coenzyme preference. This feature coincides with the fact that the respective sequences appear to form a discrete group of sequence related proteins, distinct from the known class B flavoprotein monooxygenases subclasses: the so-called flavin-containing monooxygenases (FMOs), N-hydroxylating monooxygenases (NMOs) and Type I Baeyer–Villiger monooxygenases (BVMOs). Taken together, these data reveal the existence of a new subclass of class B flavoprotein monooxygenases, which we coined as Type II FMOs, that can perform Baeyer–Villiger oxidations and accept both NADPH and NADH as coenzyme. The uncovered biocatalytic properties of the studied Type II FMOs make this newly recognized subclass of monooxygenases of potential interest for biocatalytic applications.

Purification of recombinant protein by cold‐coacervation of fusion constructs incorporating resilin‐inspired polypeptides

Polypeptides containing between 4 and 32 repeats of a resilin-inspired sequence AQTPSSYGAP, derived from the mosquito Anopheles gambiae, have been used as tags on recombinant fusion proteins. These repeating polypeptides were inspired by the repeating structures that are found in resilins and sequence-related proteins from various insects. Unexpectedly, an aqueous solution of a recombinant resilin protein displays an upper critical solution temperature (cold-coacervation) when held on ice, leading to a separation into a protein rich phase, typically exceeding 200 mg/mL, and a protein-poor phase. We show that purification of recombinant proteins by cold-coacervation can be performed when engineered as a fusion partner to a resilin-inspired repeat sequence. In this study, we demonstrate the process by the recombinant expression and purification of enhanced Green fluorescent protein (EGFP) in E. coli. This facile purification system can produce high purity, concentrated protein solutions without the need for affinity chromatography or other time-consuming or expensive purification steps, and that it can be used with other bulk purification steps such as low concentration ammonium sulfate precipitation. Protein purification by cold-coacervation also minimizes the exposure of the target protein to enhanced proteolysis at higher temperature.

Cloning, functional expression and phylogenetic analysis of plant sterol 24C-methyltransferases involved in sitosterol biosynthesis

Sterol 24C-methyltransferases (SMTs) constitute a group of sequence-related proteins that catalyze the distinct patterns of 24-alkyl sterols that occur throughout nature. Two SMT cDNAs (SMT2-1 and SMT2-2) were cloned by homology based PCR methods from young leaves of Glycine max (soybean) and the corresponding enzymes were expressed functionally in Escherichia coli. The full-length cDNA for SMT2-1 and SMT2-2 have open reading frames of 1086 bp and 1092 bp, respectively, and encode proteins of 361 and 363 residues with a calculated molecular mass of 40.3 and 40.4 kDa, respectively. The substrate preference of the two isoforms was similar yet they differed from SMT1; kinetically SMT2-1 and SMT2-2 generated k cat values for the optimal substrate 24(28)methylene lophenol of 0.8 min −1 and 1.34 min −1, respectively, compared to the activity of SMT1 that generated a k cat for the optimal substrate cycloartenol of 0.6 min −1. SMT2-2 was purified to homogeneity and the subunit organization shown to be tetrameric in similar fashion to other cloned SMTs. Analysis of the accumulated products catalyzed by the recombinant enzymes demonstrated that soybean SMT2-1 and SMT2-2 operate transalkylation activities analogous to the soybean plant SMT1. Metabolite analyses correlated with transcript profiling of the three SMT isoforms during soybean maturation clearly demonstrated that SMT isoform expression determines specific C24-methyl to C24-ethyl ratios to flowering whereas with seed development there is a disconnection such that the SMT transcript levels decrease against an increase in sterol content; generally SMT2-2 is expressed more than SMT2-1 or SMT1. These observations suggest that the genes that encode SMT1 and SMT2 in sitosterol biosynthesis may have undergone divergent evolution. In support of this proposition, the genomic organization for SMT1 of fungi and protozoa align very closely with one another and to those of the plant SMT2; both sets of SMTs lack introns. Unexpectedly, the SMT1 from Glycine max and other embryophytes of diverse origin possess disparate intron–exon characteristics that can be shown relates back to the algae. Our results suggest that the order of SMT1 appearing before SMT2 in phytosterol synthesis arose recently in plant evolution in response to duplication of a more primitive SMT gene likely to have been bifunctional and catalytically promiscuous.

Characterization of PrEST-based antibodies towards human Cytokeratin-17

Antibody-based proteomics efforts depend on validated antibodies to ensure correct annotation of analyzed proteins. We have previously argued that a low sequence identity to other proteins is a key feature for antigens used in antibody generation. Thus, a major challenge for whole-proteome studies is how to address families of highly sequence related proteins within the context of generating specific antibodies. In this study, two non-overlapping parts of human Cytokeratin-17, a protein belonging to the intermediate filament family of highly sequence-related proteins, were selected as a model system to study the specificity and cross reactivity of antibodies generated towards such a target. These recombinantly produced Protein Epitope Signature Tags (PrESTs) were immunized in five rabbits each and the batch-to-batch variations in the obtained immune responses were studied by mapping of linear epitopes using synthetic overlapping peptides. The obtained results showed a similar but not identical immune response in the respective antibody groups with a limited number of epitopes being identified. Immunohistochemical analysis of the affinity purified monospecific antibodies on tissue micro arrays resulted in a general recognition of human cytokeratins for all analyzed binders whereas antibodies identified as binding to the most unique parts of the PrESTs showed the most Cytokeratin-17 like staining. The data presented here support the strategy to use sequence identity scores as the main criteria for antigen selection but also indicate the possibility to instead produce a single antibody recognizing a defined group of proteins when the intended targets overall sequence identity score is too high. This type of group-specific antibodies would be an important tool for antibody-based projects aiming for a complete coverage of the human proteome.

Comparison of the folding mechanism of highly homologous proteins in the lipid‐binding protein family

The folding mechanism of two closely related proteins in the intracellular lipid-binding protein family, human bile acid-binding protein (hBABP), and rat bile acid-binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence. Both of these single domain proteins fit well to a two-state model for unfolding by fluorescence and circular dichroism at equilibrium. Three phases were observed during the unfolding of rBABP by fluorescence but only one phase was observed during the unfolding of hBABP, suggesting that at least two kinetic intermediates accumulate during the unfolding of rBABP that are not observed during the unfolding of hBABP. Fluorine NMR was used to examine the equilibrium unfolding behavior of the W49 side chain in 6-fluorotryptophan-labeled rBABP and hBABP. The structure of rBABP appears to be more dynamic than that of hBABP in the vicinity of W49 in the absence of denaturant, and urea has a greater effect on this dynamic behavior for rBABP than for hBABP. As such, the folding behavior of highly sequence related proteins in this family can be quite different. These differences imply that moderately sized proteins with high sequence and structural similarity can still populate quite different structures during folding.

Molecular Features Governing the Stability and Specificity of Functional Complex Formation by Mycobacterium tuberculosis CFP-10/ESAT-6 Family Proteins

The Mycobacterium tuberculosis complex CFP-10/ESAT-6 family proteins play essential but poorly defined roles in tuberculosis pathogenesis. In this article we report the results of detailed spectroscopic studies of several members of the CFP-10/ESAT-6 family. This work shows that the CFP-10/ESAT-6 related proteins, Rv0287 and Rv0288, form a tight 1:1 complex, which is predominantly helical in structure and is predicted to closely resemble the complex formed by CFP-10 and ESAT-6. In addition, the Rv0287.Rv0288 complex was found to be significantly more stable to both chemical and temperature induced denaturation than CFP-10.ESAT-6. This approach demonstrated that neither Rv0287.Rv0288 nor the CFP-10.ESAT-6 complexes are destabilized at low pH (4.5), indicating that even in low pH environments, such as the mature phagosome, both Rv0287.Rv0288 and CFP-10.ESAT-6 undoubtedly function as complexes rather than individual proteins. Analysis of the structure of the CFP-10.ESAT-6 complex and optimized amino acid sequence alignments of M. tuberculosis CFP-10/ESAT-6 family proteins revealed that residues involved in the intramolecular contacts between helices are conserved across the CFP-10/ESAT-6 family, but not those involved in primarily intermolecular contacts. This analysis identified the molecular basis for the specificity and stability of complex formation between CFP-10/ESAT-6 family proteins, and indicates that the formation of functional complexes with key roles in pathogenesis will be limited to genome partners, or very closely related family members, such as Rv0287/Rv0288 and Rv3019c/Rv3020c.

Molecular properties of two proteins homologous to PduO-type ATP:cob(I)alamin adenosyltransferase from Sulfolobus tokodaii

In the thermophilic archaeon Sulfolobus tokodaii, there are two genes homologous to PduO-type ATP:cob(I)alamin adenosyltransferase, ST1454 and ST2180. To address the structure and function of these two sequence-related proteins from one organism, we prepared them by using the Escherichia coli expression system and analyzed them by immunoblotting, matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy, circular dichroism spectrometry, ATP:cobalamin adenosyltransferase assay, and X-ray crystallography. Immunoblotting and matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy analyses showed that both these proteins are expressed in S. tokodaii cells as soluble proteins and are spontaneously digested at the N-terminal region. ATP:cob(I)alamin adenosyltransferase activity was detected for ST1454 but not for ST2180. ST2180 reduced the concentration of cob(I)alamin, suggesting that ST2180 might recognize cob(I)alamin as a ligand. The secondary structure of ST1454 was retained even in 7 M guanidine hydrochroride, whereas that of ST2180 was melted in 4.5 M guanidine hydrochloride. The X-ray crystal structural analysis revealed that the proteins shared a common structure: a trimer of five-helix bundles with a clockwise kink. There is a pocket surrounded by highly conserved residues, in which a polypropylene glycol 400 in the crystal structure of ST1454 was captured, suggesting that it is an active site. Structural comparison between these two proteins showed the difference in the number of ion pairs around the proposed active site. On the basis of these results, we propose that ST1454 and ST2180 have related but distinct functions.

Gene fusions and gene duplications: relevance to genomic annotation and functional analysis

BackgroundEscherichia coli a model organism provides information for annotation of other genomes. Our analysis of its genome has shown that proteins encoded by fused genes need special attention. Such composite (multimodular) proteins consist of two or more components (modules) encoding distinct functions. Multimodular proteins have been found to complicate both annotation and generation of sequence similar groups. Previous work overstated the number of multimodular proteins in E. coli. This work corrects the identification of modules by including sequence information from proteins in 50 sequenced microbial genomes.ResultsMultimodular E. coli K-12 proteins were identified from sequence similarities between their component modules and non-fused proteins in 50 genomes and from the literature. We found 109 multimodular proteins in E. coli containing either two or three modules. Most modules had standalone sequence relatives in other genomes. The separated modules together with all the single (un-fused) proteins constitute the sum of all unimodular proteins of E. coli. Pairwise sequence relationships among all E. coli unimodular proteins generated 490 sequence similar, paralogous groups. Groups ranged in size from 92 to 2 members and had varying degrees of relatedness among their members. Some E. coli enzyme groups were compared to homologs in other bacterial genomes.ConclusionThe deleterious effects of multimodular proteins on annotation and on the formation of groups of paralogs are emphasized. To improve annotation results, all multimodular proteins in an organism should be detected and when known each function should be connected with its location in the sequence of the protein. When transferring functions by sequence similarity, alignment locations must be noted, particularly when alignments cover only part of the sequences, in order to enable transfer of the correct function. Separating multimodular proteins into module units makes it possible to generate protein groups related by both sequence and function, avoiding mixing of unrelated sequences. Organisms differ in sizes of groups of sequence-related proteins. A sample comparison of orthologs to selected E. coli paralogous groups correlates with known physiological and taxonomic relationships between the organisms.

Ligand specificity of H-NOX domains: from sGC to bacterial NO sensors

Soluble guanylate cyclase (sGC) is a nitric oxide (NO) sensing hemoprotein that has been found in eukaryotes from Drosophila to humans. Prokaryotic proteins with significant homology to the heme domain of sGC have recently been identified through genomic analysis. This family of heme proteins has been named the H-NOX domain, for Heme- Nitric oxide/ OXygen binding domain. The key observation from initial studies in this family is that some members, those proteins from most eukaryotes and facultative aerobic prokaryotes, bind NO in a five-coordinate heme complex, but do not bind oxygen (O 2), the same ligand binding characteristics as sGC. H-NOX family members from obligate aerobic prokaryotes bind O 2 and NO in six-coordinate complexes, similar to the globins and other O 2-sensing heme proteins. The molecular factors that contribute to these differences in ligand specificity, within a family of sequence related proteins, are the subject of this review.

Sequence-related human proteins cluster by degree of evolutionary conservation

Gene duplication followed by adaptive evolution is thought to be a central mechanism for the emergence of novel genes. To illuminate the contribution of duplicated protein-coding sequences to the complexity of the human genome, we study the connectivity of pairwise sequence-related human proteins and construct a network (N) of linked protein sequences with shared similarities. We find that (i) the connectivity distribution P (k) for k sequence-related proteins decays as a power law P (k) approximately k(-gamma) with gamma approximately 1.2 , (ii) the top rank of N consists of a single large cluster of proteins ( approximately 70%) , while bottom ranks consist of multiple isolated clusters, and (iii) structural characteristics of N show both a high degree of clustering and an intermediate connectivity ("small-world" features). We gain further insight into structural properties of N by studying the relationship between the connectivity distribution and the phylogenetic conservation of proteins in bacteria, plants, invertebrates, and vertebrates. We find that (iv) the proportion of sequence-related proteins increases with increasing extent of evolutionary conservation. Our results support that small-world network properties constitute a footprint of an evolutionary mechanism and extend the traditional interpretation of protein families.

Spectroscopic characterization of the soluble guanylate cyclase-like heme domains from Vibrio cholerae and Thermoanaerobacter tengcongensis.

Soluble guanylate cyclase (sGC) is a nitric oxide- (NO-) sensing hemoprotein that has been found in eukaryotes from Drosophila to humans. Prokaryotic proteins with significant homology to the heme domain of sGC have recently been identified through genomic analysis. Characterization of two of these proteins is reported here. The first is a 181 amino acid protein cloned from Vibrio cholerae (VCA0720) that is encoded in a histidine kinase-containing operon. The ferrous unligated form of VCA0720 is 5-coordinate, high-spin. The CO complex is low-spin, 6-coordinate, and the NO complex is high-spin and 5-coordinate. These ligand-binding properties are very similar to those of sGC. The second protein is the N-terminal 188 amino acids of Tar4 (TtTar4H), a predicted methyl-accepting chemotaxis protein (MCP) from the strict anaerobe Thermoanaerobacter tengcongensis. TtTar4H forms a low-spin, 6-coordinate ferrous-oxy complex, the first of this sGC-related family that binds O2. TtTar4H has ligand-binding properties similar to those of the heme-containing O2 sensors such as AxPDEA1. sGC does not bind O2 despite having a porphyrin with a histidyl ligand like the globins. The results reported here, with sequence-related proteins from prokaryotes but in the same family as the sGC heme domain, show that these proteins have evolved to discriminate between ligands such as NO and O2; hence, we term this family H-NOX domains (heme-nitric oxide/oxygen).

Determinants in nuclease specificity of ape1 and ape2, human homologues of Escherichia coli exonuclease III

Abasic sites and non-conventional 3′-ends, e.g. 3′-oxidized fragments (including 3′-phosphate groups) and 3′-mismatched nucleotides, arise at significant frequency in the genome due to spontaneous decay, oxidation or replication errors. To avert the potentially mutagenic or cytotoxic effects of these chromosome modifications/intermediates, organisms are equipped with apurinic/apyrimidinic (AP) endonucleases and 3′-nucleases that initiate repair. Ape1, which shares homology with Escherichia coli exonuclease III (ExoIII), is the major abasic endonuclease in mammals and an important, yet selective, contributor to 3′-end processing. Mammals also possess a second protein (Ape2) with sequence homology to ExoIII, but this protein exhibits comparatively weak AP site-specific and 3′-nuclease activities. Prompted by homology modeling studies, we found that substitutions in the hydrophobic pocket of Ape1 (comprised of F266, W280 and L282) reduce abasic incision potency about fourfold to 450,000-fold, while introduction of an ExoIII-like pocket into Ape2 enhances its AP endonuclease function. We demonstrate that mutations at F266 and W280 of Ape1 increase 3′ to 5′ DNA exonuclease activity. These results, coupled with prior comparative sequence analysis, indicate that this active-site hydrophobic pocket influences the substrate specificity of a diverse set of sequence-related proteins possessing the conserved four-layered α/β-fold. Lastly, we report that wild-type Ape1 excises 3′-mismatched nucleotides at a rate up to 374-fold higher than correctly base-paired nucleotides, depending greatly on the structure and sequence of the DNA substrate, suggesting a novel, selective role for the human protein in 3′-mismatch repair.

Identification, sequence analysis and expression of transcripts encoding a putative metalloproteinase, eMDC II, in human and macaque epididymis.

The metalloproteinase-like, disintegrin-like, cysteine-rich (MDC) family is a large group of sequence-related proteins, first characterized in the male reproductive tract, but subsequently also identified in non-reproductive tissues. Their primary translation products are of approximately 90 kDa and each can be divided into distinct domains which show remarkable homology to reprolysins; snake venom haemorrhagic components possessing metalloproteinase and/or disintegrin domains. Several MDC proteins are abundantly-expressed in the male reproductive tract, suggesting functions in fertility. We now describe the cloning, sequence determination and characterization of transcripts encoding the human and macaque (Macaca fascicularis) orthologues of a novel member of the MDC family (eMDC II) which is abundantly-expressed in the epididymis. Unlike many MDC proteins expressed in the reproductive tract, eMDC II possesses the extended 'catalytic centre' consensus sequence characteristic of a reprolysin-like metalloproteinase. This suggests that eMDC II has proteolytic activity.

Identification and characterization of a human tRNA-specific adenosine deaminase related to the ADAR family of pre-mRNA editing enzymes.

The mammalian adenosine deaminases acting on RNA (ADARs) constitute a family of sequence-related proteins involved in pre-mRNA editing of nuclear transcripts through site-specific adenosine modification. We report here the identification and characterization of a human ADAR protein, hADAT1, that specifically deaminates adenosine 37 to inosine in eukaryotic tRNA(Ala). It represents the functional homologue of the recently identified yeast protein Tad1p [Gerber, A., Grosjean, H., Melcher, T. & Keller, W. (1998) EMBO J. 17, 4780-4789]. The hADAT1 cDNA predicts a protein of 502 aa whose sequence displays strongest overall homology to a Drosophila melanogaster ORF (50% similarity, 32% identity), and the catalytic domain is closely related to the other ADAR proteins. In vitro, the recombinantly expressed and purified hADAT1 protein efficiently and specifically deaminates A(37) in the anticodon loop of tRNA(Ala) from higher eukaryotes and with lower efficiency from lower eukaryotes. It does not modify adenosines residing in double-stranded RNA or in pre-mRNAs that serve as substrates for ADAR1 or ADAR2. The anticodon stem-loop of tRNA(Ala) alone is not a functional substrate for hADAT1. The enzyme is expressed ubiquitously in human tissues and is represented by a single gene. The identification and cloning of hADAT1 should help to elucidate the physiological significance of this unique modification in tRNA(Ala), which is conserved from yeast to man.