Though search tools for conserved domain databases such as Hidden Markov Models (HMMs) are sensitive in detecting conserved domains in proteins when they share sufficient sequence similarity, they tend to miss more divergent family members, as they lack a reliable statistical framework for the detection of low sequence similarity. We have developed a greatly improved HMMerThread algorithm that can detect remotely conserved domains in highly divergent sequences. HMMerThread combines relaxed conserved domain searches with fold recognition to eliminate false positive, sequence-based identifications. With an accuracy of 90%, our software is able to automatically predict highly divergent members of conserved domain families with an associated 3-dimensional structure. We give additional confidence to our predictions by validation across species. We have run HMMerThread searches on eight proteomes including human and present a rich resource of remotely conserved domains, which adds significantly to the functional annotation of entire proteomes. We find ∼4500 cross-species validated, remotely conserved domain predictions in the human proteome alone. As an example, we find a DNA-binding domain in the C-terminal part of the A-kinase anchor protein 10 (AKAP10), a PKA adaptor that has been implicated in cardiac arrhythmias and premature cardiac death, which upon stress likely translocates from mitochondria to the nucleus/nucleolus. Based on our prediction, we propose that with this HLH-domain, AKAP10 is involved in the transcriptional control of stress response. Further remotely conserved domains we discuss are examples from areas such as sporulation, chromosome segregation and signalling during immune response. The HMMerThread algorithm is able to automatically detect the presence of remotely conserved domains in proteins based on weak sequence similarity. Our predictions open up new avenues for biological and medical studies. Genome-wide HMMerThread domains are available at  The prediction of a protein's function is one of the most valuable contributions of bioinformatics to biological research. Next to providing functional prediction for experimental design, the functional annotation of entire proteomes is nowadays a basic task of genome database providers. Among the most used resources for functional annotations are conserved domains, which are distinct structural and functional units of a protein When proteins are analyzed manually, even more sensitive methods can be applied. PSI-BLAST searches The Superfamily database Alternative to sequence-based searches, structural features have been recognized as useful in detecting remote sequence similarity. As the function of a protein is to a great extent defined by its structure, its fold is generally better conserved than its sequence http://vm1-hmmerthread.age.mpg.de.Here we introduce the genome-wide HMMerThread resource of remotely conserved domains. Based on a much improved HMMerThread algorithm we have previously published Among the remote conserved domain hits in our dataset we find a number of interesting new or additional function(s) that could be assigned to proteins associated with a selected number of biological processes and human diseases. Our data allow for many predictions that can be functionally tested and thus open up completely new avenues in experimental research. In conclusion, with the HMMerThread database we have created a rich and accurate resource of remotely conserved domains of great value to experimental biological and medical research.The HMMerThread software searches for remotely conserved domains in proteins by a combination of relaxed sequence-based conserved domain searches with a subsequent fold recognition step to eliminate false positive domain hits resulting from high E-value thresholds We needed to take two factors into consideration, when choosing a threading engine for genome-wide HMMerThread searches: First, we needed an algorithm, which was parallelizable and readily adaptable for a high-performance computing setting. Second, we wanted to ensure good performance of the threading engine. Among the tools available, only few algorithms fulfilled the first criterion. Next to Threader3.5 a priori, which structural folds to look for. Therefore, we considered two factors for scoring hits from fold recognition in HMMerThread:Our attempt on unsupervised fold recognition required the development of a reliable scoring of a threading hit. The Z-score of threaded structures in OpenProspect cannot be easily interpreted and very diverse structure families can give similar Z-scores for the same sequence. Yet, as our approach combines sequence similarity searches together with fold recognition, we could take advantage of knowing We considered the Z-score from OpenProspect, as it represents a guide to the strength of a hit. Given a certain Z-score, we wanted to deduce a p-value reflecting the probability that this Z-score can be treated as significant. We therefore constructed a cumulative distribution function of the Z-scores for all structures in SCOP for a random set of 1000 human proteins. This gave us a naïve probability for each possible Z-score. The threshold at 0.5% of all threaded structures (∼top 60 hits) was determined as stringent after examining results of test sets containing known domains  and remotely conserved domains.We considered the number of structures associated with a conserved domain that were positively identified by OpenProspect. As many conserved domains have generally more than a single structural representative in the SCOP database, we used the hypergeometric probability function to determine, whether a given set of structures associated with a conserved domain was significantly overrepresented. This method enabled us to discriminate between significantly scoring structures due to their high frequency and those that are truly overrepresented and therefore a true positive hit. The top 60 hits we considered approximately equals 90% of the hits at a hypergeometric p-value threshold of 0.05 , but achieved a significant score in the newer one . We created an overlapping dataset of the Pfam10 and Pfam22 releases, resulting in 5266 conserved domain profiles that we could test. This dataset we refer to from hereafter as the Pfam10∶22 remotely conserved domain set. We ran HMMER2 searches against both profile versions of the conserved domains using the human proteome and selected those domains, which scored with an E-value>0.1 using Pfam10, while having an E-value<0.1 in Pfam22 and where the difference between the two E-values was greater than or equal to a 10-fold change. This resulted in a total of 408 conserved domains that could be considered as true positive, weakly conserved hits in Pfam10. For these domains, we performed HMMER2 searches against Pfam10, which generated 1520 possible, overlapping hits. These, we could analyze for true positive (TP), false negative (FN), true negative (TN) and false positive (FP) identification . We could also confirm most of the remotely conserved domains in proteins discussed in the original manuscript describing the ProFAT and HMMerThread server LOC84060 and Parn). The SAM domain (for Sterile Alpha Motif) was verified in the epidermal growth factor receptor pathway substrate 8 protein families EPS8 and EPS8L3 and we automatically detected the Acetyltransferase domain (Acetyltransf_1) in the LOC79969 proteins using the novel HMMerThread server (LOC79969). Interestingly, a large-scale screen of protein-protein interactions in worm revealed that the C. elegans ortholog of LOC79969, W06B11.1, interacts with a methyltransferase (C01B10.8), suggesting that this protein is part of a larger chromatin-remodelling complex Saccharomyces cerevisiaeSchizosaccharomyces pombe . HMMerThread also confirmed the presence of winged-helix domains in two proteins required for meiotic recombination, Mnd1 and Hop2 TNFRSF21), the presence of which was also shown by structural analysis .We could confirm the good performance of HMMerThread, when we searched for remotely conserved domains that have been described in literature before and which we were well familiar with: We could confirm the existence of a BAR domain (Bin-Amphiphysin-RVS) in most of the proteins that we had previously described In order to estimate the advancement of HMMerThread in predicting remotely conserved domains compared to its previous version, as well as to other existing resources, we used the Pfam10∶22 remotely conserved domain set to calculate recall, precision and accuracy of the old HMMerThread algorithm, GenTHREADER When we compared the precision, accuracy and recall of the old versus new version of HMMerThread, we find that our modifications have significantly improved the software see . While wWe decided to compare the novel HMMerThread algorithm to two existing resources that provide information on remote conservation between proteins. For one, we looked at GenTHREADER Superfamily As noted earlier, it seems to be a built-in feature of our dataset that even precise algorithms like GenThreader or Superfamily show a higher than usual false positive rate . We therefore decided that a false positive rate of 8% with our dataset was acceptable for the new HMMerThread algorithm.We conclude from this data that HMMerThread with its unique approach to consider not only sequence-, but also structural information outperforms other methods currently available in identifying remotely conserved domains.We have carried out HMMerThread searches against the proteomes of the eight most common model organisms including human and detected a total of 58330 weakly conserved domains with an E-value above 0.1 see . About 1S. cerevisiae contain summaries and GO terms from SGD. For human records, we integrate iPfam data along with domains that may explain interactions in a separate table. We also provide a “live search” feature, where sequences that have not been processed by us can be searched using the HMMerThread pipeline with an updated version of the Astral database (the currently used version is 1.73). The database is publicly available at http://vm1-hmmerthread.age.mpg.de.The results of the genome-wide HMMerThread searches are presented in a web-based relational database, which includes the results from all eight proteomes analyzed. The database can be queried by gene name, protein ID or Pfam conserved domains. The HMMerThread domains are shown along with associated annotation for the given gene from diverse sources like the NCBI, SGD, Wormbase, Flybase or HPRD. Though remote conserved domain searches were not carried out against the Pfam24 database We set out to search for novel functional predictions based on detected HMMerThread domains. To do this, we followed several strategies: 1) we searched for undiscovered, remotely conserved domains in proteins that were detected in genome-wide functional screens. We discuss the overall statistics of a genome-wide functional dataset on factors involved in Hepatitis C Virus replication in human cells and describe one more detailed example with potential mechanistic insight. 2) We looked for remotely conserved domains in proteins associated with a biological process. We focused on mitotic and meiotic genes, as well as genes associated with diseases using the OMIM resource (NCBI) We have chosen a functional screen for cofactors of Hepatitis C Virus replication in human cells Nab1 (NGFI-A binding protein 1). Nab1 is a transcriptional co-repressor that interacts directly with early growth response transcription factor 1 (Erg1) and thereby either positively or negatively modulates transcriptional activation of early response genes Among the hits that showed significant reduction of viral replication with more than two independent silencing triggers was the gene In order to provide examples of the predictive power of the HMMerThread method, we set out to search for remotely conserved domains in proteins involved in mitosis and/or meiosis, as well as genes associated with human diseases (taken from OMIM) that could elucidate their molecular mechanism.Ssp2. This protein is required for the proper formation of the prospore membrane (PSM) and the spore wall (SW) during sporulation  protein was first described as a heterochromatin organizer in fly, whose loss leads to chromosome missegregation in meiosis Wapl proteins  is member of a diverse protein family, which binds to the regulatory subunit of protein kinase A , namely the ability to bind to Protein kinase A (PKA). The Lba-GFP fusion protein translocates from cytoplasm to intracellular vesicles upon stimulation with LPS and can be found on Golgi membranes, lysosomes, ER, plasma membrane and endocytic vesicles In vitro experiments precluded binding of the PH-BEACH domain to phospholipids Lba proteins  that stimulates the response of the cells to bacterial pathogens. The protein Lba  has been identified as a gene that is expressed in response to LPS and is involved in the maturation of immune cells proteins . Though proteins  and foldproteins  . Other examples are methyltransferase_11 domains hidden in the DUF689 domains (DrXP_684963 or DrCiapin1), which are easily verifiable by PSI-BLAST searching and the DUF738 domain, which contains an Acetyltransferase domain (Acetyltransf_1) that was already described in an earlier section of this manuscript . A complete list of conserved domains with an associated function within DUF domains can be found in Supplemental Domains of unknown function (DUF) are often annotated in case a clear block of sequence similarity is found within a protein family with unknown function. Domain profiles of DUF-domains are often restrictive, as not many members have been assigned to these conserved domain families. When analyzing the data from HMMerThread searches, we frequently found remotely conserved domains of known function within DUF-domains. For example, a Calponin Homology (CH-) domain lies within the N-terminal region of the DUF1042 domain , which overlaps with the PROSITE Rab-binding pattern. The RPH3A_effector domain was initially described as a Rab3 interaction motif found in the Rabphilin-3A protein Among the HMMerThread hits we found in the human interactome was a weakly conserved RPH3A_effector domain  in the protein Als2cL, a closely related protein to Alsin (Als2), which lacks the N-terminal RCC (regulator of chromosome condensation) domain Another remotely conserved HMMerThread domain is the RhoGEF/DH domain of the protein With the HMMerThread method we attempt to provide conserved domain predictions beyond the statistical threshold of purely sequence-based methods. By relaxing significance thresholds of sequence-based conserved domain searches and selecting for true positive hits by subsequent fold recognition, we can go far into and beyond the twilight zone of sequence similarity to detect remotely conserved domain members. We can significantly improve the precision of weak conserved domain predictions by cross-species validation of HMMerThread hits. The new implementation of HMMerThread shows a clearly superior performance to our previously published version of the software. We have raised the accuracy of our predictions from 79% to 90% and in this surpass existing methods of genome-scale detection of remote conservation between proteins that are either based on sequence, or structure alone. We could positively identify a number of remotely conserved domains previously reported in literature. We have discussed a number of highly interesting examples of weak, conserved domain hits that are associated with specific functional screens, cellular processes or human diseases. Our predictions could explain in part the observed phenotypes and open up new avenues for experimental studies. In this, the HMMerThread resource provides a rich resource of sensitive, functional annotations of proteins for all major model organisms. In the human proteome, we found ∼12000 remotely conserved domains with an E-value above 0.1. Of those ∼4500 could be validated in at least one other species and ∼2700 were validated against 2 species. This data enhances greatly the ability to functionally characterize many proteins and demonstrates that our knowledge of protein functions can be increased based on more sensitive searches against the current databases.One improvement to the previous version of HMMerThread was the implementation of a reliable scoring scheme for HMMerThread hits so that the software could be applied to entire genomes without manual interference. In contrast, the Z-score of OpenProspect does not effectively discriminate between true- and false- positives and the confidence measure the authors describe for Prospect II Anopheles gambiae, Fugu rubripes or chicken. Moreover, we do not find any usable proteome information for the close relatives of some of the chosen model organisms, like Schizosaccharomyces pombe or Dictyostelium discoideum. In these cases, we do not have any validation information based on orthologous sequences. These problems will however be solved in the future, as more genomes are being sequenced and as the annotation status of genomes from non-model organisms improves over time.In order to improve the quality and the reliability of our conserved domain predictions, we chose to validate remotely conserved domains by confirming their presence in the orthologs of other, related species. We found that this is a very good measure for the reliability of weak conserved domain predictions and employed this strategy when using HMMerThread domains for annotating proteins in genome-wide screens. This procedure however is highly dependent on a) the availability of the complete and annotated genome of at least one related species and b) the quality of the genome annotations. We do not try to predict genes in genomic sequences and are relying on the predicted CDS provided by genome databases. Clearly not all genes are correctly predicted, if predicted at all, in less common model organisms such as the mosquito A second approach we take is to validate remotely conserved domains by looking for the presence of essential residues provided by the CDD resource Contrary to purely structure-based techniques, HMMerThread can only detect remotely conserved domains, whose structure has been solved. Given this fact, we limit ourselves to conserved domains that have an associated three-dimensional structure. We currently can cover about 35% of the conserved domain sequence space. With newly solved three-dimensional structures, we can update our database with low effort, as we can specifically look for newly added structures. Due to the much smaller database sizes, we can therefore greatly reduce the required processing time for updates.Another limitation we have chosen to accept is to ignore overlapping conserved domains and limit the fold recognition step to the top hit of the HMMER2 search. This was purely due to limitations in computational resources. Based on statistics from the yeast proteome, in which we have attempted to discriminate the true positive conserved domain hit in a set of overlapping domains, we estimate that we miss roughly 54% true positive hits in the other organisms, which we could only retrieve through an eight-fold increase in run-time. This data again demonstrates the power of our approach, as in more than 50% of the cases, the true positive hit is not the first one that is detected in the sequence-based search. We are currently working on an updated version of the database that includes overlaps in all organisms presented. Finally, updating of the HMMerThread database with novel software releases will result in the highest cost concerning computing time. Meanwhile, the Pfam24 database has been released and we have included this data in our resource. Live searches using HMMerThread already use the new release of the Pfam database. We will furthermore incorporate future releases of Pfam in updates of the HMMerThread database and we will do the same for the fold library, SCOP. Likewise, we will use HMMER3 for updates, once it is out of beta testing.Remotely conserved HMMerThread domains in the HMMerThread resource are a valuable guideline for further experimental studies of protein function. Often, a remotely conserved HMMerThread domain is the sole information available for a protein under study and it provides clues for experimental design to elucidate the mechanistic function of a protein. Moreover, HMMerThread has demonstrated high precision, recall and accuracy. Yet, it is clear that conserved domain prediction based on weak sequence similarity is essentially a prediction and will need further verification. Moreover, as we partly rely on fold recognition, HMMerThread predictions have to be considered as clan-based predictions of conserved domains. All remotely conserved domains that are discussed in this manuscript have been verified by independent methods like PSI-BLAST, profile-profile comparisons or pure fold recognition using algorithms other than Prospect II/OpenProspect. We suggest, when analyzing proteins in low throughput, to use remotely conserved HMMerThread domains as a starting point for functional prediction and – especially when looking at remotely conserved domains with very low sequence similarity – to proceed with an independent verification step.We are currently working on a downloadable version of the HMMerThread tool. Provided that validation data can be generated from a related species, HMMerThread will prove to be a highly useful approach for sensitive conserved domain annotation in entire genomes.HMMerThread was implemented using Pfam Release 22 and SCOP release 1.71. The pipeline was implemented in 4 components and in the Perl 5.8 scripting language without dependencies to allow for execution on various HPC platforms. The 4 components include domain searches  and the domains from the domain search step were retrieved from of the pre-processed sequence to be sent for threading.Threading was performed with OpenProspect on the input sequences from pre-processing. Searches were done on a high-performance computing (HPC) system. Settings included the use of “full” Z-scores (option -zscore_full) and 100 Z-cycles (option -zcycles 100). Runtime for an input file varied in accordance with the sequence length. The average runtime was ∼ 4 hours on a single core of a 2.6 GHz AMD x85 Opteron processor. HPC was provided by the ZIH (TU-Dresden) in the form of a PC Farm of 2,584 cores. The processing of all 8 species took ∼3,000,000 CPU hours including cross-species validation.Post-Processing was performed in 2 steps. Firstly, the results of the Threading run were processed. This involved extracting the key parameters (Z-Score and position) from the output file. These parameters were ranked producing a hit list for all SCOP domains . Secondly, scoring was performed on this ordered list based on two factors. The first was on the p-value from the naïve probability generated from a cumulative distribution function (CDF) of the Z-Scores from 1,000 OpenProspect runs (∼12 million Z-Scores) and the second is based on the cumulative probability from a hypergeometric distribution. Therefore:where pCDF is the p-value of the Z-Score from the scoring of expected structure, N  =  all structures threaded, n  =  top 60 structures threaded, m  =  expected structures threaded and k  =  expected structures in hit list. The p-value threshold for considering a domain as a hit was 0.001.Validation through orthologs relied first on the accurate detection of orthologs. This was possible through the use of the Homologene database  and Pfam22 (July 2007). Common conserved domains seen in both versions were extracted resulting in 5248 domains. The resulting HMM databases were calibrated and searched against the human proteome set  using hmmpfam. Conserved domains were selected, if they scored < 0.1 in Pfam22, >0.1 in Pfam10 and had an e-value difference of greater than 10 fold. These conserved domains were considered to be True Positives (TPs) in the following analysis. For each conserved domain region, HMMerThread was run with all overlapping domains enabled  against the Pfam10 profiles. This provided us with ∼1520 potential domains for HMMerThread to distinguish between True Positives (TPs), False Positives (FPs), False Negatives (FNs) and True Negatives (TNs). The True Negative dataset was derived from Pfam10∶22 searches that did not score significantly in either Pfam10 or Pfam22 according to our criteria. Clan members were furthermore excluded from false positive calculations.Formulas for TPs, FNs, FPs and TNs were as follows:TP  =  (BestHit ∈ 10∶22PosDS) && FN  =  (BestHit ∈ 10∶22PosDS) && FP  =  (BestHit ∉ 10∶22PosDS) && TN  =  (BestHit ∉ 10∶22PosDS) && ,whereby BestHit is the conserved domain hit discovered as top hit in HMMerThread, 10∶22PosDS are all conserved domains from the Pfam10∶22 dataset qualifying as true positives (see above), and p-value represents the combined probability developed for scoring HMMerThread hits.Accuracy, recall and precision were determined as follows:Accuracy  =  (TP+TN)/(TP+TN+FP+FN);Recall  =  TP/(TP+FN);Precision  =  TP/(TP+FP).The old version of HMMerThread was used on the same dataset with standard settings and a hit-depth of 25. The p-value of positively identified conserved domains was set to 0.0000001, negatively identified domains received a p-value of 1. The according p-values were used for calculating TPs, FPs, TNs and FNs. All other procedures were done as described as above.The local version of Genthreader (pgen 8.2) was used on the Pfam10∶22 remotely conserved domain set. PSIPred 2.5 was used with the uniref90 database for secondary structure prediction. Threading was performed against the SCOP fold library provided from 20th July 2009. In order to score conserved domains, SCOP structures that were scored as “CERTS” were mapped to Pfam domains using the PDBMAP mapping provided by Pfam. These were then compared directly with the Pfam10∶22 list as with HMMerThread (old and new versions). Calculations of TPs, FPs, TNs and FNs followed the above formulas, except that instead of the p-value, the presence (as a CERT domain) or absence of a domain was used as the second criteria. CLAN members were again excluded from false positive calculations.The local version of Superfamily was downloaded from the Superfamily website and setup according to the instructions on the site using HMMER 2.3.2 as the HMM search program and SCOP 1.73 as the models database. Processing the sequences produced 766 unique superfamily hits, most of which were scored via multiple superfamily models. In order to determine domain level hits, the SCOP IDs from the models that encompassed the hits for each superfamily were used to map to Pfam domains through the PDBMAP mapping provided by Pfam. True positives were scored, if any of the pfam-IDs associated with a superfamily were identified. All other related Pfam families found in the same region were excluded from false positive calculations next to the CLAN members also used in GenThreader and HMMerThread searches. Formulas for calculating TPs, FPs, TNs and FNs followed the above formulas, again using the presence or absence of a domain as the second criteria.The HMMerThread Database was implemented in Python 2.4 and MySQL. The web-service is provided by Apache.Annotation for the database was obtained from species specific sources either from ftp downloads or from HTTP downloads. Annotation in the form of InterPro Domains H. sapiens protein in the HMMerThread database. For each interaction partner, conserved domains (HMMerThread and Pfam) were matched with known domain-domain interactions from iPfam Protein-protein interactions from the HPRD were extracted for each live version of HMMerThread was implemented in Python 2.4. The only difference to the Perl implementation is that all of the steps for processing are combined and the handling of web jobs is added. Furthermore, the HTML output capability was added directly in a manner similar to the HMMerThread Database. The HMMerThread live runner uses the additional threading module for Python to allow for the submission of jobs simultaneously on different threads. Furthermore, it relies on the SMP capabilities of PSI-BLAST (4 CPUs) and HMMER2 (4 CPUs) along with the MPI implementation in OpenProspect (32 CPUs) to reduce the runtime of the jobs.The PSI-BLAST searches Figure S1Cumulative Distribution Function of threading Z-scores. When a hypergeometric p-value threshold < 0.05 is used, 90% of the expected conserved domain structures fall within the top 60 positions of threading hits with a Z-score <2.38.(PDF)Click here for additional data file.Figure S2ROC curve of the performance of the HMMerThread algorithm. True positives were plotted against false positive predictions of the HMMerThread algorithm. The optimal p-value range corresponds to our chosen cutoff (1E-03), resulting in 14% false positive rate and a 95% true positive rate Click here for additional data file.Figure S3Screenshots of the HMMerThread Database. (A) Overview of one entire record in the HMMerThread database, in this case showing H. sapiens APPL1 with all associated annotation. Those include links to original database entries (NCBI), interaction partners, interacting domains and literature including GeneRIFs, Gene Summaries, Gene Ontology information, as well as known sequence-based domains. (B) HMMerThread domains image with the validated BAR domain (3 species), displayed by mouse over. The associated results of remotely conserved domains are shown in the HMMerThread hits table and the HMMer alignment of all remotely conserved HMMerThread domains are provided below the hit table.(PDF)Click here for additional data file.Table S1Species used for cross-species validation of remotely conserved HMMerThread domains.(PDF)Click here for additional data file.Table S2Comparison of performance of the old and new version of HMMerThread, GenTHREADER and Superfamily.(XLS)Click here for additional data file.Table S3False positive and true positive HMMerThread predictions using different p-value settings.(XLS)Click here for additional data file.Table S4Conserved domains  of hits from a genome-wide screen for cofactors of Hepatitis C Virus replication in human cells(XLS)Click here for additional data file.Table S5HMMerThread remotely conserved domains found in DUF domains(XLS)Click here for additional data file.Table S6list of known interactors and their interacting domains based on remotely conserved domains(XLS)Click here for additional data file.Table S7Accession numbers of sequences used in -.(XLS)Click here for additional data file.


The study of the folding of single domains, in the context of their multidomain environment, is important because more than 70% of eukaryotic proteins are composed of multiple domains. The structures of the tandem immunoglobulin (Ig) domain pairs A164–A165 and A168–A169, from the A-band of the giant muscle protein titin, reveal that they form tightly associated domain arrangements, connected by a continuous β-strand. We investigate the thermodynamic and kinetic properties of these tandem domain pairs. While A164–A165 apparently behaves as a single cooperative unit at equilibrium, unfolding without the accumulation of a large population of intermediates, domains in A168–A169 behave independently. Although A169 appears to be stabilized in the tandem protein, we show that this is due to nonspecific stabilization by extension. We elucidate the folding and unfolding pathways of both tandem pairs and show that cooperativity in A164–A165 is a manifestation of the relative refolding and unfolding rate constants of each individual domain. We infer that the differences between the two tandem pairs result from a different pattern of interactions at the domain/domain interface. ► We investigate the stability and folding of titin Ig domains in a multidomain environment. ► Tandem A-band domains in A164–A165 and A168–A169 are linked by a continuous β-strand. ► At equilibrium, A164–A165 exhibits cooperativity, but A168–A169 domains do not interact. ► Modeling using kinetic data shows that an intermediate accumulates in A164–A165. ► Biophysical studies show that these homologous proteins exhibit very different properties. Many folding studies have concentrated on domains isolated from larger, multidomain proteins rather than on domains linked covalently to their natural neighbors.2 FNfn9–FNfn10).Many proteins, especially in eukaryotes, contain tandem repeats of domains from the same family.et al.In some cases, there are significant interactions between the domains in neighboring proteins.The A-band of titin is made up of Ig and FNIII domains, which are mostly arranged in repeating patterns.In studies of multidomain proteins, it is essential to ensure that the domain boundaries are chosen correctly. Interdomain stabilization might be inferred if domains are cut “too short” when being isolated from a multidomain protein.50%, of 2.8 M and 2.2 M, in 150 mM NaCl, respectively) . The unfolding of the tandem protein A164–A165 reveals a single transition, with an apparent [urea]50% the same as that of A164 (2.8 M) but very importantly with an apparent m value of almost twice that of the single domains . This is indicative of a system where the two domains are unfolding as a single cooperative unit with no significant accumulation of intermediates during equilibrium unfolding.m value  and is no longer twice that of the isolated domains  . As is e.m value . A varia domains .50% of 4.5 and 3.6 M, respectively), but again the m values of the individual domains are similar . [Note that the CD/fluorescence data (not shown) clearly demonstrate that both domains are initially fully folded in the tandem protein.] This is significantly lower than the apparent m value expected if the protein behaved as a single cooperative folding unit. The apparent [urea]50% for A168–A169 is the same as that for the more stable domain, A168, and higher than that obtained when the same experiment was performed on an equimolar mixture of the two isolated domains .m value is the same as that of the individual domains.We have previously demonstrated that equilibrium m value that is close to or slightly lower than that of the individual domains. However, at low salt, we see very different behavior from that seen for a mixture of the two domains. The midpoint for the tandem is the same as that for the most stable domain (A168) and not midway between the two domains, as is seen for the mixture. Again, the apparent m value is the same as that for a single domain; hence, we definitively say that the two domains are not unfolding in an all-or-none fashion. We have the conundrum that, in the tandem protein, A169 is apparently stabilized by A168 at low salt but not at high salt—a possible explanation for this is explored later.A168–A169 is a more complicated system. Our equilibrium experiments at high salt show that A168–A169 behaves exactly as has been predicted for a noninteracting system where the two domains are independent of each other. Where the denaturation midpoints are close, one expects to see a denaturation midpoint for the tandem between the two individual domains and an apparent The analysis of the folding kinetics of A164–A165 is facilitated by the different fluorescence behaviorWe see only a single unfolding phase for A164–A165 irrespective of the observation wavelength. In kinetics, if there are two interdependent phases, and the slow phase precedes the fast phase, then only the slow phase is observed. There are two reasons for supposing that the rate-limiting step in the unfolding of A164–A165 is the unfolding of the A165 domain, in the presence of still folded A164. First, in the isolated domains, A165 unfolds more rapidly than A164. If there are stabilizing interactions between the two domains at the interface in the native state of the tandem, then the two domains will be stabilized to the same extent and the relative rates of unfolding should remain the same—that is, A165 should unfold before A164 in the tandem. Second, when using the  350-nm cutoff filter, no unfolding is observed below ∼ 6 M urea—that is, rate constants below this value must reflect the unfolding of A165. Thus, the data suggest that in unfolding, A165 unfolds first and then the A164 domain, which is no longer stabilized by folded A165, unfolds much faster, probably with the same rate constant as A164 alone.− 1 by the folded A164 domain. Since we cannot observe the A164 domain folding in the presence of folded A165, we cannot quantify the effect of A165 on A164. A164 and A165 are marginally stable domains ; these results show that the interface between these domains contributes significantly to their overall stability.Thus, the kinetic pathway for A164–A165 folding proceeds via an intermediate with A164 folded and A165 unfolded . We can − 1—even when A168 is unfolded.Although the high-salt data suggest clearly that A168 and A169 do not interact in the tandem protein, the low-salt data were less clear. Thus, the kinetics were investigated under low-salt conditions. A168 folds and unfolds significantly faster than A169, both alone and in the tandem protein. Thus, interestingly, the folding and unfolding pathways of this tandem protein are not the reverse of each other and a different intermediate is populated in folding compared with unfolding . Such beIt is well established that if domain boundaries are selected inappropriately and domains are cut too short, then interdomain interactions might be incorrectly attributed.m values, for both tandem proteins (F–A169U) accumulates to ∼ 70%; there is a much smaller population (∼ 5%) of the intermediate with A168 unfolded and A169 folded (A168U–A169F) (F–A165U) accumulates to only ∼ 30% (the intermediate with A164 unfolded and A165 folded is not populated at equilibrium)  b. Interelibrium) a. A168F–Our results on A164–A165 lead us to suppose that the presence of a continuous β-strand between tandem Ig domains imparts cooperative folding, as was reported for the domain/domain-connecting helices in spectrin domain repeats.2 and 217 Å2, respectively), perhaps explaining, at least in part, why only A164–A165 demonstrates cooperative folding behavior. It is worth noting, however, that neither of these interfaces is particularly large,− 1. In both tandems, there is a connecting β-strand with regular β-sheet interactions in both neighboring domains . The bound protein was cleaved from the resin with thrombin and further purified by gel filtration using a G75 Superdex 26/60 column  in 10 mM phosphate buffer, pH 7.0, and 150 mM NaCl. The purified protein was dialyzed into 10 mM phosphate, pH 7.0, and stored at 4 °C in this buffer supplemented with 0.2 mM TCEP. The genes encoding A164, A165, and A164–A165 were cloned into a pETZ2-1a vector containing an N-terminal histidine tag, a z2 carrier, and a tobacco etch virus protease cleavage site; the mutant A164–G–G–A165 was generated using the Phusion Site-Directed Mutagenesis Kit (Stratagene). Protein expression was carried out in E. coli BL21(DE3) cells, and the protein was purified from the soluble fraction, after centrifugation, by affinity chromatography on Ni2+-agarose resin (Qiagen). Bound protein was eluted in 25 mM Tris, pH 7.5, supplemented with 300 mM NaCl and 400 mM imidazole. The eluted protein was digested with tobacco etch virus protease and dialyzed into 25 mM Tris, pH 7.5, and 300 mM NaCl. A second Ni2+-agarose step removed the cleaved solubility tag, and a final purification step was performed using a Hi-Load 16/60 Superdex 75 column .The genes encoding A168, A169, and A168–A169 were inserted into a modified version of the vector pRSETA (Invitrogen), which encodes an N-terminal histidine tag. Protein expression was carried out in To try to establish whether any effects could be directly attributed to surface electrostatic interactions, we investigated the equilibrium behavior of the proteins under both low-salt and high-salt conditions. Note, however, that A165 was particularly aggregation prone and so data (equilibrium and kinetics) for A164, A165, and tandem A164–A165 were collected in buffer containing 150 mM NaCl  as low-salt buffer. For all proteins, the high-salt buffer contained 500 mM NaCl. All kinetics were investigated in low-salt buffer. There are Cys residues in domain A164 (2), A168 (1), and A169 (1); hence, all buffers also contained 5 mM DTT.Equilibrium studies of all the proteins were performed in urea, using between 0.5 and 2 μM protein in phosphate buffer, pH 7.0, at 25 °C with the samples being allowed to equilibrate for at least 4 h. The equilibrium behavior of all the proteins was investigated with unfolding monitored by the change in intrinsic fluorescence, on a Varian Cary Eclipse fluorimeter; the excitation wavelength was 280 nm in all cases. Emission was followed at 320 nm for all proteins except A164, which was followed at 360 nm. The data for the isolated domains and for the tandem proteins were fitted to an equation that describes a two-state transition.− 1 were measured using an Applied Photophysics stopped-flow fluorimeter. Experiments were performed with excitation at 280 nm and emission monitored at wavelengths > 320 nm for A168, A169, and A168–A169; > 350 nm for A164 and A164–A165; and > 305 nm for A165 and A164–A165. The experiments on A164, A165, and A164–A165 were carried out in 10 mM phosphate, pH 7.0, and 150 mM NaCl with 5 mM DTT. The experiments on A168, A169, and A168–A169 were carried out in 10 mM phosphate, pH 7.0, and 5 mM DTT. For A168, A168–A169, and A164–A165, double-jump (interrupted unfolding) experiments were performed: 6 μM protein was unfolded in 25 mM NaOH in the appropriate buffer; after an unfolding delay time ranging from 1 to 500 s, the unfolded protein was refolded in 5 mM HCl in the appropriate buffer (with urea), using a 5:1 ratio of acid:alkali. A final concentration of 0.5 to 2 μM protein was used in all stopped-flow experiments, except for refolding experiments investigating the dependence of the refolding rate constant upon protein concentration below 2 M urea in A168 and A168–A169, where a final concentration of 0.2, 1, and 5 μM protein was used. The stopped-flow apparatus was maintained at 25(± 0.5) °C. Rate constants below 0.005 s− 1 were determined by monitoring the change in fluorescence, after manual mixing of protein and denaturant, in a Varian Cary Eclipse fluorimeter. Excitation was at 280 nm and emission was monitored in a  1 cm path-length cuvette thermostatted at 25 °C. For both methods, the data from between 3 and 10 experiments were averaged for each denaturant concentration. Data were fitted to equations describing a single exponential, a single exponential with a baseline drift term, or a double exponential, using KaleidaGraph (Synergy Software).Rate constants > 0.005 s


Ribonuclease H-like (RNHL) superfamily, also called the retroviral integrase superfamily, groups together numerous enzymes involved in nucleic acid metabolism and implicated in many biological processes, including replication, homologous recombination, DNA repair, transposition and RNA interference. The RNHL superfamily proteins show extensive divergence of sequences and structures. We conducted database searches to identify members of the RNHL superfamily (including those previously unknown), yielding >60 000 unique domain sequences. Our analysis led to the identification of new RNHL superfamily members, such as RRXRR (PF14239), DUF460 , DUF3010 (PF11215), DUF429 , DUF1092 (PF06485), COG5558, OrfB_IS605  and Peptidase_A17 (PF05380). Based on the clustering analysis we grouped all identified RNHL domain sequences into 152 families. Phylogenetic studies revealed relationships between these families, and suggested a possible history of the evolution of RNHL fold and its active site. Our results revealed clear division of the RNHL superfamily into exonucleases and endonucleases. Structural analyses of features characteristic for particular groups revealed a correlation between the orientation of the C-terminal helix with the exonuclease/endonuclease function and the architecture of the active site. Our analysis provides a comprehensive picture of sequence-structure-function relationships in the RNHL superfamily that may guide functional studies of the previously uncharacterized protein families. E. coli was the first protein of the superfamily for which the 3D structure was determined, revealing a new architecture of the polypeptide chain, subsequently called the RNase H fold (The ribonuclease H-like (RNHL) superfamily is a large group of evolutionarily related, but strongly diverged, proteins with different functions. Ribonuclease (RNase) H from e H fold ,2. Simile H fold , Piwi/Are H fold ,6, numere H fold  and Prp82+, but Mn2+ also supports catalysis, while Ca2+ inhibits the cleavage . These representatives were used as queries to search the Protein Data Bank (PDB) with DALI (Structures of known members of the RNHL superfamily were selected from the SCOP database (1.75 release) . For eacith DALI  to identP-values of highly scoring segment pairs obtained from an N × N BLAST search, to compute attractive and repulsive forces between each sequence pair in a user-defined data set. A 2D or 3D representation of sequence families is achieved by randomly seeding the sequences in the arbitrary distance space, then moving them within this environment according to the force vectors resulting from all pairwise interactions, and repeating the process until convergence, based on the Fruchterman–Reingold graph layout algorithm . Sequence similarity-based clustering was used three times in this analysis, first to group the sequences representing proteins with known structures, second to group representatives of Pfam, COG and KOG databases and third to group all identified RNHL-like domains.Amino acid sequences corresponding to the collected structures of RNHL superfamily members were clustered using CLANS , so thatlgorithm . The P-vSupplementary File S1), which we used as main queries in our searches and in further comparative analyses.Based on the results of clustering performed with CLANS, we selected representative sequences for each cluster. We gave preference to proteins with known function and/or classified as members of the RNase H-like superfamily in the SCOP database . Orphan sequences that did not connect to any cluster were also included in the representative sequences set, after visual validation of the corresponding structures, to confirm their similarity to RNase H fold. As a result, we obtained a set of 51 RNHL members with known structure (List 2 in Supplementary File S1) were used as queries to search a database of profile-HMMs corresponding to alignments of protein families included in the COG, KOG .Sequences corresponding to multiple sequence alignments from the COG, KOG and Pfam entries predicted to be homologous to any of the RNHL queries were downloaded and clustered using CLANS . SequencSupplementary File S2). To divide the RNHL superfamily into families, the final set of domain sequences was clustered using CLANS. Owing to a large number of sequences with different levels of similarity, to produce the qualitatively best results, the clusters were defined by visual analysis of the clustering diagram using different P-value thresholds, starting with 1e-9 for identification of the most diverged sequences. Subsequently, more stringent threshold values were introduced to elucidate subgroups within more connected cluster groups. Based on the results of the clustering, 152 families were defined (the division is indicated in Supplementary File S2). For further analysis, representative sequences were selected for each of 152 clusters (Supplementary File S3). For clusters that contained at least one RNHL domain with known structure, one structure per cluster was selected as a representative, giving 41 structurally characterized representatives in total.Based on the alignments obtained in the previous step, we mapped the position of the RNHL domains in the identified full-length protein sequences. The extracted sequences of the RNHL domains were used in further steps of the analysis. To reduce the size of the resulting data set of RNHL domains, we clustered the sequences using CD-HIT  with 80%Supplementary File S1) were first superimposed using Swiss-PdbViewer  and 3D-Jury (Supplementary File S3).Sequences of proteins with unknown structures were fitted to the structural alignment generated in the previous step based on secondary structure predictions and alignments returned by the fold recognition methods via the GeneSilico metaserver gateway  . Next, all the profiles were compared with each other, and the resulting pairwise P-values were used as distance scores for clustering in CLANS. Groups of RNHL families were defined based on the manual investigation of the cluster map obtained with the P-value threshold of 1e-2.For each of the 152 RNHL families defined in this study, a HMM profile was calculated using the HHblits package ; however, both comprise similar sequence groups. The tree calculated based on the combined sequence and nonsequence data also formed similar groups, but yielded considerably better support of the deep branches (Supplementary File S4 contains the MrBayes input file and Supplementary File S5 contains the phylogenetic tree shown on Supplementary Files S8–S10). We therefore conclude that our approach is robust and that the definition of clades presented on To balance the relative weight of the sequence and nonsequence data used for the phylogenetic reconstruction, the amount of morphological characters columns was multiplied: a single column defining interfamily sequence similarity was multiplied 20 times, whereas 20 columns describing residue conservation were multiplied three times. This arbitrary assignment of weights to different types of data was based on our experience with similar analyses in the past  and aimeTo collect all known RNase H-like families, including those without structurally characterized representatives, and to identify other potentially homologous families, we conducted extensive sequence database searches. We used the representative sequences as queries to search profile-HMMs corresponding to multiple sequence alignments of protein families obtained from the COG, KOG  and PfamTo identify a complete set of the RNHL superfamily members , we conducted clustering of the sequences retrieved from those databases, selected an extended set of representatives and used the sequences of their RNase H-like domains (with other domains removed) as queries in PSI-BLAST searches of the nr database, carried out until convergence. We retrieved all sequences reported with e-value < 1e-3 and removed obvious duplicates. We have also conducted extensive analysis of the available literature to find all potentially homologous proteins, including highly divergent proteins that were not identified by the methods described above. In the final data set, we have included transposases and their inactive derivatives, 3′–5′ exonuclease domains of numerous enzymes including DNA polymerases as well as RNases, some of which, though not all, have been included in the RNase H clan in the Pfam database.Ricinus communis GI: 255594834, 255599154 and Populus balsamifera, GI: 222834205) and fungi .Transposases constitute the most abundant group of RNHL families . We idenpol polyproteins . The former classification to the A17 protein family of Bel/Pao peptidases in MEROPS database (Members of the PF05380 family (Peptidase_A17 in Pfam) are present in Bel/Pao LTR retrotransposons. This protein family exhibits a patchy phylogenetic distribution in eukaryotes, which is a feature characteristic for mobile genetic elements. A typical member of PF05380 retains catalytic residues typical for RNase H1 and most likely functions as a nuclease of the reverse transcriptase protein, as it often follows the RVT_1 reverse transcriptase domain within hypothetical Bel/Pao database  thus seeMethanohalobium evestigatum, GI: 298674346, 298674707, 298675090, 298675173, 298675374) and from bacteria to plants .We found that two other protein families: RRXRR (PF14239) and DUF429  that encompass uncharacterized and poorly annotated proteins, should be also assigned as members to the RNHL superfamily. The genomic context of DUF429 is conserved in closely related taxa, which suggests that these proteins are unlikely to be mobile. These families display conservation of potential active site residues within the RNHL domain (in the first and fourth strand), suggesting that these proteins are likely to be active as nucleases. The RNHL domain of PF14239 proteins contains a RRXRR motif in the highly positively charged region directly preceding the first core α-helix. Genomic context of proteins belonging to this family is highly variable, not even conserved between different strains of one species . Interestingly, two HGTs could be identified from bacteria to Archaea  and DUF3010 (PF11215), not assigned to RNase H clan in Pfam, but predicted herein to belong to the RNHL superfamily, show similarity to HJRs. DUF460 is present solely in Archaea, it has conserved classic RNHL active site residues  and it is also related to the Toxin expression protein (Tex) family. DUF3010 comprises single-domain bacterial proteins (mostly from Gammaproteobacteria). It exhibits conserved active site residues similar to the active site of exonucleases.Bifidobacterium breve (GI: 291457831). Both DUF1258 and Transposase_21 have conserved active site residues similar to RNase H-like proteins, which suggests that they are functional transposases. DUF1280 (PF06918) corresponds to putative transposases of Chapaev transposons present in animals , Transposase_21 (PF02992), DUF1280 (PF06918), Tnp_P_element family (PF12017), DUF659 (PF04937) and DUF4371 (PF14291). DUF1258 (PF06869) encompasses proteins from animals, mostly nematodes. This family corresponds to the Mirage group of putative transposases described previously . DUF1258 animals .Polysphondylium pallidum, GI: 281203306) and Chromalveolata .DUF659 (PF04937) and DUF4371 (PF14291) include transposases of hAT transposons. DUF659 is found mostly in Viridiplantae and it has conserved RNHL active site residues, thus most likely is a functional transposase. DUF4371 proteins occur in transposable elements (both in DNA transposons and retroelements) and their phylogenetic distribution in eukaryotes is patchy (e.g. they are absent in plants). DUF4371 proteins are composed of an N-terminal ZnF_TTF domain (acronym for ‘zinc finger in transposases and transcription factors’) followed by an RNHL domain. DUF4371 family members usually have additional domains including other zinc fingers  located at the N-terminus and a hAT family C-terminal dimerization region (Dimer_Tnp_hAT). In Pfam database the Tnp_P_element family (PF12017) comprises sequences from insects, yet we find its distant homologs also within Amebozoa . An overview of these families is given below.Clustering of RNHL domain sequences was performed based on their pairwise BLAST similarity scores using CLANS . The cluRNases H type 1  grouped into eight clusters, strongly connected to each other. Clusters E.1, E.3, E.4 and E.5 embrace eukaryotic, bacterial and viral proteins, including those from gammaretroviruses and endogenous viruses similar to gammaretroviruses, epsilonretroviruses and spumaviruses. RNHL domain of Ty3/Gypsy retrotransposons grouped within clusters E.4 and E.5. Sequences of the RNase H domain of non-LTR retrotransposons formed a cluster E.7. RNase H sequences from lentiviruses grouped into one cluster E.8. RNase H domain of the Betaretroviruses clustered in E.11 with mammalian Alpharetroviruses, avian Alpharetroviruses and Deltaretroviruses. RNase H domain of Ty1/Copia retrotransposons formed a large cluster E.2 that is connected but clearly separated from the other seven clusters. The more diverged sequences of RNase H domain from the C-terminus of hepatitis B-type viruses P proteins (PF00336) grouped in distant cluster E.15. Most of the putative nucleases of the reverse transciptase from Bel/Pao-like elements (PF05380) formed a cluster E.6.RNase H type 2 enzymes (RNH2 and RNH3) (PF01351) grouped together into one cluster IV.1 without strong sequence similarity to the above-mentioned RNases H1. Within that cluster we distinguished three subclusters: prokaryotic RNases H2 (COG0164), RNases H2 from Eukaryota (KOG2299) together with RNases H2 from Archaea (COG0164) and RNases H3 (COG1039).The integrase core domain  sequences are present in 14 clusters  connected with each other to form a supercluster in the central part of the clustering diagram. Sequences of integrase domain of Ty3/Gypsy-related retrotransposons grouped within clusters A.2 and A.12, while Ty1/Copia retrotransposons integrase can be found in cluster A.5 that formed on the edge of the central supercluster. According to the phylogenetic analysis (see below) these clusters belong to a monophyletic group that also contains transposases . Some of these clusters contain both, integrases and transposases, further confirming strong similarity between these groups. This finding is in agreement with previous studies that revealed similarity between retroelement integrases and DDE transposases from Mariner transposons .DUF4371  and DUF659  and P element transposase  show similarity to hAT Activator transposase. Transposase_21 (PF02992) and DUF1258 (PF06869) families clustered together (III.1). Tn3 transposaes  formed cluster III.3.Supplementary Table S1.OrfB_IS605 family members  show no strong similarity to any other family in the clustering of a phylogenetic analysis. Clusters of different transposases are listed in The DDE endonuclease (PF03184) cluster A.4 can be divided into two subgroups, one comprising mainly predicted fungal proteins of unknown function, and the other comprising Tigger transposable element derived proteins, Jerky protein homologs as well as the Centromere Protein B domain (KOG3105). The latter appears to have lost the metal binding residues, thus is unlikely to have endonuclease activity .Lactococcus phage middle-3 (M3) proteins, formed small clusters D.24 and D.25, respectively, but some of the individual sequences remained spread between clusters. The YqgF/RuvX family of putative HJRs  split into two well defined and strongly connected clusters (D.1 and D.14). Both clusters are connected to cluster D.7 that represents Transcriptional accessory or Toxin expression protein (Tex) , which in turn is connected to cluster D.9 of Transcription elongation factor SPT6 . Sequences of DUF460  were also clustered close to the above-mentioned groups. Sequences of COG3513 (predicted CRISPR-associated nucleases that contain McrA/HNH-nuclease and RuvC-like nuclease domains), comprising Cns1 and Cas5e families of CRISPR-associated proteins form tight separate cluster D.16 and also showed similarity to the RuvC and RuvX HJRs. Sequences from the PF11215  family, which grouped together gammaproteobacterial proteins of unknown function also showed similarity to HJRs and Tex proteins.HJRs and related proteins formed six well-defined clusters. RuvC endodeoxyribonucleases  and the mitochondrial resolvase Ydc2 family (PF09159) formed clusters D.13 and D.2, respectively. The Poxvirus A22 protein (PF04848), a HJR of the dsDNA viruses (mostly Poxviridae), and PF07066 family that comprises several Piwi/Argonaute domain sequences (PF02171) formed a large cluster with a central part comprising eukaryotic proteins (KOG1041—Translation initiation factor 2C (eIF-2C) and related proteins, KOG1042—Germ line stem cell division protein Hiwi/Piwi), surrounded by smaller subgroups of bacterial and archaeal sequences from COG1431.The endonuclease V sequences  formed I.2 cluster localized on the edge of the clustering map, but with more permissive threshold values it showed connections to the nuclease subunit of the excinuclease complex—UvrC protein . Sequences annotated either as the Endonuclease V homologs (COG1628) or proteins of unknown function DUF99 (PF01949) formed cluster V.1.Families annotated as the nuclease domain of phage terminases  formed seven separate clusters .B. subtilis YkuK protein, already predicted to be members of the RNHL superfamily  family represented by the erfamily , formed A group of proteins assigned to the NurA domain family (PF09376) formed four clusters .Cluster V.2 comprising the RNase H-like domain of the pre-mRNA processing splicing factor 8 (prp8)  was localized on the edge of the clustering diagram and does not show significant sequence similarity to any of the other families. RAG1 proteins (products of the recombination activating gene 1) , involved in V-D-J recombination, also separated at the early stage of the clustering process.A large cluster F.3 was formed by the PF03104  with eukaryotic DNA polymerases alpha, delta, epsilon and zeta  and prokaryotic DNA polymerase II (COG0417). This cluster is tightly connected to the cluster F.12 of predicted 3′–5′ exonucleases related to the exonuclease domain of PolB . Strongly connected to both of these clusters were other three clusters with members of PF13482: the first harbored predicted exonucleases from COG3359 (F.10), the second grouped predicted nuclease (RecB family) from COG2251 (F.13) and the third grouped sequences of PF13482 (F.23) from various phages. PF13482 members were also found in a separate cluster comprising RecQ ATP-dependent DNA helicases (DEAD/DEAH-box like helicase) and T7 DNA polymerases (F.14). DNA polymerases type B from plant and fungal mitochondria and viruses (PF03175) split into four small clusters based on the organism of origin .The PF00929 family, which included a variety of exonuclease proteins, formed one large cluster surrounded by five strongly connected clusters of variable size. The largest cluster F.1 comprised a variety of exonucleases, including RNase T, epsilon and alpha subunits of the DNA polymerase III, probable ATP-dependent helicase DinG homolog, 3′ repair exonuclease 2 (TREX2)  and some sequences of PF13482. We also distinguished a cluster F.8 that comprised KOG0542 and COG5018, proteins like Enhanced RNA-interference protein 1 (Eri1), 3′ histone mRNA exonuclease 1 (THEX1), Cell death Related Nuclease 4 (CRN4), Inhibitor of the KinA pathway to sporulation (sporulation inhibitor KapD) and the Prion protein interacting protein 1 (PRNPIP). There were also two small strongly connected clusters of PF00929, F.15 with Exodeoxyribonuclease I (COG2925) and F.21 with Exodeoxyribonuclease VIII enzymes. Oligoribonucleases  formed a separate cluster F.7. The last large separate cluster of PF00929 (F.5) comprised RNA exonucleases 1, 3 and 4 , interferon-simulated gene product of 20 kDa protein (ISG20) (KOG2249), Exonuclease NEF-sp  and the deadenylating RNase PAN2 (PAB-dependent poly(A) RNase, subunit PAN2) (KOG1275).Caenorhabditis.The deadenylating enzymes  grouped into two clusters: F.9 comprising POP2 and CAF1 nucleases  and F.11 with the poly(A)-specific exoribonuclease Poly(A)-specific RNase (PARN) (KOG1990) and the TOE1 protein (target of EGR1). Members of DNA_pol_A_exo1 (PF01612) (one of the Pfam families representing 3′–5′ exonucleases) formed several clusters. DNA polymerase I 3′-5′ exonuclease domain (COG0749) formed one large cluster F.2. Next to it there was a second group representing PF01612, which we divided into four subgroups: F.6 comprised exonucleases from KOG2207 and KOG4373, including the Warner syndrome ATP-dependent helicase (WRN); F.4 comprised RNase D (COG0349) and exosome complex exonuclease Rrp6 (KOG2206); F.16 comprised uncharacterized exonucleases from KOG2405; and F.25 comprised sequences of uncharacterized exonucleases from Cluster VI.3 of maelstrom proteins (PF13017) involved in germ line piRNA pathway, at more permissive threshold values showed some connections to 3′–5′ exonucleases, mainly DNA polymerase III. An RNase H-like domain from the Poxvirus F12L protein (Pox_F12L) , assigned to DNA polymerase B-like clan in Pfam, showed remote connections to several exonucleases. Arenavirus nucleocapsid proteins  also showed similarity to exonucleases. The phylogenetic analysis (see below) confirmed that these clusters are included in (or are related to) the exonuclease clade F.To identify all members of the RNHL superfamily, extended searches of the structure and sequence databases were conducted . Comparison of the selected structural representatives required a correct superposition of the structures. Due to high divergence of the structures, the superposition had to be conducted manually, based on the knowledge of relative positions of corresponding catalytic amino acids. To compare topologies of the representative domains, we prepared topological diagrams representing the structures  are present between the β-strands in the sequence on one side of the β-sheet, while the third structurally conserved α-helix (α3) is located in the C-terminus on an opposite side of the β-sheet. The C-terminal α-helix of the catalytic core can be found either immediately after the β5 strand of the core or separated from it by an insertion of variable size. The length of the strands differs vastly, both within and between different members of the RNHL superfamily. Strands β1, β2 and β3 are typically longer (8–10 residues) than strands β4 and β5 (3–6 residues). The examples of the exceptions are the structures 1zup (TM1739) and 3e9l (RNHL domain of Prp8), where the first β-strands  are almost 20 residues long. In 1zup the extension forms an additional β sheet with two small strands inserted after the common strand β3, while in 3e9l the first two β-strands form a β-finger that protrudes from the protein. The common α-helices maintain their approximate position with respect to other secondary structure elements; however, they vary greatly in size and in the angle at which they are positioned relative to other helices and to the central β-sheet; they often cannot be simultaneously superimposed, if strongly divergent members are compared with each other.E. coli DNA polymerase II (1q8i), not only an extension of the core β-sheet is present, but also an additional β-sheet parallel to the core β-sheet.In many members of the superfamily, additional secondary structure elements (most often at least one α-helix) are present N-terminally to the core, but they are not conserved throughout the superfamily and can be found in different positions and orientations with respect to the core. The C-terminal part of the RNHL superfamily members shows even higher variability than the N-terminus. Additional α-helices can be present there; they may assume positions on both sides of the central β-sheet, but are more common on the side corresponding to the position of the C-terminal α-helix of the core (α3). Such expansion of the core is present in RNases H2 and H3 , HJRs  and numerous 3′–5′ exonucleases, especially deadenylating RNases (1uoc and 2a1r). In many members the central β-sheet is extended by additional β-strands that can be found next to β3 or β5, at both edges of the common β sheet. In the Piwi/Argonaute proteins , Tn5 transposase inhibitor (1b7e) or 3′–5′ exonuclease domain of Topological diagrams illustrate the presence of structural elements characteristic for particular groups of more closely related proteins. Insertion of α-helices between the strands β2 and β3 is characteristic for RNases H2 and H3. In other proteins, insertions between the first three β-strands or between the first α1 and β4 are relatively rare. Insertion of additional secondary structure elements, usually α-helices, between β3 and α1 is common in exonucleases. This region has been shown to carry residues important for binding of the substrate 3′-terminus, indicating its importance for exonuclease activity ,42.B. halodurans RNase H1 (2g8h) lacks a regular C-terminal α-helix, which is replaced a by a loop without secondary structure, but the catalytic Asp residue is still present and positioned similarly to other members. On the contrary, in the reverse transcriptase connection domain, the helix, the loop or the catalytic residue is not present. In this case, β5 is linked directly to the subsequent domain. Another interesting feature that distinguishes 3′–5′ exonucleases from other RNHL superfamily members is the direct transition of the fifth β-strand into an α-helix.An interesting feature observed in some of the RNHL superfamily members is an inversion of the C-terminal α-helix, which preserves the axis of the helix, but reverses the direction of the polypeptide. In RNases H, integrases, transposases as well as in Ago/Piwi, UvrC, EndoV, Prp8 and also in NurA, the direction of the polypeptide in the C-terminal helix is the same as in β1, so the C-α goes along the β1 from its N to its C terminus. In DNA polymerases, deadenylases and many other exonucleases, including RNaseT, ExoI, Eri1, but also in some endonucleases including terminases and HJRs, the C-terminal helix runs in the opposite direction. The structure of To make the comparative analysis of the entire RNHL superfamily feasible with standard tools for phylogenetic analysis, we selected a set of representative sequences. Our aim was to maximize the coverage of sequence, structure and functional diversity in this superfamily. Based on the multiple sequence alignment generated for each of the identified groups described above, we selected two representative sequences from each major cluster. In the selection of the first representative we gave preference to sequences with known structures and from the SwissProt section of the UniProt Knowledgebase, which includes human-curated functional annotations. For the second representative, we selected sequences that were relatively dissimilar to the first representative, to maximize the content of information about sequence divergence within the family. As a result, we gathered 304 representative sequences of the RNase H and RNase H-like domains extracted from 152 clusters and subjected them to further analyses.Supplementary File S3).The RNase H fold is one of the evolutionarily oldest protein folds , and in Because of strong sequence divergence of various members of the RNHL superfamily, there are no evidently conserved sequence motifs or patterns common to all the RNase H-like families, apart from the position of catalytic amino acid residues. However, even these residues are not absolutely conserved. The overall conservation of the physicochemical properties of residues in the core (i.e. hydrophobicity) is a general feature of globular proteins. Nonetheless, the alignment hints at sequence similarities between certain families of RNase H-like proteins that may be more closely related to each other than to other members.The position of some of the catalytic residues is, expectedly, highly conserved among members of the RNHL superfamily. However, there are numerous variants of the active site, characteristic for specific subgroups, differing by the position and identity of additional catalytic residues. Due to higher flexibility of the α-helices in the RNHL fold compared with β-strands, the position of the catalytic amino acid residues located in α-helices is slightly more variable. They seem to be less critical for the enzymatic activity than the ones localized in the β-strands, and the main feature determining their functionality is a proper distance from the other catalytic residues. In some cases, the active site is rearranged or incomplete, lacking one or more of the catalytic residues, which often makes these enzymes inactive. Comparison of the structures in the complexes with the substrates and metal ions shows that the position of catalytic residues and metal ions is relatively well conserved within endonucleases, and even more conserved within exonucleases. When these two groups are compared with each other, we observe a shift in the position of the active site and the metal ions with respect to the core of the RNHL fold .Figure The position of the first aspartic acid residue, located in the central part of strand β1, is conserved in all of the RNHL enzymes. In RNase H2, an additional E residue is present right next to the first catalytic D. An acidic residue in this position is also present in some of the transposases and Prp8, and E is sometimes replaced by D. The RNase H-like domain of Prp8 is an example of an incomplete active site, in which only the carboxylate chains in strand β1 (DD corresponding to DE in RNase H2), are retained in the putative active site. It has been hypothesized that this domain may form a composite nuclease active site together with the functional groups from the bound RNA substrate , the arcet al. . Phylogenetic analysis based on this combined data set yielded a tree that allowed us to group reliably most of the RNHL families into 12 clades (A–F and I–VI).Supplementary File S5) revealed clear division of the RNHL superfamily (The tree obtained based on the combined data set (erfamily  into exoClades A and B comprise endonucleases and are dominated by the presence of transposases and integrases. Among them we found Hermes (PDB: 2bw), Mutator, Mariner (PDB: 2f7t), Tigger, bacteriophage Mu (PDB: 1bco), MULE and IS1 transposases and several integrase families (PDB: 1qs4). Family DUF1092 (PF06485), identified in this study as new member of RNHL superfamily, was found in clade A, indicating its involvement in the transposition/integration activity. Transposases of hAT transposons DUF4371 (PF14291) and DUF659 (PF04937) were found in clade B together with P element transposase . A closely related clade C consists almost entirely of transposases/insertion sequences such as IS4, Tn5 (PDB: 1b7e) and ISC1217, but also includes a subclade comprising four families of archaeal and bacterial NurA, two of which are structurally characterized (PDB: 1zup and 3tai). Importantly, the NurA nucleases exhibit both a single-stranded endonuclease activity and a 5′–3′ exonuclease activity on single-stranded and double-stranded DNA (Lactococcus phage M3 (DUF3882/PF07066) proteins and newly identified RNHL superfamily members: DUF3010 (PF11215), DUF460 (COG2433/PF04312), RRXRR (PF14239) and DUF429 . Interestingly, all structures present in this clade have the reversed variant of the C-terminal α-helix, which is otherwise found only in the exonuclease clade F. Clade E contains RNases H1, including representative structures 1s1x from HIV, 2g8h from Bacillus halodurans and 2rn2 from Escherichia coli. This clade also includes YkuK (PF04308), RNHL domain from Hepatitis Protein P (PF00336) and several groups of transposases and RNasesH of retroelements such as Sola, Ty1/Copia, Bel/Pao (PF05380) and archaeal COG5558. The structures present in this clade have a variant of the C-terminal α-helix characteristic for RNase H, the same as in clades A, B and C.The exact branching points of clades D and E could not be resolved; however, both these clades are clearly more related to endonuclease clades  than to the exonuclease clade F. Clade D contains transposases  and terminases (PDB: 3cpe and 3n4q), as well as resolvases . This clade also encompasses Tex (pdb: 3bzk), Spt6, Csn1 CRISPR-associated nuclease (COG3513), In the endonuclease part of the tree, we identified three smaller clades I, II and III. Clade I encompasses UvrC (2nrt) and Endonuclease V (2w36), clade II contains RAG1 protein and DUF1280 (PF06918), whereas clade III groups together phage terminases (COG5525 and COG1783), Tn3 transposase (COG4644) and a family of putative transposases DUF1258 (PF06869) that clusters together with Transposase_21 (PF02992). The branching points of the remaining small clades  could not be predicted with confidence; however, they also seem to be more related to endonucleases than to exonucleases. Clade IV contains two transposase families (represented by IS66 transposon), uncharacterized family DUF4152 (PF13680)  and typeIn the course of evolution, the RNHL fold was affected by several rearrangements, including the reorientation of the C-terminal helix and the emergence of an additional β-strand next to the third β-strand. Although the results of the analysis of structural features were not used directly to generate the phylogenetic tree, the correlation between the structural features and the tree topology can be observed. One important feature, already emphasized in this work, is the orientation of the C-terminal α-helix of the catalytic core . The same orientation of this α-helix is observed in all 3′–5′ exonucleases (clade F), but also in the HJRs, Tex protein and other members of clade D. The opposite orientation of the C-terminal α-helix is observed for all other endonucleases, i.e. those from clades A, B, C and E. Because clade D is not the closest relative of exonucleases (clade F), it is plausible that RNHL domains characterized by the reversed C-terminal helix are not monophyletic and evolved independently in the two aforementioned groups. The hypothesis of multiple origins of C-terminal helix reversion is also supported by the structural data. According to DALI clustering , structuOne can hypothesize that the common ancestor of RNHL superfamily was an endonuclease. In this scenario, the independent emergence of clades F and D involved change of the direction of the C-terminal α-helix with respect to strand β1. Alternatively, the ancestral RNHLs could have been 3′–5′ exonucleases from which endonucleases have evolved. In that alternative scenario, the origin of the endonuclease activity is associated with a reversion of the C-terminal helix, which has later returned to the ancestral state only in clade D.Interestingly, the change in the orientation of the C-terminal helix in clade D correlates with the change in the active site: in contrast to RNHLs from clades A, B, C and E that usually have Glu in fourth β, the members of clade D have Asp at the equivalent position.Another structural feature worth discussing is the presence of an additional β-strand next to the third β-strand of the catalytic core. This structural element appeared there three times independently, as it was formed by three nonhomologous sequence regions. In the structures of 3′–5′ exonucleases from clade F: 1uoc, 2fbt, 2kfn and 2a1r, that additional β-strand is formed by the N-terminal part of the RNase H-like domain, so in the sequence it is located before the first β-strand of the catalytic core. In the structure of type B polymerase, 1q8i, localized also in clade F and in the structure 1zup, localized in a distantly related clade C, analogous β-strand is formed by an insertion between the third β-strand and the first α-helix of the core. An additional β-strand in this position and with the same orientation is also present in the structure of the yeast mitochondrial HJR Ydc2 , but in this case the element is formed by the C-terminal fragment of the protein sequence.Also the additional β-strands  next to the fifth β-strand of the catalytic core can be of different origins. In the structures of the exonuclease domains of DNA polymerase type B (1noy and 1xhx), there are two and three additional β-strands present, respectively, formed by an insertion between the second α-helix and the fifth β-strand of the core. In case of the structures 1b7e, 1zup, 2qh9 and 2w36, the β-strands were formed by an insertion between the fifth β-strand and the C-terminal α-helix. In 2qh9, there are two β-strands present (as in 1noy), but there are also two additional α-helices. In the structures 1b7e and 1zup there are three additional β-strands, of which the first two have the same orientation and topology as in all the other structures, while the orientation of the third β-strand is opposite to the orientation of the third β-strand in 1xhx. Additional β-strands, but with different topology, are also present in the structures 2nrt and 1u04, while in the structure 1w9h the β-strands are shortened in respect to the corresponding β-strands in 1u04.Supplementary Figure S1. Some of the RNHL superfamily proteins possess only the core domain, typically decorated with additional α-helices and β-strands. However, in the majority of cases, the RNase H-like domain is associated with other domains, with diverse structures and functions, fused N- and/or C-terminally. In some cases, other domains are inserted into the RNase H-like domain. In the PARN, the R3H domain is inserted, which may function as a nucleic acid binding domain, while in the Hermes DNA transposases a large all α-helical domain is inserted. DNA polymerase III PolC-type is an example of a converse situation, where the RNase H-like domain is inserted into another domain. In many cases, regions of predicted intrinsic disorder are present in the terminal extensions and between the domains.The diversity of functions and substrates in proteins containing the RNase H-like domain is reflected not only in the variability within that domain, but also in the presence and order of other domains in full-length protein sequences. To identify groups of enzymes with similar combinations of domains, we conducted additional domain and fold recognition analyses for sequence regions outside of the RNHL domain . Representative diagrams are shown in Halobacterium salinarum and GI: 375358235 from Bacteroides fragilis. In the context of transposases/integrases we detected also other domains, for instance TnsA-like endonuclease domain (PF08722+PF08721) was found in Mu-like transposon GI: 383757636 from Rubrivivax gelatinosus (family A.14). Many of those domains that do not have experimentally assigned functions (DUFs) were found to be distant relatives of HtH motifs . For others, like PF14294 (co-occurring with C.14 and C.28), PF13006 (C.15 and C.28), PF13808 (C.9) and PF13700 (III.3), we could not identify evident homologs with known structures and functions; however, they probably also adopt a HtH fold.Nearly all members of the RNase H-like superfamily are nucleases; hence, they often contain domains implicated in RNA and/or DNA binding. The domains that co-occur most commonly with the RNHL domain are the different types of Helix-turn-Helix (HtH) domains, as well as different types of zinc fingers. HtH domains are present in many of the transposases and integrases, whereas zinc fingers appear to be used less frequently in these families. We identified only one transposase family that posseses both HtH and zinc finger domains: COG3676 (PF12762), e.g. GI: 4467436 from pol polyporotein of LTR retrotransposons and retroviruses, is a tandem array of RNase H 1-like from clade E and integrase domain from clade A. In the case of Ty1/Copia transposons, the order of the two domains is reversed, i.e. integrase domain (PF00665) occurs N-terminally to RNase H 1-like domain (PF00075). In animal retroviruses the above-mentioned array is preceded by another RNHL domain (pol protein (GI: 55740237) is characterized by the presence of a connection , RNase H  and integrase  domains.Many proteins contain more than one copy of an RNHL domain. The most typical arrangement, characteristic for the L domain . For exaSome domains are specific for particular families or clades. For example, the hAT dimerization domain (PF05699) is frequently present in the context of RNHL domains from clade B, i.e. B.2 , B.3 DUF659; PF04937), B.5 and in Activator-like transposases B.1 and B.6 (KOG1121). The only families in clade B, whose members do not contain hAT domain, are P elements with a Tnp_P_element domain (PF12017) . Other e; PF04937Newly identified RNHL families are frequently characterized by specific and conserved domain composition patterns. For instance, in RRXRR proteins (PF14239) localized in family D.10, the RNHL domain is frequently accompanied C-terminally by the HNH nuclease domain. This architecture resembles the CRISPR-associated nucleases (COG3513) from family D.16. Most members of DUF429 (clade D) family are single-domain proteins with a few exceptions co-occuring with a NUDIX hydrolase domain  or a RelA/SpoT domain (in Actinobacteria). Members of DUF3010  comprise a single RNHL domain. In COG2433  also only the RNHL domain was detected; however, the average length of proteins in this family suggests presence of other domains.Komagataella pastoris.Family F.16  from the exonuclease clade F co-occurs with the Helicase-and-RNase-D C-terminal domain. This domain is also present in two other families within clade F: F.6  and F.4  families represented by WRN exonuclease and RNase D, respectively. RNHL domains in proteins from COG3359  are frequently followed by Tetratricopeptide repeats. Families F.17  and F.13  were found to contain DUF83, which resembles PD-(D/E)XK nucleases. In COG2251, a tandem repeat of AAA domains is frequently observed. Some members of family F.8  co-occur with RNA recognition motif domains, for example, GI: 254572319 from Supplementary Figure S1), for example, A/26 , D.19 , IV.3 , as well as E.9 , F.28 (PF03337). However, one should keep in mind that the PFAM annotations can be misleading, for instance, PF05876 is not a single domain but corresponds to RNHL and P-loop NTPase domains.As already mentioned, many of the families comprise only the RNHL domain , DUF460 , DUF3010 (PF11215), DUF429 , DUF1092 (PF06485), COG5558, OrfB_IS605  and Peptidase_A17 (PF05380). Altogether, our study presents a comprehensive picture of sequence-structure-function relationships among RNHL superfamily members. We hope this analysis will help to understand relationships between proteins that have been functionally characterized, and to predict functions and plan experiments for proteins that have not yet been studied.Supplementary Data are available at NAR Online.European Research Council (ERC) [StG RNA + P = 123D to J.M.B.]; Foundation for Polish Science , Polish National Science Centre ; statutory funds of the Adam Mickiewicz University ; Ministry of Science and Higher Education [grant Iuventus Plus 0376/IP1/2011/71 to A.M.]; European Social Fund [UDA-POKL.04.01.01-00-072/09-00 to K.S.]; International Early Career Scientist grant from the Howard Hughes Medical Institute ; ‘Ideas for Poland’ fellowships from the FNP (to M.N. and J.M.B.) and ‘START’ fellowship from the FNP (to S.D.H.). Funding for open access charge: ERC [StG grant RNA + P = 123D].Conflict of interest statement. None declared.