The Toll-like receptors represent a largely evolutionarily conserved pathogen recognition machinery responsible for recognition of bacterial, fungal, protozoan, and viral pathogen associated microbial patterns and initiation of inflammatory response. Structurally the Toll-like receptors are comprised of an extracellular leucine rich repeat domain and a cytoplasmic Toll/Interleukin 1 receptor domain. Recognition takes place in the extracellular domain where as the cytoplasmic domain triggers a complex signal network required to sustain appropriate immune response. Signal transduction is regulated by the recruitment of different intracellular adaptors. The Toll-like receptors can be grouped depending on the usage of the adaptor, MyD88, into MyD88-dependent and MyD88 independent subsets. Herein, we present a unique phylogenetic analysis of domain regions of these receptors and their cognate signaling adaptor molecules. Although previously unclear from the phylogeny of full length receptors, these analyses indicate a separate evolutionary origin for the MyD88-dependent and MyD88-independent signaling pathway and provide evidence of a common ancestor for the vertebrate and invertebrate orthologs of the adaptor molecule MyD88. Together these observations suggest a very ancient origin of the MyD88-dependent pathway Additionally we show that early duplications gave rise to several adaptor molecule families. In some cases there is also strong pattern of parallel duplication between adaptor molecules and their corresponding TLR. Our results further support the hypothesis that phylogeny of specific domains involved in signaling pathway can shed light on key processes that link innate to adaptive immune response. The pattern recognition receptors (PRR) of innate immunity form a large class of germ-line encoded receptors implicated in the detection of pathogen associated microbial patterns (PAMPs) and the initiation of downstream signaling cascades. Although these proteins are widely expressed, they play an important role in cell types collectively named antigen presenting cells (APC), such macrophages and dendritic cells, and specialized in sampling and presenting microbial antigens to lymphocyte Drosophila melanogaster. Originally isolated in connection with dorsal-ventral developmental patterning Nicotiana glutinosaAmong the best characterized pattern recognition receptors, the Toll-like receptors (TLRs), are named for their homology with the Toll receptor first identified in The evolution of TLR signaling is driven by the need to conserve the capability to recognize specific pathogen signature while at the same time allowing for the development of new platforms on which to build more complex signaling networks drives. To better understand this dynamic and the molecular evolution of TLRs, phylogenetic reconstructions of the TLRs have been calculated from both full-length gene or amino acid sequences Individual protein domains are the palette of structural units from which protein functions are composed TLRs also do not act alone. Although all TLRs result in the activation of the transcription factor NF-κB and IFN regulatory factors (IRFs) A second, MyD88-independent, signaling pathway is used exclusively by TLR3 and in addition to the MyD88-dependent pathway by TLR4. TLR3 and TLR4 make use of this pathway through the recruitment of the adaptor molecule TRIF Because adaptor molecules are not simply links between receptors and their effectors, but are coordinators of the signaling pathway dynamic, they are a critical factor shaping the evolution of the TLRs and their protein domains. In this study we report a molecular cophylogeny of the TLRs together with their signaling cognate adaptor molecule viewed through their shared TIR domain. We use these data to show how early gene duplications shaped extant TLR and adaptor associations, confirm the relationship between invertebrate and vertebrate TLR families, evince the ancient origin of the TIR domain and its associated adaptors, and suggest a model for the emergence of the MyD88 independent pathways. This represents a novel approach to the analysis of the TLR evolution through the phylogeny of the TIR signaling domain of related proteins in the signaling cascade.Homo sapiensinterleukin-1 receptor. Consistent with previous phylogenies of the TIR domain of the TLRs, in both reconstructions, strong boot-strap support is observed for individual branches corresponding to vertebrate TLR3, TLR4, TLR5, and invertebrate TLR9. In addition a single cluster composed of vertebrate TLR7, TLR8, and TLR9 shows strong support. As observed by Kanzok et. al. Following the protocol described in methods section, we analyzed TIR domain of TLRs family members and their adaptors. The maximum likelihood (ML) phylogeny reconstructed from the TIR domains of the TLRs and TLR adaptor molecules is shown in Homo sapiensMyD88. Again individual clusters for vertebrate TLR3, TLR4, TLR5, and invertebrate TLR9 are shown to be strongly supported in both reconstructions as is a strongly supported branch containing vertebrate TLR7, TLR8, and TLR9. Placement and support of the remaining vertebrate TLRs with no human ortholog are essentially identical to the reconstruction including the TLR adaptor molecules. Boot-strap values observed in the focused reconstruction are somewhat larger than the boot-strap values observed in the reconstruction including adaptor molecules. However, as observed previously, boot-strap supports observed in the ML reconstruction remain uniformly lower than the ME reconstruction.The phylogenies of the TIR domain of the TLRs not including the adaptor molecules and the phylogenies of the full length TLRs show receptor groupings consistent with the groupings found in the phylogeny of the TIR domain of the TLRs calculated with the adaptor molecules. This suggests that the adaptor molecules have co-speciated with the TLRs. ML and ME reconstructions of the TIR domain of the TLRs are shown in Nicotiana glutinosa gene of the full-length TLR are displayed in figures S4 and S5 respectively. Consistent with the TIR domain phylogenies reconstructed with and without TLR adaptor molecules, vertebrate TLR3, TLR4, and TLR5 form strongly supported individual branches and a single strongly supported cluster contains vertebrate TLR7, TLR8, and TLR9. As observed in all phylogenies restricted to the TIR domain, within the TLR7, TLR8, and TLR9 cluster, TLR9 is observed to associate more distantly than TLR7 and TLR8. Similarly both ME and ML have a strongly supported individual cluster for TLR2 and a strongly supported cluster for TLR1, TLR6, and TLR10 as observed in the TIR restricted reconstructions.ML and ME reconstructions of the full-length TLRs rooted by the outgroup consisting of the protein coded by the Homo sapiensTLR5. Three branches are moderately supported in both reconstructions: a MyD88 branch including both invertebrate and vertebrate MyD88, a branch containing TIRAP, and a branch composed of TRAM, TRIF, and SARM. The branching of MyD88 from the remaining adaptor molecules is well-supported and there is modest support in both reconstructions for a common ancestor to invertebrate and vertebrate MyD88 as observed in the reconstructed phylogeny of TIR including the TLRs.Reconstructions of the phylogeny of the TIR domain of the adaptor molecules along are shown in Within the TRAM, TRIF and SARM branch, there is moderate to strong support in both reconstructions for a common ancestor to the MyD88-independent pathway adaptors: TRAM and TRIF. SARM also appears to be associated to this branch although it is observed with less support, consistent with the hypothesis that SARM is a late addition to the adaptors family The data above suggest a long standing evolutionary relationship between TLRs and their adaptor molecules. To uncover the dynamics of this relationship, the TIR domain of the adaptor molecule phylogeny was embedded into the phylogeny of the TIR domain of the TLRs restricted to human and mouse . All genThese embeddings suggest an early cospeciation of the MyD88 adaptor molecule branch from the branch containing ancestors of the remaining adaptors with the MyD88 ancestor associated to the invertebrate TLR branch and the common ancestor of TIRAP, TRIF, TRAM, and SARM associated with the vertebrate TLRs. In the invertebrate TLR branch, a later cospeciation is observed with the divergence of individual invertebrate TLRs. Vertebrate MyD88 is indicated as resulting from an early host switch from the invertebrate MyD88 branch. This branch then duplicates ultimately resulting in distinct human and mouse MyD88.The adaptor molecules TIRAP, TRIF, and SARM are indicated as resulting from early duplication events associated with the vertebrate TLR phylogeny. Ancestors of the molecules specialize at a later stage into extant forms for mouse and human. A distinct pattern of cospeciation is observed with TRAM where adaptor molecule phylogeny branches for mouse and human TRAM diverge roughly contemporaneously with the divergence of TLR4 for mouse and human, the ancestor of TRAM being the result of a duplication event within the TIRAP, TRIF, and SARM.The need to conserve the capability to recognize specific pathogen signature while at the same time allowing for the development of new platforms on which to build more complex signaling networks drives the evolution of TLR signaling. The high degree of conservation of recognized ligands between orthologous TLRs can be, in part, explained by the observation that TLRs have evolved to recognize microbial PAMPs that cannot be easily and viable mutated in pathogens. Nevertheless pressure to maintain endogenous signaling networks remains a significant constraint. The difficulty in satisfying these competing requirements suggests the importance of adaptors molecules. Phylogenetic analysis of TLRs and their adaptor molecules can reveal the history and dynamic of these competing requirements on the evolution of TLRs.The inclusion of the transmembrane and extracellular domains of the receptor in the analysis shows how dominant these specific functional domains are dictating the TLR phylogeny. Comparing the reconstructions of the full-length TLRs with the TLR family phylogeny restricted to the TIR signaling domain shows that the large extracellular domain drives the topology of the phylogeny. This observation suggests that functional constraints in PAMP recognition dictate the evolution of this interface. All reconstructions support an interpretation of the clustering based on pattern recognition ligand In contrast, restriction of the phylogenetic analyses to the TIR domain highlights the evolution of TLR signaling. Phylogenetic reconstructions focusing on the TLRs and on their adaptor molecules separately result in essentially identical configurations suggesting that the need to maintain a particular suite of protein-protein interfaces between the TIR domain and the adaptor molecules is applying an evolutionary constraint. Among TLRs all reconstructions show strong support for clusters consisting of TLR3, TLR4, and TLR5 individually, and a single cluster for TLR7, TLR8, and TLR9. Moderate support is found for two clusters consisting of TLR2 and TLR1, TLR6, and TLR10. In most reconstructions these two clusters are joined into a single modest to moderately well-supported cluster.The phylogenetic analyses of the TIR domain of the TLRs together with their adaptor molecules support the hypothesis that adaptor molecule usages and signaling pathway are ancestral characteristics of the TLRs. Among the TLRs that use MyD88 exclusively, well-supported branches for TLR7, TLR8, and TLR9 and for TLR5 are observed. Similarly, TLR3, using TRIF exclusively, and TLR4 using MyD88, TIRAP, TRIF, and TRAM, form well-supported, individual branches as do the adaptor molecules used by the MyD88-independent pathway: TRIF and TRAM. The hypothesis is supported to a lesser extent by the TLRs using both MyD88 and TIRAP: TLR2 and TLR1, TLR6, and TLR10; with TLR2 and TLR1, TLR6, and TLR10, belonging moderately supported branches.Comparing to the TIR domain reconstructions including the TLR TIR domains as well as the TLR adaptor molecule TIR domains, the clustering of TRIF and TRAM is strongly supported over all reconstructions. Similarly all reconstructions support invertebrate and vertebrate MyD88 branches. Furthermore when compared in the context of TLR TIR domains, reconstructions strongly support a common ancestor for these two MyD88 branches suggesting that the evolution of MyD88 adaptor-TIR domain is ancient. Additionally, all reconstructions show at least moderate support for an individual TIRAP branch. These data shed additional light on recent results concerning the coevolution of TRIF, TRAM, and TIRAP with the respective TLR3/22 and TLR2 signaling pathway The hypothesis that adaptor molecule usage is an ancestral characteristic of the Toll receptor signaling is further supported by the reconstructions of the adaptor molecules phylogeny. Well-supported monophyletic clusters are observed for vertebrate MyD88, invertebrate MyD88, and both of the adaptor molecules required in the MyD88-independent pathway: TRIF and TRAM. The adaptor molecule TIRAP, used by TLR1, TLR2, TLR4, TLR6, and TLR10, is isolated to a single cluster in all reconstructions. There is no observed evidence for a common ancestor between the TIR domains of any of the TLRs and the TIR domains of any of the adaptor molecules. This pattern suggests that the TIR domains of adaptor molecules are not derived from the TIR domains of TLRs and that the primordial TLR may have co-opted an existing adaptor based signaling system.The cophylogeny of the TLRs with their adaptor molecules reveal how adaptor molecule parallels TLR evolution, pinpoints key gene duplication events , and in The outcome of pathogen recognition is critically dependent on the TLR-restricted utilization of TIR domain-containing adaptor molecules to drive stimulus specific responses. The phylogenetic analysis reported here indicates an independent evolutionary origin for the MyD88-dependent and MyD88-independent pathways. Furthermore this study indicates a common ancestor for vertebrate and invertebrate MyD88, therefore suggesting a very ancient origin of the MyD88-dependent pathway. Through the phylogeny of TIR domain, this work points out the evolution of a complex signaling network by the different usage of adaptor molecules, otherwise lost in the full length sequences analysis. Overall, this confirms a recently study in human, which reports a different form of selection between TLRs family members and their adaptors, and showes that the adaptors represent a more essential and no redundant component of the TLRs signaling cascade An initial set of amino acid sequences for the TLRs was obtained from NCBI GenBank Anopheles gambiae, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Takifugu rubripes, Gallus gallus, Mus musculus, Homo sapiens, was queried for the matches to the full-length HMM model resulting in the final set of full-length amino acid sequences submitted to phylogenetic analysis. These species have been chosen to provide the most phylogenetic insight into TLR signaling, in general, and the interactions of the TLRs with their adaptor molecules, in particular, across a wide spectrum of organisms. The resulting set of amino acid sequences was submitted to multiple sequence alignment by ProbCons 1.12 The TIR domain was predicted from the initial set of amino acid sequences using the appropriate Pfam proml in the PHYLIP 3.68 package protdist found in PHYLIP. Minimum evolution phylogeny was constructed from this distance matrix by fastmeseqboot and the consensus phylogeny was assembled with consense, both from the PHYLIP package. FigTree 1.2.1 (http://tree.bio.ed.ac.uk/software/figtree/) was used for both initial visualization and final production of figures of the resulting phylogenetic trees.Multiple sequence alignments constructed by ProbCons were submitted to both maximum likelihood and minimum evolution phylogeny reconstruction. Maximum likelihood reconstructions were calculated using Cophylogenetic embeddings of the TIR domain of the adaptor molecules into the phylogeny of the TIR domain of the TLRs together with empirical estimates of significance were constructed with Jane Figure S1Minimum evolution phylogeny of the TIR domain of the TLR family and TLR adaptor molecules. The tree is rooted by the outgroup TIR domain Homo sapiensinterleukin 1 receptor. The numbers indicate boot-strap support out of 1000. Only value above 500 are indicated.(TIFF)Click here for additional data file.Figure S2Maximum likelihood phylogeny of the TIR domain of the TLR family alone. The tree is rooted by the outgroup TIR domain Homo sapiensMyD88. The numbers indicate boot-strap support out of 1000. Only value above 500 are indicated.(TIFF)Click here for additional data file.Figure S3Minimum evolution phylogeny of the TIR domain of the TLR family alone. The tree is rooted by the outgroup TIR domain Homo sapiensMyD88. The numbers indicate boot-strap support out of 1000. Only value above 500 are indicated.(TIFF)Click here for additional data file.Figure S4Maximum likelihood phylogeny of the TLR family reconstructed from the complete amino acid sequence. The tree is rooted by the outgroup Nicotiana glutinosaN. The numbers indicate boot-strap support out of 1000. Only value above 500 are indicated.(TIFF)Click here for additional data file.Figure S5Minimum evolution phylogeny of the TLR family reconstructed from the complete amino acid sequence. The tree is rooted by the outgroup Nicotiana glutinosaN. The numbers indicate boot-strap support out of 1000. Only value above 500 are indicated.(TIFF)Click here for additional data file.Figure S6Minimum evolution phylogeny of the TIR domain of the TLR adaptor molecules alone. The tree is rooted by the outgroup TIR domain Homo sapiensTLR5. The numbers indicate boot-strap support out of 1000. Only value above 500 are indicated.(TIFF)Click here for additional data file.Supplementary Materials and Methods S1(DOCX)Click here for additional data file.


SCOP is a manually curated ordering of domains from the majority of proteins of known structure in a hierarchy according to structural and evolutionary relationships. Development of the SCOP 1.x series concluded with SCOP 1.75. The ASTRAL compendium provides several databases and tools to aid in the analysis of the protein structures classified in SCOP, particularly through the use of their sequences. SCOPe extends version 1.75 of the SCOP database, using automated curation methods to classify many structures released since SCOP 1.75. We have rigorously benchmarked our automated methods to ensure that they are as accurate as manual curation, though there are many proteins to which our methods cannot be applied. SCOPe is also partially manually curated to correct some errors in SCOP. SCOPe aims to be backward compatible with SCOP, providing the same parseable files and a history of changes between all stable SCOP and SCOPe releases. SCOPe also incorporates and updates the ASTRAL database. The latest release of SCOPe, 2.03, contains 59 514 Protein Data Bank (PDB) entries, increasing the number of structures classified in SCOP by 55% and including more than 65% of the protein structures in the PDB.Structural Classification of Proteins—extended (SCOPe,  Nearly all proteins have structural similarities with other proteins and, in many of these cases, share a common evolutionary origin. The Structural Classification of Proteins (SCOP) database , which mBy analogy with taxonomy, SCOP was created as a hierarchy of several levels where the fundamental unit of classification is a ‘domain’ in the experimentally determined protein structure. The hierarchy of SCOP domains comprises the following levels: ‘Species’ representing a distinct protein sequence and its naturally occurring or artificially created variants; ‘Protein’ grouping together similar sequences of essentially the same functions that either originate from different biological species or represent different isoforms within the same species; ‘Family’ containing proteins with similar sequences but typically distinct functions and ‘Superfamily’ bridging together protein families with common functional and structural features inferred to be from a common evolutionary ancestor. Near the root, the basis of classification is purely structural: structurally similar superfamilies are grouped into ‘Folds’, which are further arranged into ‘Classes’ based mainly on their secondary structure content and organization.The ASTRAL compendium  is a colThe SCOP database was first released in December 1994 and contained all 3091 entries in the PDB at that time (http://scop.berkeley.edu/) together with copies of our relational (MySQL) database and parseable files containing all SCOPe, SCOP and ASTRAL data.In this article, we describe SCOP—extended (SCOPe), a database that extends SCOP 1.75, with the aim of providing ongoing classification of new PDB structures in the context of the SCOP hierarchy, backward compatibility with SCOP, manual correction of errors and stable releases suitable for benchmarking. More than 800 new structures are now being deposited to the PDB in an average month  as were used for prior releases of SCOP and ASTRAL ,5,6, andUsing our new automation procedure (described in the next section), we have added 21 293 PDB files to SCOPe that were not classified in SCOP 1.75 (∼23% of the PDB). We have also modified some previous entries that were manually curated or classified via a previous automated method introduced in SCOP 1.73 . Based oWe have implemented a new automated classification algorithm, based on sequence similarity to previously classified entries, and benchmarked this method against all stable releases of SCOP from 1.57 to 1.75.An automated method for classifying new PDB entries based on previously classified entries was introduced in SCOP 1.73 ; howeverThe SCOP automated curation pipeline has been completely rewritten in SCOPe. We are replacing all domains that were predicted with the less accurate SCOP 1.73 automated method with newer predictions. Over the course of benchmarking our new method, we found the method would have been able to automatically assign domains for approximately half of the PDB entries that were manually curated in prior versions of SCOP, with no substantial differences (i.e. >10 residue difference in any domain boundary) between the automatically assigned and manually curated domain boundaries. Over the course of benchmarking, we found some differences that, upon expert examination, were determined to be the result of manual curation errors in prior versions of SCOP. These are discussed later in the manuscript and were corrected in SCOPe 2.03.One of the main challenges in automatically assigning domains based on previously classified homologs is that different homologs may have different observed residues (i.e. those present in the ATOM records of PDB data). SCOP domains in a structure are defined relative to the observed residues. It is sometimes challenging to correctly assign observed residues in a new structure that were not observed in a previously classified structure. Another challenge is in variance in manually curated boundaries. We found that in multi-domain chains with long linker regions, the exact boundaries between domains within a family can differ substantially. This is because fully manually curated releases of SCOP (those before 1.73) aimed to classify every residue in each PDB structure, even though it is sometimes unclear to which, if either, of the adjacent domains residues in a linker region should be assigned.−4 and the alignment covers most of the target domain (defined as missing at most 10 residues from each end). We group the alignments by the PDB chain that the targets belong to and rank the groups by the total number of residues covered by the hits on the query chain. We then use the top-ranking group of BLAST-based alignments to annotate domains in the query sequence. If the nearest PDB chain end or gap in ATOM records is within 10 residues of an alignment end, we extend the domain to include these residues. The purpose of extending the BLAST hit is to classify every observed residue in the chain; the 10-residue limitation makes it very unlikely that the extension will include a new domain. If the BLAST boundaries are outside the observed residues in the query chain, the assigned domain is shortened to include only observed residues.We now describe our algorithm for predicting domains and classifying them in the SCOP hierarchy. We first create a BLAST database containing the SEQRES-based sequences for each domain in SCOP and SCOPe. Then, for each newly released PDB chain, we BLAST its SEQRES sequence against the domain sequence database. BLAST performs local alignment, returning the start and end of the alignment (the ‘hit’) for the query sequence and the target sequence, as well as the E-value. We collect only the BLAST alignments where the E-value is at least as significant at 10After making predictions for each chain, we applied a set of criteria to determine whether each domain prediction was high confidence . We first exclude from high confidence those chains that are low-resolution (3 Å resolution or above), ribosomal or synthetic (due to those being classified outside the first seven classes of SCOP), or those that are homologous to genetic domains classified in SCOP (due to the difficulty of correctly automating predictions that include multiple PDB chains).In cases where an entire PDB chain was predicted to be a single SCOPe domain, the prediction was deemed high confidence if the target domain also comprised its entire PDB chain. For PDB chains that were divided into multiple SCOPe domains, additional criteria were used to determine whether the predicted domains were high confidence. First, we restricted the chains whose predictions we placed in the high confidence set to those which either (i) had 100% sequence identity with the target chain used to make the predictions or (ii) had exactly two domains, each composed of only one contiguous region of residues. We plan to extend the method to three or more domain chains and multi-region domains in the future. Second, if any region found in the ATOM records of the chain was longer than 10 residues and not assigned to any domain, we removed the chain from the high-confidence set. Third, we required the BLAST hits used for the two domains in the chain not be to the same target SCOP domain .To fully classify these predictions in the SCOP hierarchy, we also had to assign levels below ‘Superfamily’. Based on our benchmarking, we developed heuristic rules to classify domains at the ‘Family’, ‘Protein’ and ‘Species’ levels. In cases where the protein or family could not be reliably matched to an existing SCOP entity, we created a new protein or family called ‘automated matches’ rather than risking inaccurate classification.Thus far our focus has been on classifying chains that have high sequence similarity with previously classified chains in SCOP and therefore have relied solely on sequence data. The addition of structural information should expand the set of domains that can be classified with high confidence in the future.To validate the new automated method, we performed benchmarking against all SCOP releases with stable identifiers (i.e. releases 1.55–1.75). All PDB entries that were added between each pair of consecutive releases were automatically classified based on the earlier release and compared with the manually curated domains in the subsequent release. A predicted domain was considered identified and classified correctly if it was placed in the correct superfamily and its boundaries differed from the manually curated boundaries by no more than 10 residues. The SCOPe website offers a modest redesign of the SCOP website, presenting all SCOPe, SCOP and ASTRAL data through a single, unified web interface. Many objects that were difficult to find in the original website, such as the change history, are now available under tabs. Thumbnail images were automatically generated to show each domain on its own and in several structural contexts, and these are displayed as part of the browser. A fully JavaScript JSmol-based viewer was added to enable visitors to view domains in three-dimensions in isolation, in context of the chain or in context of the entire PDB structure. The SCOPe website can display data from all versions of SCOPe, SCOP and ASTRAL since release 1.55. All data are stored in a relational (MySQL) database, which is also available for download.Stable releases will potentially include manual curation of the SCOPe hierarchy, correction of errors in previously classified domains or changes to our classification workflow . However, in an effort to stay more closely synchronized with the PDB, we also plan to supplement these stable releases with periodic updates (approximately monthly). Our infrastructure automatically imports and classifies new PDB files on a weekly basis. Starting with SCOPe 2.02, we have begun to release periodic updates that add newly released PDB entries to the SCOPe classification. These periodic updates add new PDB entries to the current release, without affecting any previously classified domains. The newly classified entries are visible in the web interface and in downloadable files such as the SCOP-compatible parseable files and MySQL database. Sequences for the newly added chains and domains will not be added to the ASTRAL representative subsets until the subsequent stable release of ASTRAL.The periodic updates are not intended to replace stable releases; because the latter are commonly used for benchmarking, both will be available for download through the SCOPe website. Stable releases will be assigned version numbers in the current format , while updates to stable releases will be named according to the most recent stable version appended with the release date (e.g. 2.03-2013-12-01).National Institutes of Health (NIH) [R01-GM073109] through the US Department of Energy under Contract No. DE-AC02-05CH11231. Funding for open access charge: NIH [R01-GM073109].This work is supported by the Conflict of interest statement. None declared.


UBE3A. Individuals with AS suffer from intellectual disability, speech impairment, and motor dysfunction. Currently there is no cure for the disease. Here, we evaluated the phenotypic effect of activating the silenced paternal allele of Ube3a by depleting its antisense RNA Ube3a-ATS in mice. Premature termination of Ube3a-ATS by poly(A) cassette insertion activates expression of Ube3a from the paternal chromosome, and ameliorates many disease-related symptoms in the AS mouse model, including motor coordination defects, cognitive deficit, and impaired long-term potentiation. Studies on the imprinting mechanism of Ube3a revealed a pattern of biallelic transcription initiation with suppressed elongation of paternal Ube3a, implicating transcriptional collision between sense and antisense polymerases. These studies demonstrate the feasibility and utility of unsilencing the paternal copy of Ube3a via targeting Ube3a-ATS as a treatment for Angelman syndrome.Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by maternal deficiency of the imprinted gene  UBE3A, which is preferentially silenced on the paternal chromosome and expressed on the maternal chromosome in neurons due to genomic imprinting. It has been long proposed that by activating the originally silenced paternal allele of UBE3A, the disease may be cured. Here in our research, we demonstrated the feasibility of activating paternal Ube3a in mice by terminating the transcription of its antisense RNA Ube3a-ATS genetically. In the AS mouse model who additionally receives the terminated Ube3a-ATS allele from the paternal side, we observed restoration of Ube3a expression, amelioration of behavioral defects and reversal of the impaired long-term potentiation. We further studied the imprinting mechanisms of Ube3a and proposed a novel transcriptional collision model. These results provide solid in vivo evidence for a key regulatory role of Ube3a-ATS in the disease and open up an exciting possibility of a gene-specific treatment for Angelman syndrome.Angelman syndrome (AS) is a devastating neurodevelopmental disorder diagnosed in young children, currently with no effective treatments. It is characterized by absence of speech, ataxia, intellectual disability, epilepsy, and a characteristic behavior of frequent laughter and smiling. The disease is caused by loss of the maternal allele of  Angelman syndrome (AS) is clinically manifested by features of intellectual and developmental disability, absence of speech, ataxic movement, epilepsy, and unique behaviors such as frequent laughter and fascination with water UBE3A is intact. One could speculate that by correcting the expression level of UBE3A via activating the silenced paternal allele, the disease might be treated.Genetically, the disease is caused by deficiency of an E3 ubiquitin ligase termed UBE3A, which participates in many important neuronal functions such as synaptic development, signal transduction, and plasticity NDN, SNRPN, SNORD115 and SNORD116, are critical genes for Prader-Wiili syndrome (PWS) and form an 2-Mb imprinting cluster together with the AS gene UBE3A. Although not fully understood, it is generally believed that the PWS/AS region is regulated by a bipartite imprinting center composed of PWS-IC, which activates genes located in its proximity via looping and direct interacting with them, and AS-IC, which suppresses PWS-IC by transcription-mediated DNA methylation NDN and SNRPN show very distinct epigenetic patterns of DNA methylation and histone modifications Imprinted genes usually form clusters in the genome and are controlled by the imprinting center (IC). On human chromosome 15q11–q13, paternally expressed genes, including MAGEL2, UBE3A, however, is not associated with differential DNA methylation at the promoter region UBE3A-ATS, which is expressed from the paternally inherited chromosome in the brain Shng14) initiated from the Snrpn promoter in mice Ube3a-ATS expression is always negatively associated with Ube3a sense transcript. For example, when the Snrpn promoter was deleted, with or without the Prader-Willi syndrome imprinting center (PWS-IC), the Ube3a-ATS level was found to be reduced, coupling with significant up-regulation of paternal Ube3aUbe3a-ATS was activated through replacement of the mouse imprinting center (IC) with the human one, or deletion of the putative AS-IC, maternal Ube3a was found to be repressed to some extent Ube3a-ATS transcription in neuronally differentiated ES cells, we have showed that paternal Ube3a can be activated to a comparable level as maternal Ube3aUbe3a-ATS in suppressing paternal Ube3a.Imprinting of Ube3a-ATS as a potential therapeutic target for treating Angelman syndrome. By characterizing a novel mouse model expressing the truncated form of Ube3a-ATS, we provide the first in vivo evidence that eliminating Ube3a-ATS is sufficient to restore Ube3a expression and improve the abnormal behaviors in the AS mouse model. Mechanisms underlying paternal Ube3a silencing are also studied, and a hypothesis of transcriptional collision between Ube3a and Ube3a-ATS is proposed.In the present study, we continue evaluating Ube3a-ATS alone is sufficient to unsilence the paternal allele of Ube3a, mice with the ATS-stopUbe3a allele were generated by inserting the triple SV40 poly(A) cassette Snord115 and Ube3a (chr7:66573289 NCBI37/mm9)  . This deUbe3a-ATS and other genes located in the imprinting cluster. The Ube3a-ATS level downstream of the insertion site , indicating that the imprinting status of the PWS/AS region is not disrupted by the insertion. The level of Ndn was found to be approximately doubled in +/ATS-stopUbe3a mice compared to the other two genotypes. It is interesting that similar observation has been found in S-U/0.9del mice previously Ube3a-ATS at a lower level due to Snrpn promoter deletion. The reason for the observed up-regulation is unclear.We first determined the effect of the termination cassette on the expression level of ion site . ExpressUbe3a mRNA is doubled in the +/ATS-stopUbe3a mice, suggesting that paternal Ube3a may be unsilenced. To confirm this, male mice heterozygous for stopUbe3a-ATS were crossed with female mice heterozygous for KOUbe3aUbe3a knock-out allele (KO/+Ube3a (AS), +/ATS-stopUbe3a (stop), KO/ATS-stopUbe3a (AS/stop) were compared. In the AS/stop mice, Ube3a protein was found to be activated to ∼70% of the WT level in neocortex, ∼60% in hippocampus, and ∼50% in cerebellum. The incomplete activation may be due to leaky termination of Ube3a-ATS, as about 20% of Ube3a-ATS can still be detected in +/ATS-stopUbe3a mice  mouse model. To address this, male ATS-stopUbe3a heterozygous mice were crossed with female KOUbe3a heterozygous mice KO/+Ube3a (AS), +/ATS-stopUbe3a (stop), KO/ATS-stopUbe3a (AS/stop) were studied for various AS-related phenotypes.We next tested whether inheritance of KO/+Ube3a mice become overweight starting from three month of age, in both males and females ((WT vs. AS)<0.01 for 4, 5, 6 months of age, two-way ANOVA of repeated measures). Activation of paternal Ube3a in the AS/stop mice completely reversed the obese phenotype (p(AS vs. AS/stop)<0.05 for 4, 5, 6 months of age).Obesity is associated with a small portion of AS patients (WT vs. AS)<0.001, one-way ANOVA with Newman-Keuls post-hoc test). AS/stop mice showed a slight but significant improvement over AS mice <0.05).The marble burying test measures repetitive behavior as potentially analogous to an autistic phenotype. Interestingly, AS mice were found to be dramatically impaired in performing this task <0.05 for all eight trials, two-way ANOVA of repeated measures). AS/stop mice show restoration in the first few trials of accelerating rotarod, although they fail to improve in later trials (p(AS vs. AS/stop)<0.05 for trial 1–4). They also show full restoration of other motor defects during wire hanging test and dowel test, indicating a significant improvement of their motor coordination skills ((AS vs. AS/stop)<0.01 for wire-hanging test and <0.001 for dowel test, one-way ANOVA with Newman-Keuls post-hoc test). It is noted that maternal inheritance of the ATS-stopUbe3a allele does not affect the performance of the mice in all three motor tests <0.05, one-way ANOVA with Newman-Keuls post-hoc test). Remarkably, the freezing behavior displayed in the AS/stop mice is comparable to the WT mice, suggesting that long-term memory is fully restored <0.01).Individuals with AS are frequently affected with specific cognitive deficits (WT vs. AS)<0.01, one-way ANOVA). Notably, the expression of paternal Ube3a reverses the LTP deficits <0.05). The LTP rescue in AS/stop slices cannot be attributed to abnormal basal synaptic transmission, since the relation of fiber volley versus stimulation intensity, initial slope of field EPSPs versus afferent volley size, and paired pulse facilitation were unaltered in these slices  at Schaffer collateral–CA1 synapses, using high-frequency stimulation as the LTP-inducing protocol e slices .UBE3A, it is important to understand the molecular mechanism underlying genomic imprinting of Ube3a. Promoters of both the paternal and maternal UBE3A remain unmethylated in human brains UBE3A. In order to look for parent-of-origin epigenetic markers that may account for UBE3A imprinting, we first analyzed histone modifications of H3K4 trimethylation (H3K4me3) in human cerebellum tissues by ChIP-on-chip experiment . The PIC is a large protein complex composed of RNA polymerase II, TATA binding protein (TBP), TFIIB, and many other proteins assembled at the promoter of active genes. ChIP with antibody against RNA polymerase II was performed in brain samples of F1 hybrid of C57  crossed with B6.Cast.Chr7 , which carries Mus. musculus castaneus chromosome 7 on the Mus. musculus domesticus C57BL/6 background. Single nucleotide polymorphisms (SNPs) between the two lines allow detection of parental specific alleles. In contrast to the Snrpn promoter, from which only the transcriptionally active paternal allele was precipitated, both parental alleles of the Ube3a promoter can be detected in the same IP fraction  at the fraction . ChIP infraction . AltogetUbe3a shows multiple features of an active gene as we demonstrated above, we considered the hypothesis that it is actually transcriptionally active despite the absence of mature mRNA. To test this, a mouse model carrying a deletion from Ube3a to Gabrb3  is about 0.2 copy at both the 5′- and 3′-portions in U-G/+del mice . Therefore, similar to Airn and Kcnq1ot1, Ube3a-ATS remains located proximate to its transcription site after it being synthesized. nucleus  and can A signal . Such coUbe3a in mice UBE3A via suppressing its antisense RNA. Previous studies have defined Ube3a-ATS as the negative regulator of Ube3a imprinting Ube3a-ATS without modulating other epigenetic factors is sufficient to activate paternal Ube3a. This question is crucial in determining whether knock-down of Ube3a-ATS is a suitable strategy for treating AS. By generating a mouse model with Ube3a-ATS being prematurely terminated, we observed unsilencing of Ube3a in multiple brain regions, implying that the antisense RNA plays a regulatory role in modulating Ube3a imprinting. We then compared mice which express paternal Ube3a on the maternal Ube3a knock-out background (AS/stop) with AS and WT mice. The AS/stop mice exhibit complete reversal of obesity, motor tests of wire-hanging and dowel walking, fear conditioning defect, and plasticity-related electrophysiology. They also display slight but significant improvement in the tests of accelerating rotarod and marble burying. Therefore, our research confirmed the clinical benefit of activating paternal Ube3a in treating Angelman syndrome and provided a mouse model as the positive control for future drug testing. Given the conservation of the PWS/AS region between mouse and human, activation of paternal UBE3A through inhibiting UBE3A-ATS expression/transcription should be a promising strategy for developing AS therapy.Patients with Angelman syndrome suffer from developmental delay, speech impairment, and epilepsy. Therapies for AS are limited and focus mainly on symptomatic management UBE3A is how much UBE3A protein is needed to achieve phenotypic improvement in AS patients. In the mouse model of AS/stop, we observed some phenotypic reversal, such as obesity, and cognitive deficits. However, their performance during accelerating rotarod and marble bury test is only partially or moderately improved and their decreased locomotive activity is not restored. This may be due to the incomplete activation of paternal Ube3a, which is quantified to be 50–70% of the WT level in different parts of the brain by western blot. Some of the behavioral phenotypes might be more sensitive to the protein level of Ube3a and therefore are more difficult to reverse. Another possibility is the interference from the remaining neomycin cassette. However, paternal inheritance of the cassette on the WT background does not affect mouse behaviors, and maternal inheritance of the cassette does not change Ube3a expression and rotarod performance in mice, suggesting that the presence of the selection marker has no or minimal effect on Ube3a function. Among the human UBE3A mutations that have been reported so far, there is a striking preponderance of frameshift and nonsense mutations UBE3A display some, but not all, clinical features associated with AS and thus are excluded from AS diagnosis and research. A patient with C21Y missense mutation located outside the HECT domain of UBE3A has been reported to have a less classical phenotype UBE3A may be beneficial.One important question in activating paternal UBE3A imprinting. Interestingly, several pieces of evidence have suggested that the paternal allele of UBE3A/Ube3a is transcriptionally active. For example, the promoter of paternal UBE3A is unmethylated Ube3a pre-mRNA can be detected equally from the 5′-portion of both paternal and maternal alleles in mice . All human studies were performed in accordance with NIH guidelines and approved by the Baylor College of Medicine Institutional Review Board (IRB).Ube3a by gene targeting in wild-type AB2.2 ES cells. After microinjecting into blastocysts of C57/BL6 mice, high percentage male agouti chimeras were obtained and germline transmission was established. The lines were then backcrossed to C57/BL6 mice for more than six generations. PCR genotyping was developed with TS-F (TTCCCAGTGCTGAGACTAAAG), TS-R (CCACAATCTGAA-CCCTAAAAC) and SV40-R (AAAAGGGACAGGATAAGTATG).The insertion cassette composed of SV40 triple poly(A) signal and neomycin selection marker was inserted downstream of Total RNA was prepared with miRNeasy Mini Kit . On-column DNase treatment was performed for all the samples. The cDNA was generated using 0.2–1 µg of total RNA with SuperScript III First-Strand Synthesis System , and qRT-PCR was performed using Applied Biosystems StepOnePlus Real-Time PCR System and SYBR Green Master Mix . Primers used are listed in Western blot against Ube3a and β-tubulin was performed as previously described Tissue preparation and immunohistochemistry were performed by Neuropathology Core of Baylor College of Medicine, as previously described A battery of behavioral tests was performed using a protocol previously described and used in Behavioral Core facilities at Baylor College of Medicine 2 and 5% CO2) containing in mM: 124 NaCl, 2.0 KCl, 1.3 MgSO4, 2.5 CaCl2, 1.2 KH2PO4, 25 NaHCO3, and 10 glucose. Bipolar stimulating electrodes were placed in the CA1 stratum radiatum to excite Schaffer collateral and commissural fibers. Field EPSPs were recorded at 30–31°C, with ACSF-filled micropipettes. The recording electrodes were placed in the stratum radiatum and the intensity of the 0.1 ms pulses was adjusted to evoke 40–50% of maximal response. A stable baseline of responses at 0.033 Hz was established for at least 20 min. Tetanic LTP was induced by using two 1 s, 100 Hz tetani, 20 s apart at baseline stimulus intensity, as previously described Horizontal hippocampal slices (350 µm) were cut with a Leica (VT 1000S) vibratome  from brains of WT, AS and AS/stop mice in 4°C artificial cerebrospinal fluid (ACSF) and kept in ACSF at room temperature for at least one hour before recording, as previously described Postmortem brain tissues from control, PWS, and AS individuals were obtained from NICHD Brain and Tissue Bank for Developmental Disorders from University of Maryland School of Medicine.MAGEL2 to GABRB3  in the 4X44k format. Hybridization, wash and scanning were performed according to manufacturer's instructions. The image files were processed with Agilent Feature Extraction software using protocol CGH-v4_95_Feb07 and further analyzed with Agilent G4477AA ChIP Analytics 1.3 software.ChIP-on-chip analysis was performed as previously described Brain tissues of 50 mg from newborn mice was chopped into fine pieces, crosslinked with 1% formaldehyde in DMEM and lysed in SDS lysis buffer . The lysate was then sonicated (Fisher Scientific 500 Sonic Dismembrator) and centrifuged. The supernatant was collected and combined with IP buffer . Immunoprecipitation was then performed with Protein G Dynabeads (Invitrogen) coated with anti-pol II , anti-TFIIB , or anti-TBP  overnight.Snrpn or Ube3a promoter primers, purified with MinElute PCR purification kit (Qiagen) and analyzed by Sanger sequencing to identify allelic SNPs. Alternatively, unpurified PCR products were digested with BsaI for the Snrpn promoter or BbsI for the Ube3a promoter, and analyzed by eletrophoresis on a 1.5% agarose gel.Immunoprecipitated DNA was PCR amplified with Ube3a pre-mRNA in a strand-specific manner Total RNA of 200 ng from cortices of newborn mice was used as the input in the analysis. The cDNA synthesis was performed using tagged gene-specific primers in the RT reaction to detect in-vitro transcription and corresponded to chr7:66,530,657–66,531,391(NCBI37/mm9). Primers for DNA template synthesis are SP6-Ube3a-int5.1F ATTTAGGTGACACTATAGAAGCGAAGATGAGTCAG-TTTGGTTTT and T7-Ube3a-ex6.1R TAATACGACTCACTATAGGGAGATTCTGAGTCTTCTTCCATA-GC). The T7 promoter was used to generate Ube3a-ATS probe. Hybridized probes were detected by a dual amplification strategy and visualized by Alexa488 conjugated streptavidin Tissue preparation and RNA FISH were carried out by RNA In-Situ Hybridization Core at Baylor College of Medicine as previously described After RNA FISH, the slides were washed in 2XSSC at 37°C for 15 min, dehydrated in 70%, 85%, 95% ethanol at −20C for 2 min each and denatured in 70% formamide/2XSSC at 70°C for 2 min. After washing with 70%, 85% and 100% ethanol, the slides were air-dried before hybridization with the DNA FISH probe. The probe was prepared from Ube3a BAC clone bMQ311i10  or Lepre1 (chr 4) with FISH Tag DNA Red Kit (Invitrogen) and hybridized to the sections at 37°C overnight. The slides were washed in 50% formamide/2X SSC solution twice for 8 min at 42°C, once in 2XSSC for 8 min at 37°C and mounted with SlowFade Gold antifade reagent (Invitrogen).Statistical analysis was performed with GraphPad Prism 5 . One-way ANOVA with Newman–Keuls post-hoc test and two-way ANOVA with repeated measures were used.Figure S1+/ATS-stopUbe3a mice terminates the transcription of Ube3a-ATS. Total RNAs prepared from cerebrum of WT and +/ATS-stopUbe3a mice are subject to custom designed strand-specific microarray analysis. The normalized signal intensity is plotted over genomic coordinates (NCBI37/mm9 build), with the moving average of 10 probes.Insertion of poly(A) cassette on the paternal chromosome in (TIF)Click here for additional data file.Figure S2A) and cerebral cortex (B).Paternal Ube3a is unsilenced in AS/stop mice. Brain sections from adult WT, AS, stop, and AS/stop mice were analyzed by immunohistochemistry with anti-Ube3a. Shown here in the figures are hippocampus ((TIF)Click here for additional data file.Figure S3ATS-stopUbe3a leads to biallelic expression of Ube3a, while maternal inheritance of the allele has no effect on Ube3a expression. (A) Male ATS-stopUbe3a heterozygous mouse was crossed with female YFPUbe3a heterozygous mice to generate progeny of YFP/+Ube3a and YFP/ATS-stopUbe3a mice. The reciprocal cross was also carried out. (B) The scheme shows the two alleles of ATS-stopUbe3a and YFPUbe3a. YFPUbe3a carries a C-terminal YFP tag, which leads to expression of the Ube3a-YFP fusion protein with a higher molecular weight. (C) Western blot with anti-Ube3a was performed with cerebrum from YFP/+Ube3a, YFP/ATS-stopUbe3a, +/YFPUbe3a, and ATS-stop/YFPUbe3a mice. β-tubulin is used as the loading control. (D) The amount of Ube3a-YFP and Ube3a protein normalized to β-tubulin was quantified in four groups of mice. (E) The ratio of paternal to maternal Ube3a was calculated and plotted. Pat: paternal; Mat: maternal. Data are averages ± range.Paternal inheritance of (TIF)Click here for additional data file.Figure S4ATS-stopUbe3a allele on activating cis Ube3a is compared with 0.9del and 4.8del. (A) RNA levels of Ube3a were quantified by qRT-PCR in cerebrum of newborn mice and compared among different genotypes. It is normalized to the internal control of Pgk1. (B) Protein levels of Ube3a was measured by western blot and normalized to β-tubulin. (C) Ube3a-ATS RNA was analyzed by qRT-PCR. (D) RNA levels of paternal Ube3a  were plotted over Ube3a-ATS. Non-linear regression of exponential decay was performed with the best fitting curve shown in red (R2 = 0.997). Data are averages ± range. N = 3 mice per genotype group.The effect of paternally inherited (TIF)Click here for additional data file.Figure S5AS/stop mice display normal behaviors in wire-hanging test and dowel test. A second batch of WT, AS, stop, and AS/stop mice were analyzed for wire-hanging and dowel test. WT = 14, AS = 11, stop = 13, AS/stop = 9. Both male and females are included. Data are averages ± SEM. ***p<0.001, ns: not significant.(TIF)Click here for additional data file.Figure S6ATS-stopUbe3a allele does not affect motor performance in mice. (A) Accelerating rotarod. (B) Wire-hanging test. (C) Dowel test. There is no statistical significance between the two groups in these tests.Maternal inheritance of the (TIF)Click here for additional data file.Figure S7A) Pesynaptic fiber volley as a function of stimulus intensity showed no difference in fiber excitability among WT, AS, and AS/stop mice. The mean afferent volley at a given stimulation strength was fitted by linear regressions. (B) Input-output relation of fEPSPs as function of presynaptic fiber volley amplitude over a wide range of stimulus intensities was also similar for WT, AS, and AS/stop slices. (C) Paired-pulse facilitation of fEPSPs did not significantly differ between WT, AS and AS/Stop slices, as demonstrated by the plots of the paired-pulse ratio (fEPSP2/fEPSP1) for various intervals of paired stimulation.WT, AS, and AS/Stop mice have similar basal synaptic transmission in CA1 slices. ((TIF)Click here for additional data file.Figure S8Ube3a are bound by TFIIB, a component of the preinitiation complex. Cerebral cortices from F1 hybrid of C57 and Cast.Chr7 mice were used for ChIP analysis against TFIIB. Precipitated DNA was PCR amplified for Ube3a and Snrpn promoters and subject to restriction enzyme digestion to identify parental alleles. Snrpn promoter from C57 but not Cast.Chr7 can be cut by BsaI. Ube3a promoter from Cast.Chr7 but not C57 can be cut by BbsI.The promoters of both paternal and maternal (TIF)Click here for additional data file.Table S1Q-PCR primers used in the expression analysis.(DOCX)Click here for additional data file.Table S2Ube3a transcription initiation.RT and q-PCR primers used in analyzing (DOCX)Click here for additional data file.Text S1(DOCX)Click here for additional data file.


Analysis of structural variation in domain superfamilies can reveal constraints in protein evolution which aids protein structure prediction and classification. Structure-based sequence alignment of distantly related proteins, organized in PASS2 database, provides clues about structurally conserved regions among different functional families. Some superfamily members show large structural differences which are functionally relevant. This paper analyses the impact of structural divergence on function for multi-member superfamilies, selected from the PASS2 superfamily alignment database. Functional annotations within superfamilies, with structural outliers or ‘rebels’, are discussed in the context of structural variations. Overall, these data reinforce the idea that functional similarities cannot be extrapolated from mere structural conservation. The implication for fold-function prediction is that the functional annotations can only be inherited with very careful consideration, especially at low sequence identities. The availability of protein three-dimensional structures repeatedly confirms that a limited number of folds are shared by large number of protein sequences. This limitation is imposed by the physical chemistry of the polypeptide Currently, it is quite uncommon to discover a new fold, while it is possible to observe a subtle conformational difference arising from some very common structural motifs α atoms of superimposed proteins. There is an increasing evidence that, in some superfamilies, domains have undergone significant structural changes during evolution Analysis of protein domains at the superfamily level is biologically significant to study the association of evolutionary, functional and structural perspectives of domains. Structure alignment is the method of choice for comparing the superfamily members of minimal sequence identity i.e., threading a sequence to a framework structure, derived from common structural feature of a superfamily PASS2 http://www.nature.com/protocolexchange/protocols/604) to identify the structurally deviant members with family-specific functional differences within a superfamily. The aim of this paper is to provide a detailed description of functional variations of outliers in protein domain superfamilies and to illustrate that the structural divergence is found in certain domains which may be related at the superfamily level. Sometimes, large structural differences are introduced with a functional importance.In this paper, we are mainly focusing on multi-member superfamilies which exhibit one or two structurally deviant members. We show that, it is possible to employ structure alignment protocol . These outliers are also verified by TMSCORE The structural alignment of multi-member superfamilies is performed using the standard protocol of PASS2. The initial alignment is performed using MATT Functional similarity of gene products could be estimated by controlled biological vocabularies, such as Gene Ontology (GO) Here, we emphasize that using an appropriate structure alignment protocol even on protein domains with low sequence identity, one can identify structural differences which occur due to a functional reason. After the structural alignment of 731 multi-membered superfamilies, 159 superfamilies show one or more structurally deviant members within the superfamily. http://caps.ncbs.res.in/download/pass2_outliers/outliers_tables/). 41 superfamilies from the category of single and two outlier superfamilies are highly interesting, since they retain outliers which are family-specific in nature suggesting a functional context. These 159 superfamilies are characterized as single, two and multiple-outlier superfamilies  . We discuss these reasons more elaborately using one illustrative superfamily each  phosphatases II, Prim-pol domains, Porins, ADC-like, LeuD/IlvD-like, Methyl-coenzyme M reductase subunit. Since larger number of superfamilies fall into this category of family specific architecture and topology, perhaps these major structural changes could ultimately lead to the functional diversity of the domains. An additional four superfamilies  have domains with family-specific difference in the conformations of secondary structures, but, they did not show any topological difference.5). ADC is observed to be present in bacteria, fungi and plants ADC-like superfamily consists of 16 domains with the topology of β-barrel with cross-over loops. All these 16 domains contain six β-strands and two α-helices. Using structure-based sequence alignment, two domains have been observed as outliers with interesting topological differences and they belong to pyruvoyl dependent aspartate decarboxylase family (ADC). ADC is an unusual enzyme, as its catalysis depends on the pyruvoyl group formed as a result of self-processing In the current dataset, a single superfamily, of peroxidases, could be observed where high structural variations reside between domains within the superfamily, where the domains are from different taxa. Peroxidases are heme-containing enzymes which use hydrogen peroxide as electron acceptors to catalyse a number of oxidative reactions 8 topology  and proline dehydrogenase domain of bifunctional PutA protein, both sharing a common TIM-barrel fold. Proteins in these two families are the only known structures for FAD cofactors bound to a TIM barrel, the PutA PRODH domain and methylene tetrahydrofolate reductase. One out of three members from the family, proline dehydrogenase exhibits large structural difference which is reflected as high RMSD. The outlier, PutA PRODH barrel, exhibits three deviations from the classic (α/β)topology . First, This kind of circular permutation problem could be treated by stringent structure alignment protocol, but we might lose the identification of functional differences that occur between families. Apart from circular permutation, the differences in EC numbers confirm that the outliers and non-outliers have different enzymatic function. Helix α8 plays essential roles in PutA’s PRODH function, whereas the corresponding region in MTHFR does not participate directly in binding to substrates or cofactors E. coli can bidirectionally catalyze the interconversion of succinate and fumarate and each can functionally replace the other to support growth The α-helical ferredoxin superfamily is represented by four domains in the PASS2 database, derived from two different families, namely C-terminal domain of fumarate reductase/succinate dehydogenase iron-sulfur protein and N-terminal domain of dihydropyrimidine dehydrogenase (DPD). The members of the fumarate reductase/succinate dehydrogenase, have high structural conservation and enzymes from Bacillus subtilisWithin the dataset of 41 superfamilies with structural outliers, there are five superfamilies, PAP/OAS2 substrate binding domains, ParB/Sulfiredoxin, UBA-like, LigT-like and TrpR-like, where the outlier retains difference in secondary structural content and distinct topology. The superfamily of PAP/OAS2 substrate binding domain has five members in the PASS2 database. The superfamily contains domains from families Poly(A) polymerase and 2′-5′-oligoadenylate synthetase. The structural alignment of these five domains revealed that one domain belongs to AadK C-terminal domain-like domain family is structurally different. This Amino glycoside 6-adenylyltransferase (AdaK) domain is a modifying enzyme associated with bacterial resistance by adenylating streptomycin in Domain swapping is an important phenomenon involved in many biological processes such as in protein molecular evolution, functional regulation and in the formation of protein conformational/deposition diseases, such as amyloid and prion diseases in vitro and in a crystal environment, self-associates in vivo and localizes to mitotic structures. The conservation of the hydrophobic core and dimer interface residues, the presence of two copies of the polo domain in most Polo-like kinases and the covariance across tandem polo domains in most Plks suggest that the ability to adopt a dimeric conformation may be a general characteristic feature of all polo domains and that domain swapping may occur in an intramolecular manner for some family members The ‘polo-box duplication region’ family has two domains arising from the same protein chain. The outlier is a domain-swapped polo-box. It is already reported that the polo domains form dimers both We noticed that N-terminal and C-terminal embellishments of outliers occur as insertions and could meaningfully add functional variety within superfamilies. There are seven superfamilies that come under this group, namely, SGNH hydrolases, alkaline phosphatase-like domains, PAP/Archaeal CCA-adding enzyme , GAF-like domains CYTH-like phosphatases, GatB/YqeY motif and Sialidases. Apart from this, there are two superfamilies, heme-dependent catalase-like, bacterial luciferase-like which have domains with incomplete core structures due to deletion events. SGNH hydrolase superfamily has 13 protein domains in the PASS2 database and they have similar fold to flavoproteins, namely a three-layer α/β/α structure, where the β-sheets are composed of five parallel strands. The superimposed view of all the domains is shown in via an anti-parallel interaction between β-strands 4 and 8 In the current analysis, two superfamilies  retain outliers that acquire internal repeats or clear duplication of the domain fold, and therefore acquire difference in biological function. MTH1187/YkoF-like superfamily consists of six members from two families. The family MTH1187-like contains only hypothetical protein domains and the family putative thiamin/HMP-binding protein YkoF has putative thiamin/HMP-binding protein domains. Thiamin/HMP-binding protein domain is involved in the hydroxymethyl pyrimidine (HMP) salvage pathway and the other family (MTH1187-like members) contains domains of unknown function. The superfamily members retain a ferredoxin-like fold with α+β barrel with anti-parallel β-sheet topology .The supehttp://caps.ncbs.res.in/download/pass2_outliers/GO_semantics for similar plots for the other superfamilies discussed). Outliers exhibit lower GO semantics similarity scores with the other members of the superfamily in comparison to the other members with each other.As described above, changes accumulated in a protein structure, are being used by the living machinery for related but slightly different biological functions, contributing to a general evolutionary pressure to preserve these structural changes. The paper explains that structure-based sequence alignment methods are reliable for the identification of structural variations within a superfamily. All the family-specific outliers from 41 superfamilies have been examined critically. We have observed that major structural variations occur due to differences in the structural topology, domain swapping, circular mutation, irregular elaborations, duplication and insertion. High structural variations within domains of the same superfamily could be accompanied by functional differences. A quantitative comparison of Gene Ontology terms of functional characterization (as described in Methods) shows that structural variations are accompanied by diversity in protein function Click here for additional data file.Figure S2Lipocalin superfamily (50814) outliers subgroups. This superfamily has total of 14 outliers and interestingly they forms subgroups among themselves. RMSD based phylogeny and their subgroups superposition are shown.(TIF)Click here for additional data file.Figure S3Superimposed view of ADC-like superfamily. (a) Superimposed view of all the 16 domains of ADC-like superfamily. (b) Superposed figure of all the non-outliers. (c) Superposed view of two outliers.(TIF)Click here for additional data file.Figure S4Superimposed view of domains of Heme-dependent peroxidases superfamily. (a) Superimposed view of outliers .The helix is highlighted in red. (b) Superimposed view of all the non-outliers. They superimpose with low RMSD.(TIF)Click here for additional data file.Figure S5Superimposed view of all domains of Alpha-helical ferredoxin (46548) superfamily. The N-terminus to C-terminus is coloured from Blue to Red.(TIF)Click here for additional data file.Figure S6Superimposed view of all the domains of PAP/OAS1 substrate-binding domain (81631) superfamily. The outlier is shown in pink colour.(TIF)Click here for additional data file.Figure S7The superimposed view of all the members of SGNH hydrolase (52266) superfamily. All the non-outliers are coloured by pymol spectrum colouring and the outlier is in pink. (b) The structure of haemagglutinin-esterase glycoprotein monomer.(TIF)Click here for additional data file.Figure S8Superimposed view of all the members of MTH1187/YkoF-like (89957) superfamily. The outlier is highlighted in pink colour.(TIF)Click here for additional data file.


Combined in one molecule, both intradomain disulfide bonds led to an increase of the mT of about 15°C. All of these mutations had no impact on the thermal stability of the CH2 domain. Importantly, the binding of neonatal Fc receptor was also not influenced by the mutations. Overall, the stabilized CH3 domains described in this report provide an excellent basic scaffold for the engineering of Fc fragments for antigen-binding or other desired additional or improved properties. Additionally, we have introduced the intradomain disulfide bonds into an IgG Fc fragment engineered in C-terminal loops of the CH3 domain for binding to Her2/neu, and observed an increase of the mT of the CH3 domain for 7.5°C for CysP4, 15.5°C for CysP2 and 19°C for the CysP2 and CysP4 disulfide bonds combined in one molecule.We report the stabilization of the human IgG1 Fc fragment by engineered intradomain disulfide bonds. One of these bonds, which connects the N-terminus of the CH3 domain with the F-strand, led to an increase of the melting temperature of this domain by 10°C as compared to the CH3 domain in the context of the wild-type Fc region. Another engineered disulfide bond, which connects the BC loop of the CH3 domain with the D-strand, resulted in an increase of  Monoclonal antibodies with high affinity and specificity are now well established therapeutics and invaluable tools for biological research. Among them, the IgG class is the most abundant one. This heterotetrameric protein is composed of two light chains and two heavy chains. Using proteolytic digestion, the IgG antibody can be divided into 2 antigen binding fragments (Fabs) and the Fc fragment. The Fc part itself is composed of two CH2 domains and two CH3 domains that form a homodimeric region at the C-terminal end of the antibody molecule. These regions associate with each other with two disulfide bridges in the hinge region which is located between the CH1 and CH2 domains and by strong non-covalent interactions between the two CH3 domains The structure of antibody constant domains is similar to that of variable domains, consisting of beta-strands connected by loops, some of which contain short alpha-helical stretches. The framework is mostly rigid and the loops are comparatively more flexible, as can be seen from the b-factors of various Fc crystal structures (e.g. 1HZH.PBD and 1OQO.PDB). In the CH3 domain, the loops at the C-terminal tip of the globular domain are the sites which are most remote from the effector molecule binding sites and hence are the most attractive sites for engineering additional functionalities into the Fc fragment.Recently, we have shown that it is possible to engineer antigen binding sites into C-terminal loops connecting beta-strands of CH3 domains, and that Fc fragments containing such modified CH3 domains, so-called Fcabs, possess all attractive properties of a classical IgG, at a size of approximately only 50 kDa per se is a very stable protein, with the CH3 domain being even more stable than the CH2 domain. Additionally, the two domains are generally believed to be independent from each other in their folding and structure The plasticity of the Fc fragment that allows it to serve as a scaffold and to retain all antibody properties while accommodating the mutations in the loop regions is based on the fact that an Fc fragment Pichia pastoris. Although yeast glycosylation has an impact on the structure of CH2 domains and the activation of effector ligands , Pro343 and Ala431 (CysP2), Ser375 and Phe404 (CysP3), Ser375 and Pro396 (CysP4) and Val348 and Lys439 (CysP5). All residue numberings in this paper are according to Kabat In CysP2, the N-terminus of the A strand and the C-terminus of the F strand are disulfide-bonded while in CysP4 the engineered disulfide bond connects the BC loop and the D-strand. The location of the stabilizing disulfide bridges is shown in a 2D presentation of the secondary structure of the CH3 domain in P. pastoris expression vector pPICZalphaA, expressed and purified by Protein A affinity chromatography as described in The gene coding for human Fc was cloned in the The thermal denaturation of human IgG1-Fc in PBS, pH 7.4 over the temperature range 5–100°C is characterised by two endothermic transitions, at 65°C and 81.9°C. The first transition has been described to be reversible as shown by heating up to 75°C, cooling and again heating to 90°C, whereas the second transition is irreversible. However, beyond 75°C, the CH2 transition also becomes irreversible P. pastoris, both Differential Scanning Calorimetry (DSC) and temperature-dependent Circular Dichroism (tCD) were used. The results of these measurements are shown in mT of the CH2 transition of wild-type Fc was at 65°C, which is 4°C lower than that of Fc expressed in mammalian cells. This can be attributed to differences in the glycosylation pattern between the two expression systems. The thermal transition of the wild-type CH3 domain could be fitted with two non-2-state transitions at 77.8°C and 82.6°C, the first event corresponding likely to the melting of CH3 dimer and the second transition representing the denaturation of CH3 domains. As measured by CD, the secondary structure of the Fc fragment at 25°C is populated primarily of beta-strands; therefore thermal denaturation was followed by tCD at 218 nm. Unfolding started at 54°C and was completed at 82.5°C. A double sigmoid model was used to obtain mT values at 65.2°C and 77.0°C.To test the thermal stability of Fc fragments expressed in mT of the CH2 domain was slightly decreased by 1.5°C, and in the double mutant it was decreased by 4°C.In all cases, the three mutants CysP2, CysP4 and CysP24 were remarkably more stable than wild-type Fc. Notably, 50% unfolding of the CH3 domains of CysP2, CysP4 and CysP24 mutants occurred at temperatures that were significantly higher than that of wild-type Fc. In the case of CysP4, the second and the third transition appeared at temperatures 5°C higher than with wild-type Fc. The stabilization effect was even more pronounced in the case of CysP2, where the second and the third transition appeared at 9°C higher temperature than with wild-type Fc. The DSC profile of the Fc fragment CysP24, in which the CysP2 and CysP4 mutations are combined exhibited the second transition at 92.8°C and the third transition at 97.8°C, which is 15°C above the ones characteristic for wild-type Fc. Interestingly, in all cases thermal unfolding of the CH2 domain was reversible up to the temperature point where melting of the CH3 domain started, which was probed by annealing scans . In bothmT values, which have shown the same trend in stabilization as the data obtained from DSC measurements , this interaction was studied using Biolayer Interferometry . Association to FcRn, the binding site of which maps to the interface between the CH2 and CH3 domains, was assessed at pH 6.0, whereas the biologically important dissociation was measured by shifting the pH to 7.4. None of the mutants show any observable difference to wild-type Fc .For the Fc fragment H10-03-6, which binds to Her2 with about 70 nM affinity, a decrease in thermal stability comparing to wild-type Fc has been observed. In this case irreversible thermal unfolding can be deconvoluted with two transitions, corresponding to melting of the CH3 domain at 61.6°C and of the CH2 domain at 65.3°C. Each of the three mutants H10-03-6-CysP2, -CysP4 and -CysP24 was more stable than the parent clone. In the case of H10-03-6-CysP4, the transitions were still overlapping and by deconvolution appeared at 62.9°c and 69.0°C. The stabilization in the case of H10-03-6-CysP2 was more effective as the transitions appeared at 62.7°C and 77.0°C. The mutant with combined novel disulfide bridges H10-03-6-CysP24 has shown transitions at 60.0°C and 80.3°C, which is 19°C above the Tm of the CH3 domain of the unmodified H10-03-6 CH3 domain .in vitro directed evolution mT of a human isolated IgG1 CH2 domain was described. However, it remains to be demonstrated that this stabilized CH2 domain is also suitable as a module in an Fc or in a complete immunoglobulin.A wide variety of strategies and methods has been successfully applied to increase the thermal stability of proteins, including random mutagenesis combined with We have recently shown m to an unfolded CH2 domain. Stabilization of CH3 dimer, for example due to “compacting” at low pH values, however can weaken the cis interactions The introduction of the additional disulfide bonds described in this report has led to a significant increase in thermostability of the Fc, while leaving all other measured properties unchanged. Expression characteristics were identical for all proteins studied, and structural parameters as assessed by size exclusion chromatography as well as far- and near-UV CD measurements were indistinguishable. Binding to neonatal Fc receptor also remained unchanged. A slight decrease in the temperature of the first transition  was however observed. It has been proposed that the intensity of the first heat absorption peak is dependent on melting of the CH2 domain polypeptide, interaction of the CH2 domains and of the cis interaction of CH2 and CH3 domains in vivo half-life. For example, it has been shown for a Pichia expressed scFv-Fc fusion protein that its half-life was extended to 93 h in mice in comparison to 3.5 h for a typical stand-alone scFv Pichia in general attach high-mannose structures, most commonly Man14GlcNAc2 as compared with even longer (over 50 mannose residues) chains in S. cerevisiae proteins The binding of FcRn was used as an indicator of correct folding of the Fc and of the retention of its remarkably long mT 10°C), in which the N-terminus of the A strand and the C-terminus of the F strand are disulfide-bonded showed a higher degree of stabilization in the CH3 domain than CysP4 (deltamT 5°C), in which the BC loop and the D-sheet were connected. The contribution of disulfide bonds to protein stability is presumed to be mainly due to loss of conformational entropy in the unfolded state CysP2  is estimated to be −3.2 kcal/mol whereas in the case of CysP2, there are 85 residues in the loop forming the disulfide bond, yielding an estimate of −4.5 kcal/mol.mT of the denaturation of the CH3 domains, also the mT of the second CH3-associated transition has increased. The most common explanation for this transition is the event of dimer dissociation. We therefore conclude that the newly introduced disulfide bridges not only stabilized the conformation of the monomeric CH3 domain but also enhanced the CH3/CH3 homodimer interaction. This is in accordance with reports that have shown that native disulfide bond formation in human CH3 domains is critical for both, stability and dimerization Another interesting observation is that with the increase in in vivo half-life. Such mutations may cause destabilizing effects due to slight alterations in the interaction network of chemical bonds within the protein domain. Stabilization efforts such as the ones described here can counteract such unwanted effects and can therefore allow a higher degree of freedom for mutagenesis.The results presented in this paper have implications for the development of stabilized Fc fragments which can be used as stand-alone scaffolds or as parts of whole antibodies. Mutations are routinely introduced in antibodies in order to enhance their effector functions or to achieve longer in vivo.All stabilizing mutations described here resulted in stabilized Fc fragments with wild-type like expression properties, displaying homodimeric proteins without tendency for aggregation, which is an important prerequisite for potentially therapeutic proteins. Importantly, the engineered disulfide bridges also exerted their stabilizing effects on a model antigen-binding Fcab selected in our lab. Interestingly, it was possible also in this case to combine the stabilization effect of both novel disulfide bonds. Further studies will be performed to investigate whether higher thermal stability also correlates with greater stability Under Austrian law and under the internal rules of the University of Natural Resources and Life Sciences, Vienna, the work described here did not require permission by an ethics committee. No animal experiments were performed.The necessary declaration of work involving the use of genetically modified microorganisms was performed at the Committee for Biological Safety of the Department of Biotechnology, which subsequently did not forbid the described experiments (declaration number IAM 2007_03).http://caps.ncbs.res.in/dsdbase/dsdbase.html). 1OQO.PDB was used as a structure of the Fc fragment of human IgG1. The DSDBASE algorithm identifies residue pairs with Calpha-Calpha distances of less than or equal to 6.5 Å and Cbeta-Cbeta distances of less than or equal to 4.5 Å and evaluates the proposed disulfide bridges using limits for the structural parameters of the various torsion angles and S-S bond length. For the monomeric CH3 domain, 29 potentially suitable positions were found based on these parameters. All proposed disulfide bridges were evaluated by visual inspection of the crystal structure of the Fc fragment (1OQO.PDB) and 5 (CysP1–CysP5) were subsequently chosen for experimental evaluation.Positions in the CH3 domain sequence that could be mutated to cysteine in order to create disulfide bridges were predicted using DSDBASE Pichia pastoris strain X33 after linearization with SacI. The amino acid sequence of the human IgG1 Fc fragment is as follows:The Fc fragment of human IgG1 was cloned in the expression vector pPICZalphaA (Invitrogen) and transformed to the AOX locus of EFTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKEcoRI site used for cloning. The threonine at position 3 corresponds to amino acid position 225 according to Kabat EU numbering.The cloned sequence contained 2 extra amino acid residues (EF) at the N-terminal site due to the P. pastoris X33 in the presence of 1% methanol and induction was continued up to 5 days. The supernatant was harvested by centrifugation, loaded onto a Protein A HP column , eluted with 0.1 M glycine, pH 3.5, and subsequently dialysed against PBS at 4°C. The protein concentration was determined by UV spectroscopy using an absorption coefficient at 280 nm of 1.397 for wild-type Fc dimer.The recombinant protein was expressed in Mutagenesis was performed using the QuikChange® Multi Site-Directed Mutagenesis Kit (Stratagene), according to the manufacturer's instructions. The primers used are listed in In order to prevent the potential scrambling reactions of disulfide bonds during the proteolytic degradation, the samples were digested at acidic pH. For this, 20 µL of the respective Fc sample (0.5 mg/mL) were adjusted to 1% formic acid final concentration, by the addition of 20 µL of 2% formic acid in water, containing 10 µg/mL pepsin . Prior to the digestion at 37°C, over night, the resulting pH (i.e. approx. pH 2) was briefly checked on pH paper.The peptic peptide maps were produced by LC-ESI-MS/MS, using a Dionex Ultimate 3000 LC-system, coupled to a Waters Q-TOF Ultima equipped with its standard ESI-source. 5 µL aliquots of the proteolytic digests were directly loaded onto a BioBasic C18 column  at a flow rate of 6 µL 60 mM ammonium formate, pH 3.0, per minute, and separated by developing a linear gradient from 0 to 80% AcCN , over 80 minutes, at a flow rate of 6 µL per minute.For MS analysis of the peptic peptides, the capillary voltage was set to 3.2 kV, the cone voltage to 80 V, the source temperature to 100°C and the desolvation temperature to 120°C. Positively charged ions were recorded in the m/z range from 50 to 2000. The MS/MS experiments were performed in the data dependant acquisition mode, automatically selecting the respective ions, as well as CID energies. MS and MS/MS data were manually evaluated using the MassLynx 4.0 software.4HCO3, containing 10 mM DTE. The reduction was then allowed to stand for 30 minutes at 56°C, before the reduced samples were directly subjected to LC-ESI-MS/MS analysis.In order to identify the elution time points of the disulfide-bonded di-peptides in the peptide maps based on their susceptibility to reducing conditions, 5 µL aliquots of the peptic digests were dried in a speed vac, and taken up in 5 µL 100 mM NHDifferential Scanning Calorimetry experiments were performed using an automated VP-Capillary DSC Microcalorimeter , using 5 µM protein solutions, diluted in PBS at pH 7.4 The heating rate was 1°C/min, and the heating was performed from 20°C to 110°C. Fitting was performed using the non-2-state transition mechanism. The highest temperature that allowed thermal denaturation of the CH2 domain was first explored using annealing scans, with the first one from 20°C to the temperature point at which the start of melting of the CH3 domain was observed as indicated by the deconvolution of the thermogram, followed by cooling of the sample in situ, and a second scan from 20°C to 110°C. Data were confirmed by controls runs in which the highest permissible temperature was exceeded by 1.5°C.The circular dichroism (CD) spectra were measured on a Chirascan spectropolarimeter (Applied Photophysics). A 1 mm path-length quartz cuvette was used for far-UV spectra measurement, and a 10-mm path-length quartz cuvette was used for the near-UV spectra measurement. The protein concentration was 570 µg/ml, and the buffer used was PBS. The CD spectra of the buffer solution were subtracted from the sample spectra before conversion to absolute CD values.Thermal unfolding was monitored by measurement of temperature-dependent circular dichroism (tCD) on a Chirascan spectropolarimeter (Applied Photophysics). A 1 mm quartz cuvette was used at a wavelength of 218 nm. All measurements were performed form 25 to 98°C with 0.2°C increment at a heating rate of 1°C/min.A Shimadzu LC-20A Prominence system equipped with a diode array detector and a refractive index detector was used to perform size-exclusion HPLC with A TSK G3000 SWXL column (7.8×30 mm). The mobile-phase buffer used was PBS, 200 mM NaCl. Chromatography was conducted with a constant flow-rate of 1 ml/min. A total of 50 µg protein at about 1 mg/ml were loaded on the column for analysis.In vivo biotinylated FcRn was refolded from bacterial inclusion bodies and was a kind gift of Sune Justesen (University of Copenhagen).ForteBio Octet was used for observation of the interaction of disulfide-stabilized Fc mutants with FcRn. Streptavidin-coated tips (ForteBio) were loaded with biotinylated FcRn at 5 µg/ml. Fc fragments at a concentration of 4.8 µM were allowed to associate in PBS, pH 6.0, and dissociation from FcRn was monitored in PBS, pH 7.4, at 30°C and with shaking at 1000 rpm. Binding of Fc fragments to an uncoated tip was used as a reference. Data were analysed with ForteBio Data Analysis package 6.2. Mutagenesis was performed using the QuikChange® Multi Site-Directed Mutagenesis Kit (Stratagene), according to the manufacturer's instructions. The primers used are identical to the primers used for Fc wild-type, except for the mutation S375C, and are listed in Figure S1SDS-PAGE of purified wild-type Fc (lane 1), CysP4 (lane 2), CysP2 (lane 3) and CysP24 (lane 4). Molecular weight standard (lane M).(TIF)Click here for additional data file.Figure S2MS/MS data enabling prediction of disulfide bonds based on comparative analysis of the reduced and non-reduced peptide-sample. A: data derived for CysP2, B: data derived for CysP4.(TIF)Click here for additional data file.File S1Primers used for site directed mutagenesis.(DOC)Click here for additional data file.