Conserved Amino Acid Sequence Motifs Research Articles

MotifQuest: An Automated Pipeline for Motif Database Creation to Improve Peptidomics Database Searching Programs.

Endogenous peptides are an abundant and versatile class of biomolecules with vital roles pertinent to the functionality of the nervous, endocrine, and immune systems and others. Mass spectrometry stands as a premier technique for identifying endogenous peptides, yet the field still faces challenges due to the lack of optimized computational resources for reliable raw mass spectra analysis and interpretation. Current database searching programs can exhibit discrepancies due to the unique properties of endogenous peptides, which typically require specialized search considerations. Herein, we present a high throughput, novel scoring algorithm for the extraction and ranking of conserved amino acid sequence motifs within any endogenous peptide database. Motifs are conserved patterns across organisms, representing sequence moieties crucial for biological functions, including maintenance of homeostasis. MotifQuest, our novel motif database generation algorithm, is designed to work in partnership with EndoGenius, a program optimized for database searching of endogenous peptides and that is powered by a motif database to capitalize on biological context to produce identifications. MotifQuest aims to quickly develop motif databases without any prior knowledge, a laborious task not possible with traditional sequence alignment resources. In this work we illustrate the utility of MotifQuest to expand EndoGenius' identification utility to other endogenous peptides by showcasing its ability to identify antimicrobial peptides. Additionally, we discuss the potential utility of MotifQuest to parse out motifs from a FASTA database file that can be further validated as new peptide drug candidates.

Functional Roles of the Conserved Amino Acid Sequence Motif C, the Antiporter Motif, in Membrane Transporters of the Major Facilitator Superfamily.

The biological membrane surrounding all living cells forms a hydrophobic barrier to the passage of biologically important molecules. Integral membrane proteins called transporters circumvent the cellular barrier and transport molecules across the cell membrane. These molecular transporters enable the uptake and exit of molecules for cell growth and homeostasis. One important collection of related transporters is the major facilitator superfamily (MFS). This large group of proteins harbors passive and secondary active transporters. The transporters of the MFS consist of uniporters, symporters, and antiporters, which share similarities in structures, predicted mechanism of transport, and highly conserved amino acid sequence motifs. In particular, the antiporter motif, called motif C, is found primarily in antiporters of the MFS. The antiporter motif's molecular elements mediate conformational changes and other molecular physiological roles during substrate transport across the membrane. This review article traces the history of the antiporter motif. It summarizes the physiological evidence reported that supports these biological roles.

Uncovering DNA-PKcs ancient phylogeny, unique sequence motifs and insights for human disease

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a key member of the phosphatidylinositol-3 kinase-like (PIKK) family of protein kinases with critical roles in DNA-double strand break repair, transcription, metastasis, mitosis, RNA processing, and innate and adaptive immunity. The absence of DNA-PKcs from many model organisms has led to the assumption that DNA-PKcs is a vertebrate-specific PIKK. Here, we find that DNA-PKcs is widely distributed in invertebrates, fungi, plants, and protists, and that threonines 2609, 2638, and 2647 of the ABCDE cluster of phosphorylation sites are highly conserved amongst most Eukaryotes. Furthermore, we identify highly conserved amino acid sequence motifs and domains that are characteristic of DNA-PKcs relative to other PIKKs. These include residues in the Forehead domain and a novel motif we have termed YRPD, located in an α helix C-terminal to the ABCDE phosphorylation site loop. Combining sequence with biochemistry plus structural data on human DNA-PKcs unveils conserved sequence and conformational features with functional insights and implications. The defined generally progressive DNA-PKcs sequence diversification uncovers conserved functionality supported by Evolutionary Trace analysis, suggesting that for many organisms both functional sites and evolutionary pressures remain identical due to fundamental cell biology. The mining of cancer genomic data and germline mutations causing human inherited disease reveal that robust DNA-PKcs activity in tumors is detrimental to patient survival, whereas germline mutations compromising function are linked to severe immunodeficiency and neuronal degeneration. We anticipate that these collective results will enable ongoing DNA-PKcs functional analyses with biological and medical implications.

Functional and Structural Roles of the Major Facilitator Superfamily Bacterial Multidrug Efflux Pumps.

Pathogenic microorganisms that are multidrug-resistant can pose severe clinical and public health concerns. In particular, bacterial multidrug efflux transporters of the major facilitator superfamily constitute a notable group of drug resistance mechanisms primarily because multidrug-resistant pathogens can become refractory to antimicrobial agents, thus resulting in potentially untreatable bacterial infections. The major facilitator superfamily is composed of thousands of solute transporters that are related in terms of their phylogenetic relationships, primary amino acid sequences, two- and three-dimensional structures, modes of energization (passive and secondary active), and in their mechanisms of solute and ion translocation across the membrane. The major facilitator superfamily is also composed of numerous families and sub-families of homologous transporters that are conserved across all living taxa, from bacteria to humans. Members of this superfamily share several classes of highly conserved amino acid sequence motifs that play essential mechanistic roles during transport. The structural and functional importance of multidrug efflux pumps that belong to the major facilitator family and that are harbored by Gram-negative and -positive bacterial pathogens are considered here.

Structures of teixobactin-producing nonribosomal peptide synthetase condensation and adenylation domains.

The recently discovered antibiotic teixobactin is produced by uncultured soil bacteria. The antibiotic inhibits cell wall synthesis of Gram-positive bacteria by binding to precursors of cell wall building blocks, and therefore it is thought to be less vulnerable to development of resistance. Teixobactin is synthesized by two nonribosomal peptide synthetases (NRPSs), encoded by txo1 and txo2 genes. Like other NRPSs, the Txo1 and Txo2 synthetases are large, multifunctional, and comprised of several modules. Each module is responsible for catalysis of a distinct step of teixobactin synthesis and contains specific functional units, commonly including a condensation (C) domain, an adenylation (A) domain, and a peptidyl carrier protein (PCP) domain. Here we report the structures of the C-A bidomains of the two L-Ser condensing modules, from Txo1 and Txo2, respectively. In the structure of the C domain of the L-Ser subunit of Txo1, a large conformational change is observed, featuring an outward swing of its N-terminal α-helix. This repositioning, if functionally validated, provides the necessary conformational change for the condensation reaction in C domain, and likely represents a regulatory mechanism. In an Acore subdomain, a well-coordinated Mg2+ cation is observed, which is required in the adenylation reaction. The Mg2+-binding site is defined by a largely conserved amino acid sequence motif and is coordinated by the α-phosphate group of AMP (or ATP) when present, providing some structural evidence for the role of the metal cation in the catalysis of A domain.

Pacing across the membrane: the novel PACE family of efflux pumps is widespread in Gram-negative pathogens

The proteobacterial antimicrobial compound efflux (PACE) family of transport proteins was only recently described. PACE family transport proteins can confer resistance to a range of biocides used as disinfectants and antiseptics, and are encoded by many important Gram-negative human pathogens. However, we are only just beginning to appreciate the range of functions and the mechanism(s) of transport operating in these proteins. Genes encoding PACE family proteins are typically conserved in the core genomes of bacterial species rather than on recently acquired mobile genetic elements, suggesting that they confer important core functions in addition to biocide resistance. Three-dimensional structural information is not yet available for PACE family proteins. However, PACE proteins have several very highly conserved amino acid sequence motifs that are likely to be important for substrate transport. PACE proteins also display strong amino acid sequence conservation between their N and C-terminal halves, suggesting that they evolved by duplication of an ancestral protein comprised of two transmembrane helices. In light of their drug resistance functions in Gram-negative pathogens, PACE proteins should be the subject of detailed future investigation.

Defining Electron Bifurcation in the Electron-Transferring Flavoprotein Family.

Electron bifurcation is the coupling of exergonic and endergonic redox reactions to simultaneously generate (or utilize) low- and high-potential electrons. It is the third recognized form of energy conservation in biology and was recently described for select electron-transferring flavoproteins (Etfs). Etfs are flavin-containing heterodimers best known for donating electrons derived from fatty acid and amino acid oxidation to an electron transfer respiratory chain via Etf-quinone oxidoreductase. Canonical examples contain a flavin adenine dinucleotide (FAD) that is involved in electron transfer, as well as a non-redox-active AMP. However, Etfs demonstrated to bifurcate electrons contain a second FAD in place of the AMP. To expand our understanding of the functional variety and metabolic significance of Etfs and to identify amino acid sequence motifs that potentially enable electron bifurcation, we compiled 1,314 Etf protein sequences from genome sequence databases and subjected them to informatic and structural analyses. Etfs were identified in diverse archaea and bacteria, and they clustered into five distinct well-supported groups, based on their amino acid sequences. Gene neighborhood analyses indicated that these Etf group designations largely correspond to putative differences in functionality. Etfs with the demonstrated ability to bifurcate were found to form one group, suggesting that distinct conserved amino acid sequence motifs enable this capability. Indeed, structural modeling and sequence alignments revealed that identifying residues occur in the NADH- and FAD-binding regions of bifurcating Etfs. Collectively, a new classification scheme for Etf proteins that delineates putative bifurcating versus nonbifurcating members is presented and suggests that Etf-mediated bifurcation is associated with surprisingly diverse enzymes.IMPORTANCE Electron bifurcation has recently been recognized as an electron transfer mechanism used by microorganisms to maximize energy conservation. Bifurcating enzymes couple thermodynamically unfavorable reactions with thermodynamically favorable reactions in an overall spontaneous process. Here we show that the electron-transferring flavoprotein (Etf) enzyme family exhibits far greater diversity than previously recognized, and we provide a phylogenetic analysis that clearly delineates bifurcating versus nonbifurcating members of this family. Structural modeling of proteins within these groups reveals key differences between the bifurcating and nonbifurcating Etfs.

Identification and characterization of two selenium-dependent glutathione peroxidase 1 isoforms from Larimichthys crocea

Glutathione peroxidases, a vital family of antioxidant enzymes in oxybiotic organisms, are involved in anti-pathogen immune response. In this study, two complete selenium-dependent glutathione peroxidase 1 cDNAs (designated as LcGPx1a and LcGPx1b) were obtained from the large yellow croaker Larimichthys crocea by rapid amplification of cDNA ends. The full-length sequence of LcGPx1a was 917 bp with a 5′-untranslated region (UTR) of 52 bp, a 3′-UTR of 289 bp, and an open reading frame of 576 bp encoding 191 amino acid (aa) polypeptides. The cDNA of LcGPx1b was composed of 884 bp with a 5′-UTR of 59 bp, a 3′-UTR of 258 bp, and an open reading frame of 567 bp encoding 188 aa polypeptides. The conserved selenocysteine insertion sequence was detected in the 3′-UTR of both isoforms, which can classify types I and II. Protein sequence analysis revealed that both isoforms included a selenocysteine encoded by an opal codon (TGA) and formed the functioning tetrad site with glutamine, tryptophan, and asparagine. Three conservative motifs, including one active site motif (“GKVVLIENVASLUGTT”) and two signature site motifs (“LVILGVPCNQFGHQENC” and “V(A/S)WNFEKFLI”), were conserved both in sequence and location. Multiple alignments revealed that they exhibited a high level of identities with GPx1 from other organisms, especially in the abovementioned conserved amino acid sequence motifs. Tissue expression analysis indicated that LcGPx1a and LcGPx1b had a wide distribution in nine tissues with various abundances. The transcript level of LcGPx1a was not significantly different among the nine tissues, whereas that of LcGPx1b was higher in the kidney and head kidney than in the other tissues. After Vibrio parahaemolyticus stimulation, the expression levels of LcGPx1a and LcGPx1b were unanimously altered in the liver, spleen, kidney, and head kidney but with different magnitudes and response time. LcGPx1a and LcGPx1b showed distinct expression trends in the liver, where LcGPx1b was induced and LcGPx1a was depressed in response to pathogen infection. These results indicate that LcGPx1a and LcGPx1b display functional diversities and play crucial roles in mediating the immune response of fish.

Ion and inhibitor binding of the double-ring ion selectivity filter of the mitochondrial calcium uniporter

The calcium (Ca2+) uniporter of mitochondria is a holocomplex consisting of the Ca2+-conducting channel, known as mitochondrial calcium uniporter (MCU), and several accessory and regulatory components. A previous electrophysiology study found that the uniporter has high Ca2+ selectivity and conductance and this depends critically on the conserved amino acid sequence motif, DXXE (Asp-X-X-Glu) of MCU. A recent NMR structure of the MCU channel from Caenorhabditis elegans revealed that the DXXE forms two parallel carboxylate rings at the channel entrance that seem to serve as the ion selectivity filter, although direct ion interaction of this structural motif has not been addressed. Here, we use a paramagnetic probe, manganese (Mn2+), to investigate ion and inhibitor binding of this putative selectivity filter. Our paramagnetic NMR data show that mutants with a single carboxylate ring, NXXE (Asn-X-X-Glu) and DXXQ (Asp-X-X-Gln), each can bind Mn2+ specifically, whereas in the WT the two rings bind Mn2+ cooperatively, resulting in ∼1,000-fold higher apparent affinity. Ca2+ can specifically displace the bound Mn2+ at the DXXE site in the channel. Furthermore, titrating the sample with the known channel inhibitor ruthenium 360 (Ru360) can displace Mn2+ binding from the solvent-accessible Asp site but not the inner Glu site. The NMR titration data, together with structural analysis of the DXXE motif and molecular dynamics simulation, indicate that the double carboxylate rings at the apex of the MCU pore constitute the ion selectivity filter and that Ru360 directly blocks ion entry into the filter by binding to the outer carboxylate ring.

Pore Architecture and Ion Selectivity Filter of the Mitochondrial Calcium Uniporter

The calcium uniporter of mitochondria is a holocomplex consisting of the calcium-conducting channel, known as the MCU channel, and a score of regulatory proteins. Previous electrophysiology study found that a preeminent property of the uniporter is the high calcium selectivity and conductance and this has been shown to critically depend on the highly conserved amino acid sequence motif, DXXE, in the MCU channel. Our recent NMR data of the MCU channel indicate that the DXXE forms two parallel carboxylate rings at the channel entrance that appear to serve as the ion selectivity filter. We used paramagnetic probes to show that mutants with a single carboxylate ring can bind divalent cation specifically, while in the wild type, the two rings bind the ion cooperatively, resulting in drastically higher apparent affinity. Our new data, together with structural analysis of the DXXE motif, indicate that the double carboxylate rings at the apex of the MCU pore constitute the ion selectivity filter of the channel and that Ru360 directly blocks ion entry into the filter.

The early history and emergence of molecular functions and modular scale-free network behavior.

The formation of protein structural domains requires that biochemical functions, defined by conserved amino acid sequence motifs, be embedded into a structural scaffold. Here we trace domain history onto a bipartite network of elementary functional loop sequences and domain structures defined at the fold superfamily level of SCOP classification. The resulting ‘elementary functionome’ network and its loop motif and structural domain graph projections create evolutionary ‘waterfalls’ describing the emergence of primordial functions. Waterfalls reveal how ancient loops are shared by domain structures in two initial waves of functional innovation that involve founder ‘p-loop’ and ‘winged helix’ domain structures. They also uncover a dynamics of modular motif embedding in domain structures that is ongoing, which transfers ‘preferential’ cooption properties of ancient loops to emerging domains. Remarkably, we find that the emergence of molecular functions induces hierarchical modularity and power law behavior in network evolution as the network of motifs and structures expand metabolic pathways and translation.

Transcriptomics-based identification of WRKY genes and characterization of a salt and hormone-responsive PgWRKY1 gene in Panax ginseng.

WRKY proteins belong to a transcription factor (TF) family and play dynamic roles in many plant processes, including plant responses to abiotic and biotic stresses, as well as secondary metabolism. However, no WRKY gene in Panax ginseng C.A. Meyer has been reported to date. In this study, a number of WRKY unigenes from methyl jasmonate (MeJA)-treated adventitious root transcriptome of this species were identified using next-generation sequencing technology. A total of 48 promising WRKY unigenes encoding WRKY proteins were obtained by eliminating wrong and incomplete open reading frame (ORF). Phylogenetic analysis reveals 48 WRKY TFs, including 11 Group I, 36 Group II, and 1 Group III. Moreover, one MeJA-responsive unigene designated as PgWRKY1 was cloned and characterized. It contains an entire ORF of 1077 bp and encodes a polypeptide of 358 amino acid residues. The PgWRKY1 protein contains a single WRKY domain consisting of a conserved amino acid sequence motif WRKYGQK and a C2H2-type zinc-finger motif belonging to WRKY subgroup II-d. Subcellular localization of PgWRKY1-GFP fusion protein in onion and tobacco epidermis cells revealed that PgWRKY1 was exclusively present in the nucleus. Quantitative real-time polymerase chain reaction analysis demonstrated that the expression of PgWRKY1 was relatively higher in roots and lateral roots compared with leaves, stems, and seeds. Importantly, PgWRKY1 expression was significantly induced by salicylic acid, abscisic acid, and NaCl, but downregulated by MeJA treatment. These results suggested that PgWRKY1 might be a multiple stress-inducible gene responding to hormones and salt stresses.

OsSIDP366, a DUF1644 gene, positively regulates responses to drought and salt stresses in rice.

Domain of unknown function 1644 (DUF1644) is a highly conserved amino acid sequence motif present only in plants. Analysis of expression data of the family of DUF1644-containing genes indicated that they may regulate responses to abiotic stress in rice. Here we present our discovery of the role of OsSIDP366, a member of the DUF1644 gene family, in response to drought and salinity stresses in rice. Transgenic rice plants overexpressing OsSIDP366 showed enhanced drought and salinity tolerance and reduced water loss as compared to that in the control, whereas plants with downregulated OsSIDP366 expression levels using RNA interference (RNAi) were more sensitive to salinity and drought treatments. The sensitivity to abscisic acid (ABA) treatment was not changed in OsSIDP366-overexpressing plants, and OsSIDP366 expression was not affected in ABA-deficient mutants. Subcellular localization analysis revealed that OsSIDP366 is presented in the cytoplasmic foci that colocalized with protein markers for both processing bodies (PBs) and stress granules (SGs) in rice protoplasts. Digital gene expression (DGE) profile analysis indicated that stress-related genes such as SNAC1, OsHAK5 and PRs were upregulated in OsSIDP366-overexpressing plants. These results suggest that OsSIDP366 may function as a regulator of the PBs/SGs and positively regulate salt and drought resistance in rice.

In Silico Analysis for Five Major Cereal Crops Phytocystatins.

Five major cereal crops such as rice, wheat, maize, barley and sorghum are continuously threatened by a multitude of pathogens and other disorders. Cystatins offers a pivotal role in deciding the promising plant response. The use of bioinformatics tools for phylogenetic relationships of five major cereal crop (rice, wheat, maize, barley and sorghum) phytocystatins based on amino acid sequence information was elucidated, and their secondary and tertiary structures were investigated for structural comparisons. Twenty-eight distinct phytocystatins from 28 plant species were investigated. Phytocystatins could be divided into five distinct phylogenetic groups. Five major cereal crops their structural features were highly conserved, and their amino acid sequence similarities ranged from 48 to 86 %. A new highly conserved amino acid sequence motif, YEAKxWxKxF, in the C-terminal end being unique to phytocystatins was identified. The predicted 3D homology models showed a high conservation of the general central structure of the phytocystatins, i.e., the 4-5 anti-parallel [Formula: see text]-sheets, wrapping halfway round a single central [Formula: see text]-helix and particularly the three active site regions, the N-terminal, the first and second hairpin loops. Any structural differences seem to be mainly in the length of the N- and C-terminal, the length of the second hairpin loop and the fifth [Formula: see text]-sheet. Via docking experiments, small heterogeneities were observed in the vicinity of the OC-I active sites that seemed to be influential in the binding process and stability of the resultant inhibitor-protease complex.

Annotation and expression analysis of cuticular proteins from the tobacco hornworm, Manduca sexta

The insect cuticle is a unique material that covers the exterior of the animal as well as lining the foregut, hindgut, and tracheae. It offers protection from predators and desiccation, defines body shape, and serves as an attachment site for internal organs and muscle. It has demonstrated remarkable variations in hardness, flexibility and elasticity, all the while being light weight, which allows for ease of movement and flight. It is composed primarily of chitin, proteins, catecholamines, and lipids. Proteomic analyses of cuticle from different life stages and species of insects has allowed for a more detailed examination of the protein content and how it relates to cuticle mechanical properties. It is now recognized that several groups of cuticular proteins exist and that they can be classified according to conserved amino acid sequence motifs. We have annotated the genome of the tobacco hornworm, Manduca sexta, for genes that encode putative cuticular proteins that belong to seven different groups: proteins with a Rebers and Riddiford motif (CPR), proteins analogous to peritrophins (CPAP), proteins with a tweedle motif (CPT), proteins with a 44 amino acid motif (CPF), proteins that are CPF-like (CPFL), proteins with an 18 amino acid motif (18 aa), and proteins with two to three copies of a C-X5-C motif (CPCFC). In total we annotated 248 genes, of which 207 belong to the CPR family, the most for any insect genome annotated to date. Additionally, we discovered new members of the CPAP family and determined that orthologous genes are present in other insects. We established orthology between the M. sexta and Bombyx mori genes and identified duplication events that occurred after separation of the two species. Finally, we utilized 52 RNAseq libraries to ascertain gene expression profiles that revealed commonalities and differences between different tissues and developmental stages.

Plastid proteome prediction for diatoms and other algae with secondary plastids of the red lineage.

The plastids of ecologically and economically important algae from phyla such as stramenopiles, dinoflagellates and cryptophytes were acquired via a secondary endosymbiosis and are surrounded by three or four membranes. Nuclear-encoded plastid-localized proteins contain N-terminal bipartite targeting peptides with the conserved amino acid sequence motif ‘ASAFAP’. Here we identify the plastid proteomes of two diatoms, Thalassiosira pseudonana and Phaeodactylum tricornutum, using a customized prediction tool (ASAFind) that identifies nuclear-encoded plastid proteins in algae with secondary plastids of the red lineage based on the output of SignalP and the identification of conserved ‘ASAFAP’ motifs and transit peptides. We tested ASAFind against a large reference dataset of diatom proteins with experimentally confirmed subcellular localization and found that the tool accurately identified plastid-localized proteins with both high sensitivity and high specificity. To identify nucleus-encoded plastid proteins of T. pseudonana and P. tricornutum we generated optimized sets of gene models for both whole genomes, to increase the percentage of full-length proteins compared with previous assembly model sets. ASAFind applied to these optimized sets revealed that about 8% of the proteins encoded in their nuclear genomes were predicted to be plastid localized and therefore represent the putative plastid proteomes of these algae.

In silico analysis for five major cereal crops phytocystatins.

Five major cereal crops like rice, wheat, maize, barley and sorghum are continuously threatened by a multitude of pathogens and other disorders. Cystatins offers a pivotal role in deciding the promising plant response. The use of bioinformatics tools for phylogenetic relationships of five major cereal crops (rice, wheat, maize, barley and sorghum) phytocystatins based on amino acid sequence information was elucidated and their secondary and tertiary structures were investigated for structural comparisons. Twenty eight distinct phytocystatins from 28 plant species were investigated. Phytocystatins could be divided into five distinct phylogenetic groups. Five major cereal crops their structural features were highly conserved their amino acid sequence similarities ranged from 48 to 86%. A new highly conserved amino acid sequence motif, YEAKxWxKxF, in the C-terminal end being unique to phytocystatins was identified. The predicted 3D homology models showed a high conservation of the general central structure of the phytocystatins i.e. the 4-5 anti-parallel β-sheets, wrapping halfway round a single central α-helix and particularly the three active site regions, the N-terminal, the 1st and 2nd hairpin loops. Any structural differences seem to be mainly in the length of the N and C terminal, the length of the 2nd hairpin loop and the 5th β-sheet. Via docking experiments, small heterogeneities were observed in the vicinity of the OC-I active sites that seemed to be influential in the binding process and stability of the resultant inhibitor-protease complex.

Conserved motifs of MutL proteins

The MutL protein is best known for its function in DNA mismatch repair (MMR). However, there is evidence to suggest that MutL is not only the linker connecting the functions of MutS and MutH in MMR, but that it also participates in other repair systems, such as Very Short Patch (VSP), Base Excision (BER) and Nucleotide Excision Repair (NER). This study set out to identify the most highly conserved amino acid sequence motifs in MutL proteins.We analyzed 208 MutL amino acid sequences of 199 representative prokaryotic species belonging to 28 classes of bacteria and archaea. The analysis revealed 16 conserved motifs situated in the ATPase and endonuclease domains, as well as within the disordered loop, and in the MutL regions interacting with the β clamp of DNA polymerase III. The conserved sequence motifs thus determined constitute a structural definition of MutL and they may be used in site-directed mutagenesis studies. We found conserved residues within the potential regions where binding with MutS occurs. However, the existing data does not provide clues as to the possible sites of MutL interactions with the proteins involved in other DNA repair systems such as NER, BER and VSP.We determined the 57 most highly conserved amino acid residues, including 43 which were identical in all the sequences analyzed. The greater part of the most predominantly conserved amino acid residues identified in MutL are identical to the corresponding residues reported as mutational hot-spots in one of its human homologues, MLH1, but not in the other, PMS2.This is the first study to present the conserved sequence motifs of MutL widespread in bacteria and archaea and the classification of MutLs into five groups distinguished on the basis of differences in the C-terminal region. Our analysis is of use in better understanding MutL functions.

Novel arrangement and comparative analysis of hsp90 family genes in three thermotolerant species of Stratiomyidae (Diptera)

The heat shock proteins belonging to the Hsp90 family (Hsp83 in Diptera) play a crucial role in the protection of cells due to their chaperoning functions. We sequenced hsp90 genes from three species of the family Stratiomyidae (Diptera) living in thermally different habitats and characterized by extraordinarily high thermotolerance. The sequence variation and structure of the hsp90 family genes were compared with previously described features of hsp70 copies isolated from the same species. Two functional hsp83 genes were found in the species studied, that are arranged in tandem orientation at least in one of them. This organization was not previously described. Stratiomyidae hsp83 genes share a high level of identity with hsp83 of Drosophila, and the deduced protein possesses five conserved amino acid sequence motifs characteristic of the Hsp90 family as well as the C-terminus MEEVD sequence characteristic of the cytosolic isoform. A comparison of the hsp83 promoters of two Stratiomyidae species from thermally contrasting habitats demonstrated that while both species contain canonical heat shock elements in the same position, only one of the species contains functional GAF-binding elements. Our data indicate that in the same species, hsp83 family genes show a higher evolution rate than the hsp70 family.

Characterization of the Intramolecular Interactions in the Circularly Permuted GTPase Nucleostemin

Nucleostemin is a unique protein that plays a role in cell cycle regulation, cellular stress sensing, telomere maintenance, and tumor suppression. Similar to other nucleolar proteins, compartmentalization plays a large role in regulating the activity of NS. The mechanism behind this redistribution is poorly defined but it is linked to the GTPase properties of the protein and an uncharacterized inhibitory region that directs complex formation between NS and various cellular partners. We have been studying the structural and biochemical properties of drosophilia NS1 to better understand the molecular basis of this dynamic localization process. The canonical GTPase core contains a six-stranded β sheet surrounded by 5 α helices. The nucleotide-binding site of the protein is defined by four conserved amino acid sequence motifs, the G-1 through G-4 boxes. cpGTPases are characterized by a reordering of the characteristic G boxes specifically to a G4-G1-G3 pattern, thus a circular permutation of the classic GTPase arrangement. There is little known about how altering the positioning of the G-boxes affects the nucleotide binding and regulatory properties of the protein. Work from our group and other laboratories demonstrated that cpGTPases have broad substrate specificity and can hydrolyze GTP and ATP. We have also identified several structural domains in dNS1 using limited proteolysis. Truncation constructs have been made to elucidate the role of each domain and how the physical interactions between these domains contribute to the biological properties of the protein. Taken together, these data point to a mechanism of action that differs from the larger GTPase family.