In this paper we describe an analysis of the size evolution of both protein domains and their indels, as inferred by changing sizes of whole domains or individual unaligned regions or "spacers". We studied relatively early evolutionary events and focused on protein domains which are conserved among various taxonomy groups.We found that more than one third of all domains have a statistically significant tendency to increase/decrease in size in evolution as judged from the overall domain size distribution as well as from the size distribution of individual spacers. Moreover, the fraction of domains and individual spacers increasing in size is almost twofold larger than the fraction decreasing in size.We showed that the tolerance to insertion and deletion events depends on the domain's taxonomy span. Eukaryotic domains are depleted in insertions compared to the overall test set, namely, the number of spacers increasing in size is about the same as the number of spacers decreasing in size. On the other hand, ancient domain families show some bias towards insertions or spacers which grow in size in evolution. Domains from several Gene Ontology categories also demonstrate certain tendencies for insertion or deletion events as inferred from the analysis of spacer sizes. Proteins evolve through gene duplication, diversification and domain shuffling, which allow novel proteins to emerge via different domain combinations. Many evolutionary mechanisms shaping protein sequence and structure can be probed by studying the length distributions of proteins and protein domains. Fusion of single domain proteins and domain accretion, for example, play an important role in the evolution of eukaryotic proteins -3; as a Diverse multi-domain proteins may consist of homologous domains and knowledge of protein domain evolution may considerably help in reconstructing the evolutionary history of entire proteins. Changes in protein domains result mostly from point mutations, insertion and deletion processes. Although amino acid insertion and deletion (indel) events in proteins are less frequent than amino acid substitutions -12, theyThe mechanisms of indel events are not very well understood and there are only few statistical models describing these events in evolution -20. TradIn this paper we analyze the size evolution of whole protein domains and indels in protein domains, as judged by changing sizes of whole domains or individual unaligned regions. We study intra-domain events which are not affected by domain shuffling and domain accretion in multidomain proteins. To examine these events on a wide scale of evolutionary distances we use the Conserved Domain Database (CDD)  that proProtein domain size evolution was studied by examining the correlation between the domain size  and the evolutionary distance from the root of the tree to a given node. We note that there are other domain classifications ,25 whichThe first example represents the Ribonuclease A family  will exhibit an increasing pattern and another half will decrease in size. However, we observed 71 cases instead of 51 and the probability to observe such bias given the above assumption can be estimated from the binomial distribution and is very small (P < 0.00005). We also estimated an average rate of domain size change which was found to be 7.2 (5.7) residues per domain per unit of evolutionary distance for domains of increasing and decreasing size respectively. Unlike for the number of domains, there is no statistically significant difference between the two average rates.-6 for enzymatic domains and a p-value < 3*10-5 for ancient domains. Thus, ubiquitous domains with enzymatic activity have a tendency to accommodate very long indels, a similar observation for enzymes having been reported earlier [Analyzing domain families with large spacers of more than 50 residues long , we investigated the functional and taxonomic assignments for these domains and found that 27 of them represent enzyme domains and 31 of them belong to the "Root" taxonomic category . The probability to observe such bias, given the assumption that families with "inserted domains" are distributed equally among different functional/taxonomic classes, can be estimated from the binomial distribution, which yields a p-value of < 10-11). Although the number of spacers which increase in size exceeds the number of spacers which decrease in size, the rate of spacer size change is approximately equal, 1.8(1.9) amino acids per spacer per unit of evolutionary distance.We assigned individual spacers to the functional and taxonomic categories of corresponding CDs and performed an analysis similar to that described in the previous section with the only difference that instead of domain sizes, the individual spacer lengths were mapped to the nodes of phylogenetic trees. Using this mapping we can calculate the regression coefficients for each individual spacer in each domain. Figure 2 statistics, Figure 2 value attributed to the eukaryotic spacers accounts for 46% of the overall χ2 value of the test. For bacterial families (which are underrepresented in our test set), the situation is reversed and the majority of spacers decrease in size  is not correlated with the sign of the regression coefficients. We showed that the null hypothesis is rejected with p-value < 0.0003, which indicates that different taxonomic groups overall have different tendencies in spacer size evolution. For example, the spacers from ancient domain families show some bias toward the spacers which grow in size  or shortage (blue) of observed families are highlighted. As can be seen from this table, proteins with functions related to transferase activity, replicative cell aging, positive regulation of cell proliferation and transcription all have a statistically significant excess of spacers increasing in size. On the other hand, domains from proteins located in the endoplasmic reticulum membrane have an excess of spacers decreasing in size. Proteins participating in chemotaxis, protein folding and G-protein coupled signaling pathways have mostly "stable" domains while proteins from the ubiquitin cycle and ubiquitin dependent catabolism, membrane fraction, mitosis and G-protein coupled signaling pathways are deprived of spacers decreasing in size. We also investigated whether spacer size increase/decrease can be attributed to the number of interactions with other proteins/domains. The analysis performed using conserved binding modes [Different protein domains can have different tolerances to insertion or deletion events depending on domain function and localization in a cell. We investigated this by annotating protein domains with respect to the Gene Ontology (GO) classification  and testng modes  did not Drosophila melanogaster pseudogenes [The collection of accurate, curated multiple sequence alignments from CDD gives us an opportunity to study the evolution of domain and spacer sizes on a wide scale of evolutionary distances. Thus far the indel events have been studied on sets of relatively closely related species such as human, mouse and rat, where indels can be defined explicitly. It has been found that there exists a 2–3 fold excess of deletions over insertions in non-coding regions (and pseudogenes in particular) from human and murids ,30-32 anudogenes . Moreoveudogenes . Proteinudogenes ,35. AlonFor the entire test set we observed a certain pattern for domains and spacers to increase in size on average, with a two-fold difference between the number of domains/spacers growing in size over those diminishing in size. The overall evolutionary scenario which we can portray based on our study is the following. It has been argued that it is unlikely that early proteins represented long peptide chains. On the contrary, various data suggest that the first protein domains emerged through the recombination of short peptides or a limited vocabulary of structural units ,36,37. AE. coli are 8 times more frequent than insertions [For relatively "modern" proteins, however, the trend of domain size evolution was rather different. Eukaryotic proteins started losing residues in spacers, indicating that in eukaryotic evolution deletions played as important a role as insertions. Eukaryotic novel proteins seem to evolve mainly through acquiring new domains and through domain shuffling which could result in longer proteins with slightly shorter individual domains. At the same time, in bacteria the deletion trend was even more pronounced  and, indeed, it was shown earlier that deletions in sertions . Such bisertions .There are different factors which would favor shorter or longer proteins or spacers in evolution -43. EffiThe analysis was performed on a set of protein families with curated alignments from the NCBI Conserved Domain Database (CDD). CDD comprises diverse non-redundant sequences and alignments are refined using three-dimensional structures and structure-structure alignments . CD aligThe domain families from the test set encompass a large spectrum of functional and taxonomic groups. Protein function was categorized by the Gene Ontology (GO) terms . Gene onPhylogenetic trees were constructed from the aligned block regions (in case of sequence repeats only one instance was kept) by the neighbor-joining method  with thespacer as a non-aligned segment between two consecutively aligned block elements. To analyze how spacer lengths change in the course of evolution, the spacer lengths for all CDD sequences between two consecutive blocks were individually mapped to the external nodes of the phylogenetic tree for the corresponding domain family. To study the evolution of domain sizes, the sum of all spacer lengths in a CDD alignment  was mapped to the external nodes of the phylogenetic tree.Taking advantage of CDD block structure where multiple alignments are anchored at certain conserved positions, we define a The values of spacer lengths were inferred for the internal nodes of the phylogenetic trees using the following models. According to model #1, the spacer length for an internal node was inferred to be the same as the phylogenetically closest external node. Model #2 si is the spacer length at internal node i; s1 and s2 the spacer lengths assigned to the direct descendants of node i; and h1 and h2 the heights of the descendant subtrees. The height of a subtree is, in turn, recursively defined as the branch length plus the average height of two descendant subtrees .Where Using these models we calculated the Pearson correlation coefficients between the distance from the root to a given internal/external node and the value of the spacer length at a given node for all nodes in a tree. It should be noted that using just internal nodes in the correlation analysis does not change the results significantly. For the domain length analysis, the correlation was calculated between the evolutionary distance from the root and the sum of spacer length. The p-values for the correlation coefficients were estimated under the null hypothesis of being equal to zero; correlations for those families with p-values less than 0.01 were considered significant. The regression coefficients were calculated by linear regression analysis.YW and AP have made major contributions to conception, design analysis and interpretation of data. AP conceived of the study and wrote the first draft of the manuscript. TM was involved in the data analysis and revising the manuscript. VB participated in the statistical analysis and BS carried out comparisons with protein interactions. All authors have read and approved the final manuscript.


Specific protein associations define the wiring of protein interaction networks and thus control the organization and functioning of the cell as a whole. Peptide recognition by PDZ and other protein interaction domains represents one of the best-studied classes of specific protein associations. However, a mechanistic understanding of the relationship between selectivity and promiscuity commonly observed in the interactions mediated by peptide recognition modules as well as its functional meaning remain elusive. To address these questions in a comprehensive manner, two large populations of artificial and natural peptide ligands of six archetypal PDZ domains from the synaptic proteins PSD95 and SAP97 were generated by target-assisted iterative screening (TAIS) of combinatorial peptide libraries and by synthesis of proteomic fragments, correspondingly. A comparative statistical analysis of affinity-ranked artificial and natural ligands yielded a comprehensive picture of known and novel PDZ ligand specificity determinants, revealing a hitherto unappreciated combination of specificity and adaptive plasticity inherent to PDZ domain recognition. We propose a reconceptualization of the PDZ domain in terms of a complex adaptive system representing a flexible compromise between the rigid order of exquisite specificity and the chaos of unselective promiscuity, which has evolved to mediate two mutually contradictory properties required of such higher order sub-cellular organizations as synapses, cell junctions, and others – organizational structure and organizational plasticity/adaptability. The generalization of this reconceptualization in regard to other protein interaction modules and specific protein associations is consistent with the image of the cell as a complex adaptive macromolecular system as opposed to clockwork. Approximately 90 amino acids long, PDZ domain was first discovered as sequence repeats in the primary structures of the post-synaptic density 95  is an archetypal member of the synapse-associated protein (SAP) family of scaffolding molecules comprising PSD95/SAP90, SAP97, SAP102 and PSD93/chapsyn110. SAP proteins function as key organizers that control synaptic composition, organization and function Over the 15 years since the discovery of PDZ domains, the biochemistry and structural basis of PDZ domain recognition as well as the biology of PDZ domain-containing proteins have been subjects of numerous studies, which are summarized in a number of reviews The first uncertainty is illustrated by the continual but so far failed attempts to classify PDZ domains in accord with their specificities  or enzyme-linked immunosorbent assays (ELISA) in vivo situation better and what affinities should be considered as physiologically relevant for PDZ domain-mediated interactions The widely diverse affinities reported for PDZ domain-peptide interactions, spanning more than three orders of magnitude, represent another source of confusion. Because in-solution methods, such as fluorescence polarization (FP), tend to estimate PDZ domain-peptide interaction affinities in the low micromolar range, well within the affinity range expected from protein interaction domains mediating transient specific associations inside the cell, they are generally perceived as more trustworthy than the It should be emphasized that the ambiguities detailed above for the PDZ domain family are common, to a larger or smaller degree, to all the peptide recognition domain families To address the above-mentioned questions in a comprehensive manner, we applied a number of novel biochemical and statistical approaches to generate and analyze large populations of peptide ligands for a number of well-studied PDZ domains. The results of this study reveal a hitherto unappreciated combination of specificity and adaptive plasticity inherent to PDZ domain recognition. The complexity of PDZ domain recognition and the seemingly contradictory and/or confusing observations accumulated in the field are reconciled within a novel, if unexpected, image of the PDZ domain emerging as a complex adaptive system evolved to ensure both structure and organizational plasticity of higher order dynamic macromolecular systems such as synapses, cell junctions, and others.This study capitalizes on distinct advantages of the novel screening format for phage-displayed peptide libraries, target-assisted iterative screening (TAIS), introduced recently and described elsewhere Visual examination of binding histograms suggests that 1) the recognition specificities of all six domains examined are very similar, albeit not identical; 2) the second domains of both proteins are noticeably more promiscuous than their first and third domains; and 3) the specificities of homologous domains across different proteins appear to be more similar than the specificities of the domains belonging to the same protein. The first two observations are in agreement with the established body of experimental evidence residue-frequency-patterning (RFP) algorithm described recently To delineate recognition preferences of the target PDZ domains, the last sixteen C-terminal amino acids of the peptide ligands selected in TAIS screens were analyzed using the The relative frequencies of four residues, valine, arginine, threonine and serine are twice as high as expected. Since the probability of such frequencies arising by chance is vanishingly low , it is fair to hypothesize that V, R, T and S are the ligand residues that are preferred at the domain-ligand interaction interface and thus are likely to be important for binding to the target PDZ domains. The relative overrepresentation of V, T and S does not come as a surprise, as the known minimal recognition consensus of the PSD95 PDZ domains is X-(S/T)-X-(V/I/L)-COOH To explore the relationships between amino acid frequencies at specific ligand positions and the strength of PDZ domain-ligand interactions we arranged peptide ligands into four groups in accord with their relative affinities: 1) best binders ; 2) good binders ; 3) moderate binders  and 4) weak binders  see . IndepenAs the described positional patterns of overrepresented residues hold for all six target domains (not shown), it is fair to conclude that the general recognition consensus of the PSD95 and SAP97 PDZ domains is X-R-E-(T/S)-X-V-COOH. Indeed, this inferred consensus represents a refinement of the well-known minimal recognition consensus of the class I PDZ domains, X-(S/T)-X-(V/I/L)-COOH, first defined for the PSD95 PDZ domains through analysis of C-terminal sequences in the PSD95 interacting partners in vitro, using the peptide ELISA assay A search of SWISS-PROT and TrEMBL databases with the described above queries gave 126 potential interacting partners for the PSD95 PDZ domains  receptor subunits NR2A and NR2B with their C-terminal sequences KKMPSIESDV-COOH and EKLSSIESDV-COOH, respectively, or voltage-gated potassium channel subunits Kv1.1, Kv1.2 and Kv1.3 , bind to the first two PSD95 PDZ domains but fail to interact with the third PSD95 PDZ domain In order to gain insight into the pattern recognition differences of the PDZ domains that belong to the same class but are able to differentiate between peptide ligands sharing the class-defining C-terminal consensus, we investigated a particular case of this general puzzle, namely, the differences in recognition preferences between the second and third PDZ domains of PSD95, which are classified as type I PDZ domains but are known to discriminate between various X-(S/T)-X-(V/I/L)-COOH ligands To generalize, we suggest that by increasing statistical power one is likely to detect and define robust differences in pattern recognition between any pair of individual PDZ domains. Indeed, the comparison of the natural ligands exhibiting several-fold preference, in terms of relative affinity, for the PSD95-PDZ2 domain over the PSD95-PDZ1 domain with the best binders of the latter clearly suggests that these domains are also capable of differential recognition . It appeSummarizing the recognition patterns of the PSD95 and SAP97 PDZ domains inferred from the comparative analysis of amino acid organization of their affinity-ranked artificial and natural ligands, the PSD95 and SAP97 PDZ domains prefer positively charged residues, lysine and arginine, in the positional window “−4 to −7”, while strongly favoring lysine or arginine at the position “−4”, glutamate at the position “−3”, threonine at the position “−2” and valine at the position “0”. Even though the ligand position “−1” appears to accept various residues, it is apparently used for discrimination between individual PDZ domains – lysines and arginines are disfavored in this position by the first two PDZ domains . All the constructs were verified by sequencing. Expression and protein purification of GST fusions were performed in accord with manufacturer's instructions. The detailed protein purification protocols used in this work can be found at http://www.buckinstitute.org/TAIS. The synthetic biotinylated peptides corresponding to natural ligand sequences were purchased from JPT Peptide Technologies GmbH . Rabbit polyclonal anti-T7 antibodies were a generously gift from Dr. F. W. Studier, Brookhaven National Laboratory. Donkey anti-rabbit antibodies conjugated to horseradish peroxidase were purchased from Amersham Biosciences.The 16-mer random peptide library was generated in-house using the T7 phage display library construction kit from Novagen. The human brain cDNA library was purchased from Novagen. The GST fusion protein expression constructs of the PSD95-PDZ2, PSD95-PDZ3, SAP97-PDZ1 and SAP97-PDZ2 domains were kindly provided by Dr. B. K. Kay (The University of Illinois at Chicago). The GST fusion constructs of the PDS95-PDZ1 and SAP97-PDZ3 domains were generated by PCR amplification of the corresponding PDZ domain coding regions from SAP cDNAs ) followed by cloning into the pGEX2TK expression vector (Amersham Pharmacia). The PDZ domain borders were defined by the SMART software tools (20). The TAIS flowchart and protocols can be found on the Internet (http://www.buckinstitute.org/TAIS). Briefly, 30 µg of a GST-PDZ domain fusion immobilized on sepharose beads were blocked with 0.5% bovine serum albumin (BSA) in TBS-T  and incubated with a phage-displayed peptide library aliquot (approx. 108–109 pfu). After 90 minutes of incubation at room temperature (RT) the beads were thoroughly washed with TBS-T and bound phages were eluted with 200 µl of 1% SDS for 15 min at RT. Following elution, the phages were immediately mixed with a molten 0.6% top agarose containing host cells and plated onto two pre-warmed 150 mm agar plates. When phage plaques became visible, the plates were cooled down for 30 min at 4°C and overlaid with 132 mm nitrocellulose membranes (Schleicher & Schuell) for 5 min. Following plaque lift, the membranes were blocked in 1% BSA in TBS for 1 hour at RT and incubated overnight in 25 ml of TBS-T on a rocker at 4°C with 10 µg of the target PDZ domain that had been cleaved from the GST moiety, biotinylated and complexed with streptavidin – alkaline phosphatase (STRAP) at a ratio of 4:1. After extensive washing with TBS-T, positive plaques were developed on the membranes with insoluble alkaline phosphatase (AP) substrate BCIP/NBT (Sigma). Individual positive plaques were identified on the plates and phages from these plaques were propagated separately in the appropriate host for production of individual phage lysates. The identities of phage-displayed peptides were inferred by sequencing the library-specific DNA inserts amplified by PCR from the T7 phage display vector.A detailed description of the TAIS method is presented in Kurakin et al.  times with TBS-T. One hundred µl of freshly prepared individual phage lysate was added to the ELISA plate wells and incubated for 1 hour at RT. Following incubation, unbound phages were washed away with TBS-T and the amount of retained phages was determined with polyclonal T7 phage-specific antibodies followed by monoclonal anti-rabbit antibodies conjugated to horseradish peroxidase (HRP) (Amersham Pharmacia). The individual phage ELISA kinetics were followed and quantified colorimetrically using soluble HRP substrate (ABTS/H2O2). ELISA readings were taken on a SpectraMAX190 plate reader (Molecular Devices) at 405 nm. To ensure reproducibility, each of the individual phage ELISA binding histograms presented was obtained at least three times in at least three separate experiments performed by two different experimenters. The representative sets of binding histograms are shown in GST fusion-coated microtiter ELISA plates (COSTAR) were prepared by passive immobilization of 1 µg of the indicated GST fusion proteins  per well2O2) and measuring ELISA kinetic slopes. ELISA readings were taken on a SpectraMAX190 plate reader (Molecular Devices) at 405 nm. To ensure reproducibility, all peptide ELISA experiments presented were repeated at least three times in at least three separate experiments performed by two different experimenters. The representative sets of binding histograms are shown in Wells of microtiter plates were coated with 1 µg of the indicated GST-PDZ domain fusions, washed with TBS-T and blocked with 1% BSA in the same way as described above for phage ELISA. Individual biotinylated peptides (30 ng) were pre-incubated with 1 µg of streptavidin-HRP conjugate (Pierce) in 300 µl of TBS-T for 30 min at RT. One hundred µl of the peptide-streptavidin-HRP conjugate were added to 100 µl of TBS-T left in each coated well after the final wash of the protein immobilization/blocking procedure. Microtiter plates were incubated for 1 hour at RT, and then washed 5 (x1mL) times with TBS-T. The amounts of peptides retained were quantified colorimetrically by adding soluble HRP substrate  and from random peptide library (another 50%), we assumed that sampling was done from a population where individual amino acids are equally represented. We believe it is a reasonable, albeit coarse-grained, approximation both for random peptide library and for the cDNA library used, considering that about 90% of the peptides isolated from the latter represented frameshifts. It was assumed that natural ligands were sampled from a population where individual amino acid are distributed in accord with their average occurrence in the SWISS-PROT database (the v. 51.1 issue statistics). The chi-square and binomial tests Figure S1ADistribution of charged residues within affinity-ranked artificial ligands of the PSD95-PDZ2 and PSD95-PDZ3 domains. The aligned sequences of artificial peptide ligands are arranged in four groups based on their relative affinities to the indicated PDZ domains. The numbers in parentheses indicate the range of normalized phage ELISA values within a given affinity group. Arginines and lysines are highlighted green, while aspartic and glutamic acids are red. Upper panel - PSD95-PDZ2 ligands; lower panel - PSD95-PDZ3 ligands.(0.86 MB TIF)Click here for additional data file.Figure S1BDistribution of charged residues within affinity-ranked artificial ligands of the SAP97-PDZ1 and SAP97-PDZ2 domains. The aligned sequences of artificial peptide ligands are arranged in four groups based on their relative affinities to SAP PDZ domains. The numbers in parentheses indicate the range of normalized phage ELISA values within a given affinity group. Arginines and lysines are highlighted green, while aspartic and glutamic acids are red. Upper panel - SAP97-PDZ1 ligands; lower panel - SAP97-PDZ2 ligands.(0.80 MB TIF)Click here for additional data file.Figure S1CDistribution of charged residues within affinity-ranked artificial ligands of the SAP97-PDZ3 domain. The aligned sequences of artificial peptide ligands are arranged in four groups based on their relative affinities to SAP PDZ domains. The numbers in parentheses indicate the range of normalized phage ELISA values within a given affinity group. Arginines and lysines are highlighted green, while aspartic and glutamic acids are red.(0.41 MB TIF)Click here for additional data file.Table S1Artificial peptide ligands isolated from phage-displayed random peptide and cDNA libraries by TAIS using various SAP PDZ domains as targets(0.05 MB DOC)Click here for additional data file.Table S2Putative natural peptide ligands of SAP PDZ domains(0.06 MB DOC)Click here for additional data file.


Domains containing the β-grasp fold are utilized in a great diversity of physiological functions but their role, if any, in soluble or small molecule ligand recognition is poorly studied.Using sensitive sequence and structure similarity searches we identify a novel superfamily containing the β-grasp fold. They are found in a diverse set of proteins that include the animal vitamin B12 uptake proteins transcobalamin and intrinsic factor, the bacterial polysaccharide export proteins, the competence DNA receptor ComEA, the cob(I)alamin generating enzyme PduS and the Nqo1 subunit of the respiratory electron transport chain. We present evidence that members of this superfamily are likely to bind a range of soluble ligands, including B12. There are two major clades within this superfamily, namely the transcobalamin-like clade and the Nqo1-like clade. The former clade is typified by an insert of a β-hairpin after the helix of the β-grasp fold, whereas the latter clade is characterized by an insert between strands 4 and 5 of the core fold.Members of both clades within this superfamily are predicted to interact with ligands in a similar spatial location, with their specific inserts playing a role in the process. Both clades are widely represented in bacteria suggesting that this superfamily was derived early in bacterial evolution. The animal lineage appears to have acquired the transcobalamin-like proteins from low GC Gram-positive bacteria, and this might be correlated with the emergence of the ability to utilize B12 produced by gut bacteria.This article was reviewed by Andrei Osterman, Igor Zhulin, and Arcady Mushegian. The β-grasp fold (β-GF) was first recognized in ubiquitin and the immunoglobulin-binding (IG-binding) domains of Gram-positive cocci ,2. SinceAs part of our larger effort to understand the evolutionary and structural basis for the functional versatility of this widespread fold (for example see reference ) we wereTo identify potential novel versions of the β-GF that bind soluble ligands, we initiated comprehensive structural comparisons with various previously characterized members of the fold (see β-grasp fold in SCOP database) using the DALI program. Several of these searches retrieved the C-terminal domain of the transcobalamin protein with significant Z-scores. For example, searches initiated with MoaD proteins  retrieved the C-terminal domain of transcobalamin (PDB: 2bbc) with Z-scores of ~7. Transcobalamin is an animal-specific protein that binds cobalamin (vitamin B12), and is involved in its uptake by animal cells . TranscoTetraodon nigroviridus, gi: 47226456, region: 325–425) recovered closely related eukaryotic orthologs and paralogs fused to N-terminal α/α toroid modules (iteration 1), solo transcobalamin C-terminal domains with predicted signal peptides , and several prokaryotic proteins . In order to exhaustively recover all divergent homologs, we conducted transitive searches with all above-detected members and also evaluated all hits below the threshold of PSI-BLAST searches for the presence of potentially homologous domains. We also prepared HMMs and PSSMs from the alignment of this region of all proteins recovered with significant expect-values  and used these to search all completely sequenced genomes. These searches consistently retrieved hits to multiple sequence repeats in a group of bacterial cell-surface/secreted sugar-binding proteins involved in polysaccharide export with significant e-values . Inclusion of polysaccharide export proteins in profiles for further searches additionally recovered the N-terminal region of the ComEA family of DNA uptake receptors of Gram positive bacteria , PduS-like cobalamin reductases , the middle domain of the 51 kD subunit (F chain) of the NADPH-quinone oxidoreductase complex I  and the RnfC subunit of the oxidoreductases encoded by the bacterial Rnf operons [Rhodobacter RnfC, E: 10-6; iteration 14). This latter set of proteins was more similar to the homologous region recovered in the polysaccharide export proteins than to the transcobalamin C-terminal domain  searches for homologs using PSI-BLAST and the HMMER package respectively. In addition to orthologs of transcobalamin, intrinsic factor and solo C-terminal domains from fishes, these searches retrieved numerous prokaryotic proteins, which were either present as stand-alone β-GF domains or in large multidomain proteins. For example, a search initiated with the β-GF domain of puffer fish transcobalamin  as many members of this superfamily are known or predicted to bind soluble ligands (See below for further details).We hereinafter refer to the homologous β-GF domains found in all these proteins as the A comprehensive multiple alignment for the SLBB superfamily Figure  was prepThe alignment also helped us to classify the SLBB superfamily into several distinct families. The Transcobalamin C-terminal domain clade is unified by the presence of the above-described β-hairpin insert within the β-GF that plays an important role in contacting the ligand Fig . This β-The Nqo1-like clade includes at least two distinct families: 1) the first includes the NADPH-quinone oxidoreductase complex I subunit Nqo1 (51 kD/F chain), the RnfC oxidoreductase subunit, and the PduS-like cobalamin reductases. 2) The second family contains polysaccharide export proteins and the DNA receptor ComEA. This clade is unified by the presence of a small, often α-helical insert, in the "connector arm" between the fourth and fifth strands of the domain Figure . In someWhile the interaction between B12 and the transcobalamin-like SLBB domain involves the unique β-hairpin insert, key contacts are also contributed by the core fold  or C-terminal  to the SLBB , the FIVAR (Pfam entry: PF07554) sugar-binding domain , the fasciclin domain  and a β-propeller domain . Given that many of these domains are often involved in interactions with polysaccharides, they might play a role in tethering these proteins to the cell surface by binding peptidoglycan or capsular polysaccharides [Syntrophomonas the SLBB domain is found in putative extracellular enzymes fused to sulfite oxidase-like molybdopterin cofactor binding domain (e.g. gi: 71491441) [Most members of the transcobalamin C-terminal domain clade of the SLBB superfamily contain signal peptides, and several also contain the C-terminal Gram-positive anchor motif , suggestB Figure . As the charides -23. Oftecharides . These o1491441) . It is lMethanosarcina) and AraC-like families   Figure . These pIn the Nqo1-like clade, polysaccharide export proteins are predicted to be secreted or periplasmic proteins and contain an absolutely conserved N-terminal β-strand-rich domain followed by 1–8 repeats of the SLBB domain Figure . They apThe remaining three groups of proteins, namely NqoI, RnfC and PduS, within the Nqo1-like clade of the SLBB superfamily share a common architectural core consisting of a fusion between an N-terminal Rossmannoid domain and an SLBB domain. Unlike classical Rossmann fold domains of oxidoreductases with 5–7 strands, the Rossmannoid domain of these proteins has a 4-stranded core in a 3214 order  coupled The roles of these versions of the SLBB remain enigmatic; however there is evidence that the PduS protein might bind soluble ligands. The PduS gene typically belongs to a large mobile operon coding proteins required for the biogenesis of carboxysome/polyhedral bodies, which contains enzymes involved in propanediol degradation. The PduS has been shown to strongly bind cob(I)alamin and was characterized as a bifunctional cob(II)alamin and hydroxycobalamin (cob(III)alamin) reductase catalyzing the formation of cob(I)alamin. Cob(I)alamin is the immediate precursor of Ado-cobalamin, which serves as an essential coenzyme for the diol dehydratase in degradation of 1,2-propanediol ,31. It iThe phyletic and domain-architecture distributions show that the SLBB superfamily is well-represented and has diversified across the entire bacteria superkingdom. Their sporadic presence in archaea and the stronger affinity of the different eukaryotic versions to their bacterial counterparts suggests that the SLBB superfamily was derived early in evolution of bacteria, most probably from one of the many ancient β-grasp fold domains. The two major families in the NqoI-like clade appear to have a pan-bacterial distribution suggesting that this clade had already differentiated into versions associated with cell wall related functions (ComEA-Polysaccharide export protein family) and intracellular oxido-reductase related functions . Of the latter group the NqoI protein of the respiratory complex-I is seen across bacteria and was transferred to the eukaryotic lineage during the primary endosymbiotic event that generated the eukaryotic cell with mitochondria. RnfC and PduS proteins have more restricted phyletic distributions and are likely to be late derivatives of the more ancient Nqo1 lineage. The transcobalamin C-terminal clade is very divergent in sequence and appears to be a lineage-specific innovation in Gram positive bacteria that was recruited specifically for transporting extracellular B12-like cofactors or their precursors. Subsequently, a specific version that combined the α/α toroid domain with the SLBB domain appears to have been laterally transferred to the animal lineage early in its evolution. This event probably conferred on animals the ability to directly absorb cobalamin synthesized by bacteria in the gut. In parallel, there appear to have been sporadic transfers of large extracellular multidomain versions as well as intracellular versions fused to DNA-binding HTH domains to certain euryarchaeal lineages.In this context, it is of interest to note that the discovery of soluble ligand binding versions of the β-GF points to a noteworthy structure-function analogy with the RNA-recognition motif (RRM)-like fold. In functional terms, the RRM-like fold has long been known to bind a range of soluble ligands such as amino acids, sugars and co-factors. Notable examples of these include the ACT domain superfamily and the amino acid-binding domain of the LRP-like transcription factors ,35,36. BIn conclusion, we show that the β-GF domains found in transcobalamin, polysaccharide export proteins, ComEA, PduS, and RnfC and Nqo1-like oxidoreductases define a novel superfamily, several of which might interact with different soluble ligands. This investigation provides the possible evolutionary scenario for the origin of the vitamin B12 uptake in animals via transcobalamin and intrinsic factor. It also provides leads for new investigations into B12 metabolism in bacteria and other aspects of protein-ligand interaction in competence, cell-surface biochemistry, and respiratory electron transfer.Searches of the PDB database with query structures were conducted using the DALI program . StructuThe manuscript "A novel superfamily with the β-grasp fold involved in binding diverse soluble ligands" by A. M. Burroughs is an excellent and insightful comparative genomics study of a very interesting domain family broadly conserved across all kingdoms of Life. The authors took on a very challenging task of classifying multiple representatives of this superfamily on the edge of detectable homology. Such a task is particularly daunting for non-enzymatic proteins, where the divergence of sequence driven by adaptation to new biological tasks and contexts is much more pronounced than for enzymes. This study tracks down a complex evolution of the family and reveals an apparent functional theme, binding of small molecule ligands, such as B12 and its analogs, that "runs in the family" from bacteria to mammals. In addition to sensitive and sophisticated homology-based methods, the authors broadly used genome context analysis, which was one of the key success factors in pursuing this challenge. Of particular interest is the observation that in course of a fascinating evolutionary reshuffling, not only the ligand binding specificity, but the actual functional context of this domain could have changed on a number of occasions. One of the most remarkable events is a proposed recruitment of this domain into the context of transcriptional regulation in some bacteria. The latter theme of recruiting former enzymes and, as we see now, other types of proteins with natural affinity to certain ligands, is being recognized as one of the most important strategies of "natural engineering" of effector-binding domains in transcriptional regulators. Although this bioinformatics analysis alone does not allow to precisely identifying specific functions for all SLBB subfamilies, it certainly provides a perfect starting point for many case studies driven by specific research interests of various experimental groups. This constitutes a broad impact of this paper, which goes beyond the innovative bioinformatics methodology and obvious implications in the field of domain classification and evolution. The paper is written very clearly, with sufficient details of methods and key results and with helpful illustrations. The Supplementary material providing the entire list of SLBB domain superfamily including the information about their genome context, is highly valuable. This reviewer strongly supports the publication of this paper "as is" in Biology Direct, a perfect home for this wonderful study.We appreciate the comments, and particularly would like to emphasize that we feel the findings in this paper can be used as starting points for experimental work that will contribute significantly to the study of several important biological pathways.In this study, the authors report a novel domain superfamily within the β-grasp fold. The predicted distinct property of this domain is binding diverse soluble ligands, thus it is termed Soluble-Ligand-Binding β-grasp or SLBB. Motivation for this work came from expectation  that some members of the ubiquitous β-grasp fold are involved in binding small ligands.First, the authors searched the PDB database with known structures to retrieve related structures and then used an array of sensitive sequence-based searches (hidden-Markov-model- or position-specific-scoring-matrix-based) to identify remote homologs. Technically, it is very well done and described in sufficient detail. The authors took advantage of available structures to guide editing of the multiple alignment, which in turn allowed them to associate conserved positions with the structural features.Biological function prediction came primarily from the contextual analysis, which was quite thorough. The most interesting prediction is that the primary role of one of the two SLBB clades is binding vitamin B12 and its precursors.Overall, this is an interesting, well-executed study and defining novel domain families and assigning potential biological functions is very important. I would like to add that it is also important to deposit the newly described domain to leading domain databases, such as Pfam. I have not seen plans for doing so in this study. This reviewer is guilty of not always doing so either, but still it should be a rule rather than exception. Perhaps, two version of SLBB could be produced capturing characteristics of the two clades.We could not agree more regarding the importance of domain databases, and we will be submitting two versions of the SLBB to Pfam.Essentially, there is only one major concern with respect to generalization of biological function prediction. I think the members of the domain clade are divergent enough to worry about B12 and its precursors being the only or even the main ligand. The statement in the abstract seems to address this issue – the authors rightly indicate that "members of this superfamily are likely to bind a range of soluble ligands, including B12"; however in the text it becomes more stringent. For instance, on page 3, when describing SLBB fusions with HTH domains in transcription factors, it is suggested that these proteins "respond to B12 or its precursors". I would suggest toning down the claim that all members of the transcobalamin clade bind exclusively B12 and its precursors. Perhaps, the authors did not intend to make it sounds like that, but it does.We do admit that there might be a greater diversity of bound ligands, rather than B12 alone. Only in the cases where there was contextual evidence for involvement in B12 metabolism do we suggest it as a most likely ligand. Following the suggestion of the reviewer, we have altered the wording in the text to clarify the possibility of greater diversity in terms of bound ligands, especially with the HTH-fused versions.I have no concerns about the main observation of the extended beta-grasp SLBB superfamily, or about functional inferences from the contextual analysis of genes that contain various versions of this domain. I think, however, that the analogy with the RRM fold early in the manuscript is a distraction at that point of the authors' reasoning. Generally, I suppose that this type of domain/fold recognition work may benefit from being more logical and less chronological: that is, even if analogy with RRM played a role in authors' own thinking about the SLBB superfamily, I want to first focus on what has been actually observed. On the other hand, I would like to see a discussion of RRM and other analogous folds at the end of the manuscript, when it could serve a more useful purpose of establishing some trends in evolution .We admit this might provide a better flow for the article. Accordingly, we have now shifted the discussion of the parallels between the β-grasp and RRM folds to the Results and Discussion section.LA and SB made the initial discovery reported here. The complete sequence and structure analysis and preparation of figures and supplementary material was performed by AMB, LMI and SB. The paper was written by AMB and LA.Cartoon representations of SLBB domains. File 1 contains cartoon representations of the transcobalamin and the Nqo1 middle domain showing the structural similarity and innovations associated with the potential binding site for soluble ligands.Click here for fileComprehensive list of proteins of the SLBB superfamily, their gene neighborhoods, and domain architectures. Alignments of the SLBB domain will be submitted to the Pfam domain database [database .Click here for file


The mechanism by which the signals are transmitted between receptor and effector domains in multi-domain signaling proteins is poorly understood.Using sensitive sequence analysis methods we identify a conserved helical segment of around 40 residues in a wide range of signaling proteins, including numerous sensor histidine kinases such as Sln1p, and receptor guanylyl cyclases such as the atrial natriuretic peptide receptor and nitric oxide receptors. We term this helical segment the signaling (S)-helix and present evidence that it forms a novel parallel coiled-coil element, distinct from previously known helical segments in signaling proteins, such as the Dimerization-Histidine phosphotransfer module of histidine kinases, the intra-cellular domains of the chemotaxis receptors, inter-GAF domain helical linkers and the α-helical HAMP module. Analysis of domain architectures allowed us to reconstruct the domain-neighborhood graph for the S-helix, which showed that the S-helix almost always occurs between two signaling domains. Several striking patterns in the domain neighborhood of the S-helix also became evident from the graph. It most often separates diverse N-terminal sensory domains from various C-terminal catalytic signaling domains such as histidine kinases, cNMP cyclase, PP2C phosphatases, NtrC-like AAA+ ATPases and diguanylate cyclases. It might also occur between two sensory domains such as PAS domains and occasionally between a DNA-binding HTH domain and a sensory domain. The sequence conservation pattern of the S-helix revealed the presence of a unique constellation of polar residues in the dimer-interface positions within the central heptad of the coiled-coil formed by the S-helix.Combining these observations with previously reported mutagenesis studies on different S-helix-containing proteins we suggest that it functions as a switch that prevents constitutive activation of linked downstream signaling domains. However, upon occurrence of specific conformational changes due to binding of ligand or other sensory inputs in a linked upstream domain it transmits the signal to the downstream domain. Thus, the S-helix represents one of the most prevalent functional themes involved in the flow of signals between modules in diverse prokaryote-type multi-domain signaling proteins.This article was reviewed by Frank Eisenhaber, Arcady Mushegian and Sandor Pongor. Reviewed by Frank Eisenhaber, Arcady Mushegian and Sandor Pongor. For the full reviews, please go to the Reviewers' comments section.Comparative genomics has revealed several similarities as well as differences between signaling systems of prokaryotes and eukaryotes ,2. At thThe distinctness of prokaryote-type signaling systems  is underscored by the deployment of a unique bi-helical module, the HAMP domain, which usually occurs immediately C-terminal to a TM segment of prokaryotic signaling receptors. It is central to transmission of sensory inputs from the extracellular sensor domains to the downstream intracellular domains -17. The The CC is a simple yet versatile structure, which has been widely used as a protein-protein interaction interface throughout the evolutionary history of life . Well-stSurvey of previous experimental studies and our own anecdotal observations gathered in course of systematic analysis of signaling proteins indicated that there might be other conserved CC modules, distinct from other classes of CC segments including the DHp, in prokaryotic signaling proteins and their eukaryotic relatives -30 whichPseudomonas aeruginosa two-component signaling protein  recovered at least 1000 significant hits within 10 iterations, wherein it was found combined to the above mentioned globular signaling domains. Aided by the boundaries of associated globular signaling domains, which were precisely established using several recently available structures or structure predictions, we delineated the region associated with the conserved motif as potentially spanning a stretch of 40–45 residues. Preliminary structural predictions for this region using the JPRED and COILS program [In course of our systematic surveys of signaling proteins we observed a conserved sequence motif present upstream of several histidine kinases, including several previously characterized sensors such as Sln1p , BarA 3, TorS 3, GacS 3, LetS 3 and NarQ program ,37 stron program ,17 and s program -26 showeIn order to investigate the distribution of this motif and define it more precisely we adopted an iterative search procedure with the HMMER package . We prepPseudomonas aeruginosa protein as the seed) did not draw in any commonly encountered attractors such as myosin tails, CCs of cytoskeletal filament proteins or Rad50/SMC-like ATPases with significant e-values. To further test the distinctness of this motif, we generated "cross-hit" plots using position specific score matrices (PSSMs) for this motif and several other CC regions. To generate these plots, proteins containing a given module are queried against PSSMs for the same module as well as a PSSM for another test module. Then e-values for self-hits (protein with a given module against their own PSSM) and cross-hits (protein with a given module against the PSSM of test module) for both this motif and test modules were plotted as X-Y scatters. In these plots, we noted a strong segregation of this motif from several other tested CC regions like the DHp module, myosin and bZIPs, supporting the distinctness of the motif from both generic CCs and well-conserved CC regions . These bstrates , and 7-Thydrates  and PAS  sensing . Likewiscyclases ,30. ThesTo understand the functional significance of the S-helix we carried out a systematic analysis of the domain architecture contexts in which it is found. To do this we firstly used sensitive PSI-BLAST-derived PSSMs and HMMs for a range of domains that are known to occur in signaling proteins (refer Methods) and systematically detected all their occurrences in proteins containing the S-helix. We then collated substantial regions in these proteins that did not map to any of these known domains and scanned them for transmembrane regions, signal peptides, and compositionally biased stretches (refer Methods for details). Any regions that did not contain these compositional features were used as seeds in PSI-BLAST searches to determine if there were any divergent copies of previously known domains or uncharacterized protein domains. We randomly checked the architectures of several proteins determined through the above semi-automatic procedure using manual case by case analysis and found at least 90% recovery of correct domain architectures. This procedure allowed us to arrive at reasonably reliable domain architectures for all the S-helix proteins detected in a search of a database of 255 organisms with completely sequenced genomes. We represented this information in the form of an ordered graph Fig. , where tThe domain architecture graph of the S-helix underscores its general significance to prokaryote-type signaling systems, because most major signaling domains prevalent in prokaryotes are connected to it within two-degrees of the graph Fig. . Some imThe S-helix occurs most commonly upstream of a histidine kinase but is never observed upstream of a eukaryote-type S/T kinase. About 10% of the histidine kinases from completely sequenced genomes show an S-helix upstream of the DHp and catalytic domains. Only in animal receptor guanylyl cyclases such as Atrial natriuretic peptide receptors and Sea-urchin sperm peptide receptors the S-helix occurs downstream of the S/T kinase domain Fig. ; howeverbona fide 'eukaryote-type' signaling systems [These observations imply that the S-helix is relevant across numerous prokaryote-type signaling contexts, both membrane-associated and soluble, irrespective of the two signaling domains it links. Often these domains may sense and deliver very different kinds of signals. However, it appears to be entirely incompatible with most  systems , especiaTo obtain a clearer picture of the actual role of the S-helix in signaling we combined the inferences drawn from sequence conservation and domain architectures with previously available experimental data on proteins containing the S-helix. The best experimental leads for the possible function of the S-helix is available from mutational data on the yeast Sln1p kinase and human receptor guanylyl cyclases -29. In tTo better explore this functional proposal we constructed a model of the S-helix using other parallel CCs as a template (e.g. pdb 1ysa ). While The model also suggests that the acidic residues preferred in the external 'b' and 'c' positions are likely to form external ridges of negative charge along the surface of the S-helix Fig. . These nIdentification of the S-helix helps in delineating the distribution and specific roles of different CCs in prokaryote-type signaling proteins. An earlier computational study detected CC regions in upstream of numerous histidine kinases , but at The S-helix from different proteins is typically embedded between two flanking globular domains, of which one or both domains found in other proteins similarly associated with an S-helix. Hence, it is likely that the corresponding S-helices are duplicated and evolutionarily mobile along with their flanking homologous globular domain/s. This is a more parsimonious explanation for the proliferation of the S-helix than extensive multiple sequence convergences of connector segments between globular signaling domains. Due to the frequent presence upstream of histidine kinases, it is possible that the S-helix originally arose as an N-terminal extension of the same coiled-coil segment that contains the DHp module, and acquired a distinct regulatory function. The advantage conferred by this regulatory function resulted in natural selection operating to preserve it as a distinct module. Due to the rampant domain swapping of catalytic and sensor domains of signaling proteins that occurred in course of their evolution , the S-hIn summary, we present evidence for a special structural feature shared by numerous prokaryote-type signaling proteins, which might function as a switch in the communication between two globular domains that prevents constitutive activation of signal transduction in the absence of an appropriate stimulus from an adjacent domain.The non-redundant (NR) database of protein sequences  was searched using the BLASTP and PSI-BLAST programs . ProfileThe multiple alignment was used create a HMM using the Hmmbuild program of the HMMER package . It was Calcium channel and Chemotaxis proteins) [Histidine kinase-Associated Sensing Extracellular domain) [Histidine kinases, Adenylyl cyclases, Methyl-accepting proteins and Phosphatases) [Drosophila Period clock proteins, vertebrate Aryl hydrocarbon receptor nuclear translocator and Drosophila Single minded proteins) [), PSI-Blast searches and Pfam searches [Helix-Turn-Helix DNA binding domain) [Signal Transduction ATPases with Numerous Domains) [K-Homology RNA binding domain), and NIT  domains [To determine the domain architecture of the S-helix containing proteins query sequences and profiles of the following domains were used to search the proteins: NMP cyclase, CACHE (extracellular domain found in roteins) , CHASE ( domain) ,10, CHAS domain) , cNMPBD  domain) , EAL (EA domain) , GAF (cG domain) , GGDEF-m domain) , HAMP (dhatases) , HD-GYP hatases) , Histidihatases) , HNOB (Hhatases) , HPT (Thhatases) , MCP-N [hatases) , MEDS [5hatases) , PAS (Peroteins) ,59, PBPIroteins) ,60, PocRroteins) , Receiveroteins) , S/T/Y Kroteins) . The bousearches . Any newsearches , CBS  , TARH, H domain) , Globin  domain) , SF-II h domain) , STAND ADomains) , KH  and simple secondary structure , the S-helix domain appears to be amenable to sensitive and specific detection using probabilistic sequence comparisons and database searches.Phyletic patterns and architectural contexts of the S-helix/para 4 and elsewhere: what is 'syntactical' – is there such a thing as agreed-upon definition of grammar of protein domains? Maybe 'positional' or 'domain order' would be less pretentious. Also, obviously, biological function occurs in 3-D, where perhaps it does not matter much whether the domain fusion is N-terminal or C-terminal. The order of fused domains might be a useful synapomorphy, however, but this section does not seem to be focused on that.In Results and discussion/Currently we are using the term syntax only in one place and specifically as a descriptor for the general rules in domain architecture, which are seen in the S-helix proteins. While there are no universal grammatical principles for protein domains, we do observe that several domains show strong rules in terms of the positions in the primary structure and we are merely using the term syntax for that. The S-helix is clearly one such domain – for example it occurs rather strictly N-terminal to the histidine kinase and nucleotide cyclase domains (like a "preposition"). While we do agree that biological function occurs in 3D, there is a strong polarity in terms of 3D domain arrangement of many of the signaling proteins. For example, there are specific regions located outside the cell linked to intracellular parts by means of a relatively rigid TM helix. Likewise, the S-helix and DHp are relatively rigid helical segments that impart the protein an "extended" configuration wherein the location at the N- or C-terminus of a structurally rigid segment would matter.The homology concept is traditionally applied to protein sequence segments having no bias towards few amino acid types or short, simple, repetitive patterns and representing complete globular domains. The requirement of matching hydrophobic patterns and the larger number of alignment positions ensured significance of annotation transfer within such segment families. In this context, the existence of a common ancestor can be reliably postulated even for some cases of sequence pairs that have extremely diverged to essentially zero sequence identity.These insurances are no longer valid if shorter sequence motifs or repetitive patterns such as in coiled coils are the subject for sequence similarity searches. Both concerns are valid in this study. I find it interesting what kind of additional arguments have been brought up by the authors to support the significance of the relatedness of sequence segments within the S-helix family . The comparison with major families of CCs is instructive, although this argument is not exhaustive as well as the architecture consideration. I agree with the authors that the sequence similarity within the family might indicate similarity of function. I am not so sure whether it also indicates evolutionary relatedness in all instances since it is not improbable that short functional motifs originate de novo from diverse ancestors .We admit that in the case of sequences with lower entropy than typical globular domains the possibility of convergent sequence similarity exists. However, the sequence profile searches seeded with different starting sequences  show that despite being a coiled-coil the S-helix does not tend to promiscuously recover diverse functionally unrelated CC segments. It is also seen that the S-helix is typically embedded in the context of two flanking domains, of which one or both domains are homologous across different proteins with S-helices. The globular domains, such as the histidine kinase, PAS, GAF and the like, with the S-helix is closely associated in these architectures are clearly the product of divergent evolution following duplication. Hence, the explanation that the S-helices of the proteins are homologous and evolutionarily mobile, along with their flanking homologous domain/s is clearly a more parsimonious explanation than extensive multiple convergences of a connector or flanking region associated with such domains.I find this work of great interest and of importance for understanding protein evolution also beyond the specific functional module S-helix that is considered here.It would be convenient for the reader if the authors could make available some additional information via an FTP site, in the text or on request. The exact starting sequence segments of the searches (beginning of Results section) would be helpful for people who wish to reproduce the data. The alignments and RPS-BLAST libraries of signaling domains listed in the Methods section would be of interest to other researchers.We have added a few new sentences to the section "Identification of the Signaling Helix motif" providing an example of the starting point for the searches. We are also providing in additional file 1the entire list of S-helix proteins detected in large-scale searches across various complete genomes along with their alignment from which the RPS-BLAST profiles and HMMs were prepared.This is an interesting piece of work and a well-written paper that I suggest to be published in its present form.Comprehensive alignment and architectures of S-Helix.Click here for file


The Hsp40 chimeras containing J-domains originating from soluble (cytosolic or endoplasmic reticulum (ER)-lumenal) Hsp40s were able to reverse the thermosensitivity of E. coli OD259. In all cases, modified derivatives of these chimeric proteins containing an His to Gln substitution in the HPD motif of the J-domain were unable to reverse the thermosensitivity of E. coli OD259. This suggested that these J-domains exerted their in vivo functionality through a specific interaction with E. coli Hsp70, DnaK. Interestingly, a Hsp40 chimera containing the J-domain of ERj1, an integral membrane-bound ER Hsp40, was unable to reverse the thermosensitivity of E. coli OD259, suggesting that this J-domain was unable to functionally interact with DnaK. Substitutions of conserved amino acid residues and motifs were made in all four helices (I–IV) and the loop regions of the J-domains, and the modified chimeric Hsp40s were tested for functionality using the in vivo assay. Substitution of a highly conserved basic residue in helix II of the J-domain was found to disrupt in vivo functionality for all the J-domains tested. We propose that helix II and the HPD motif of the J-domain represent the fundamental elements of a binding surface required for the interaction of Hsp40s with Hsp70s, and that this surface has been conserved in mammalian, parasitic and bacterial systems.Both prokaryotic and eukaryotic cells contain multiple heat shock protein 40 (Hsp40) and heat shock protein 70 (Hsp70) proteins, which cooperate as molecular chaperones to ensure fidelity at all stages of protein biogenesis. The Hsp40 signature domain, the J-domain, is required for binding of an Hsp40 to a partner Hsp70, and may also play a role in the specificity of the association. Through the creation of chimeric Hsp40 proteins by the replacement of the J-domain of a prokaryotic Hsp40 (DnaJ), we have tested the functional equivalence of J-domains from a number of divergent Hsp40s of mammalian and parasitic origin . An in vivo functional assay was used to test the functionality of the chimeric proteins on the basis of their ability to reverse the thermosensitivity of a  The divThe major molecular chaperone, Hsp70, consists of an N-terminal ATPase domain and a C-terminal substrate-binding domain. The affinity of Hsp70 for protein client is modulated by ATP binding and hydrolysis. In the ATP bound state, affinity of the substrate-binding domain for the client protein is low and exchange rates are high. Hydrolysis of ATP to ADP results in high affinity for the substrate and low exchange rates, effectively locking the substrate into the binding pocket . This inHsp40s are defined by the presence of an approximately 70 amino acid region known as the J-domain, which is essential for interaction with Hsp70. The J-domain is a highly conserved α-helical structure that interacts with the Hsp70 ATPase domain and possibly also with the Hsp70 substrate-binding domain . Hsp40s E. coli DnaJ , and has been proposed to be part of a network of residues on helix II (and possibly helix III) that form an Hsp70-binding site on the J-domain  in an attempt to identify system specific and common factors in Hsp40–Hsp70 interactions. The similarities and differences in the structure and function of Hsp40s of parasites and their hosts have yet to be determined, and therefore from this broader perspective we were interested in a comparative analysis of the J-domains of parasitic and human origin. An in vivo functional assay was used to assess the ability of the J-domains to substitute for the J-domain of a prokaryotic type I Hsp40. Furthermore, the functional importance of specific residues was addressed through single amino acid substitution analysis. The data suggested that cytosolic and ER J-domains of mammalian and parasitic origin can interact with DnaK using a common mechanism, and that a fundamental binding surface appears to be conserved in J-domains of Hsp40s of mammalian, parasitic and bacterial origin.22.1E. coli OD259 (MC4100 araD139 Δara714 ΔcbpA::kan dnaJ::Tn10-42) was kindly provided by Dr. Olivier Deloche . The pGEX-4T-ERj1, pGEX-4T-ERj3 and pGEX-4T-ERj5 plasmids encode mouse ERj1, human ERj3 and human ERj5. The pET23b-Tcj3 construct encoding Tcj3 has been described  and Pfj4 (residues 1–79) J-domains into the pRJ-B vector resulted in the creation of the expression constructs encoding the chimera Pfj1-J-Agt-DnaJ and Pfj4-J-Agt-DnaJ, respectively. The coding region for the Tcj3 (residues 1–74) J-domain was amplified by PCR from pET23b-Tcj3 with BamHI and BstBI restriction sites to allow subcloning into pRJ-B. Insertion of the coding region for the Tcj3 J-domain into the pRJ-B vector resulted in the creation of the expression construct encoding the Tcj3-J-Agt-DnaJ chimera. Similarly, the coding regions for the J-domains of Hsj1a (residues 1–72), ERj1 (residues 56–128), ERj3 (residues 23–97) and ERj5 (residues 35–100) were PCR amplified from pCMV-Tag3a (Hsj1a), pGEX-4T-ERj1, pGEX-4T-ERj3 and pGEX-4T-ERj5, respectively, and subcloned into pRJ-B to give expression constructs encoding Hsj1-J-Agt-DnaJ, ERj1-J-Agt-DnaJ, ERj3-J-Agt-DnaJ and ERj5-J-Agt-DnaJ. All mutations were produced by the whole-plasmid linear amplification approach using complementary oligonucleotides . Primers were designed so as to introduce or eliminate a restriction endonuclease site to facilitate the identification of successful mutants by restriction analysis. Mutations were confirmed by subsequent DNA sequencing.J-domain B and fulJ-domain  were synJ-domain . The gen2.3E. coli dnaJ cbpA strain, OD259  broth containing 100 μg/ml ampicillin for plasmid selection and 50 μg/ml kanamycin for strain selection. The cultures were grown overnight at 30 °C, before being diluted 1:100 with YT broth containing 100 μg/ml ampicillin and 50 μg/ml kanamycin, and grown further at 30 °C until an A600 of approximately 2.0 was reached. Cultures were diluted to an A600 of 0.3 and serial dilutions were performed to a final dilution of 1 × 10−8. Aliquots (3 μl) of each of these dilutions were spotted onto agar plates containing 50 μM IPTG. Plates were grown at 30 °C, 40 °C and 42 °C, respectively, to determine the ability of Agt DnaJ, Agt DnaJ-H33Q and the chimera proteins to reverse the thermosensitivity of E. coli OD259.Plasmids were transformed into competent 2.4E. coli OD259 and its transformants producing His6-tagged Agt DnaJ and chimera proteins. Proteins were resolved on a 12%  sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to nitrocellulose membrane. Proteins of interest were detected using a mouse anti-His antibody  and horseradish-peroxidase-conjugated anti-mouse secondary antibody  using chemiluminescence-based detection . Images were captured using a Chemidoc chemiluminescence imaging system .Western analysis was performed on whole cell lysates of 2.5The pGEX-4T-1-based construct containing the coding region for the J-domain of murine ERj1 . GST was bound to the immobilized antibodies in the reference cell, while the GST-ERj1-J fusion proteins were immobilized separately in the measuring cell . Subsequently, solutions containing increasing concentrations of purified BiP (0.25–2 μM) were passed over the chip in the presence of ATP (flow rate 20 μl/min). Each BiP application was followed by the application of running buffer until baseline was reached. The analysis of the data were carried out using the BIAevaluation software version 2.2.4.Surface plasmon resonance spectroscopy was carried out in a BIAlite upgrade system. Monoclonal goat anti-GST-antibodies  were immobilized on a sensor chip CM5  by amine coupling according to the manufacturer's protocol. The sensor chip was equilibrated with running buffer , suggesting that it was capable of functionally replacing DnaJ and CbpA . The lack of reproducibility of complementation assays using the E. coli DnaJ chimeras could possibly be related to the toxic effects that have been observed for the overproduction of E. coli DnaJ , certain parasitic Hsp40s (Pfj1 and Tcj3) and certain mammalian Hsp40s . The J-domain of Pfj4 appeared to be relatively divergent from all the other J-domains. In this study, the amino acid residues targeted for substitution were chosen based on their conservation , growth at 30 °C occurred up to the highest dilution (10−8), indicating that Agt DnaJ was not toxic to the cells. Furthermore, the growth profile observed at 40 °C was similar to that observed at 30 °C, suggesting that Agt DnaJ was able to reverse the thermosensitivity of E. coli OD259 (E. coli OD259 and reverse the thermosensitivity of this strain (E. coli OD259 producing Agt DnaJ-H33Q (negative control), was able to grow at 30 °C, but was unable to grow at 40 °C. E. coli OD259 transformants producing the chimeras HSJ1-J-Agt-DnaJ, Pfj1-J-Agt-DnaJ, Pfj4-J-Agt-DnaJ, Tcj3-J-Agt-DnaJ, ERj3-J-Agt-DnaJ and ERj5-J-Agt-DnaJ were all able to grow at 30 °C, and reversed the thermosensitivity of this strain at 40 °C similar to the control  resulted in a mutant Pfj1-J-Agt-DnaJ protein that was only partially functional in the in vivo assay . FurtherRRK motif of Helix IV in Pfj4, corresponding to the QKRAA motif of Agt DnaJ) was found to be functional, while in contrast the Pfj1-J-Agt-DnaJ-K63A mutant protein (substitution of Lys63 of the KKKEF motif of Helix IV in Pfj1) was only partially functional. Mutation of the Pfj1 KKKEF motif to the more conserved QKRAA motif produced a functional Pfj1-J-Agt-DnaJ mutant protein.The roles of key conserved residues (e.g. D59) and motifs (e.g. the “QKRAA” motif) in helix IV were also investigated in this study . The equE. coli OD259 often vary between mutants and do not necessarily correlate with levels of functional recovery.Production of all the mutant chimeric proteins was detected by Western analysis, suggesting that the inability of certain mutant proteins to reverse thermosensitivity was the result of a lack of protein functionality rather than a lack of protein production . As has 3.4Since it was not possible to characterize the ERj1 J-domain any further through an in vivo assay, an in vitro analysis was conducted. Using a GST-ERj1-J fusion protein and mutant derivatives, GST-ERj1-J-R26A and GST-ERj1-JH33Q, affinity pull-down assays were conducted to evaluate the interaction of the J-domains with BiP in the presence and absence of ATP A. While 4E. coli Hsp70, DnaK. Therefore, we propose that cytosolic and ER J-domains of mammalian and parasitic origin can interact with DnaK using a common mechanism. Furthermore, we found that substitution of a basic residue at position 26 of the helix II of the J-domain compromised functionality in all the J-domains in which this mutation was investigated . Similar results have been found for E. coli DnaJ  is part of the functionally critical pentapeptide (HPD-R/KN) predicted by E. coli DnaJ resulted in loss of function , and may reflect slight differences in affinity or specificity of these J-domains for DnaK. These differences need to be probed further using quantitative in vitro assays, and incorporating an analysis of the less-conserved J-domain residues shown by NMR analysis to occur at J-domain-DnaK/Hsp70 binding interfaces. Furthermore, the possibility that there are specialized features unique to the J-domains of integral-membrane-bound Hsp40s needs to be investigated more extensively.