A disintegrin and metalloproteinase (ADAM) family proteins constitute a major class of membrane-anchored multidomain proteinases that are responsible for the shedding of cell-surface protein ectodomains, including the latent forms of growth factors, cytokines, receptors and other molecules. Snake venom metalloproteinases (SVMPs) are major components in most viper venoms. SVMPs are primarily responsible for hemorrhagic activity and may also interfere with the hemostatic system in envenomed animals. SVMPs are phylogenetically most closely related to ADAMs and, together with ADAMs and related ADAM with thrombospondin motifs (ADAMTS) family proteinases, constitute adamalysins/reprolysins or the M12B clan (MEROPS database) of metalloproteinases. Although the catalytic domain structure is topologically similar to that of other metalloproteinases such as matrix metalloproteinases, the M12B proteinases have a modular structure with multiple non-catalytic ancillary domains that are not found in other proteinases. Notably, crystallographic studies revealed that, in addition to the conserved metalloproteinase domain, M12B members share a hallmark cysteine-rich domain designated as the “ADAM_CR” domain. Despite their name, ADAMTSs lack disintegrin-like structures and instead comprise two ADAM_CR domains. This review highlights the current state of our knowledge on the three-dimensional structures of M12B proteinases, focusing on their unique domains that may collaboratively participate in directing these proteinases to specific substrates. A disintegrin and metalloproteinase (ADAM) family proteins, also known as metalloproteinase-disintegrins or metalloproteinase/disintegrin-like/cysteine-rich (MDC) proteins, are type-I transmembrane and soluble glycoproteins that have diverse functions in cell adhesion, migration, proteolysis and signaling ,2,3. TheC-terminal thrombospondin type-1 motifs in place of the ADAM transmembrane and cytoplasmic domains and thus function as secreted proteinases . R. R2+-binjunction ,91. In A2+ ions at site I, another bound Ca2+ ion has been observed in the M domain in close proximity to the active site , respectively, at different stages of sperm maturation [2+-binding, may also play a role in differential proteolytic processing of precursor proteins, giving rise to the functional complexity of snake venoms, as well as in the post-translational regulation of ADAMs’ functions, probably by modifying the capabilities of protein–protein interactions.In some instances, substantial amounts of processed DC fragments of P-IIIb SVMPs have been identified in venoms alongside their unprocessed counterparts ,95. Althturation ,96. A fls, residues 403–436 in catrocollastatin/VAP2B sequence) and the “arm“  [N-terminal region of the C domain designated as the “wrist“  subdomain (s/Da/Cw) itself seems to be rigid because it is stabilized by a number of disulfide bonds and structural Ca2+ ions. There are three disulfide bonds in each Ds and Da, and one in Cw, with the subdomains  connected by single additional disulfide bonds. The numbers and spacing of the cysteine residues involved in these disulfide bonds are strictly conserved among ADAMs and P-III SVMPs [Naja atra. The K-like proteinase lacks the 17-amino acid segment at the junction of the Ds and Da subdomains, resulting in a different disulfide-bond pattern in the D domain. Consequently, the K-like proteinase has a different orientation between the Ds and Da subdomains when compared to that of catrocollastatin/VAP2B   3EXGEXCD, in which the side-chains of underlined residues are involved in Ca2+-binding) and the main-chain carbonyl oxygen atoms of Val405 and Phe410 are involved in pentagonal bipyramid coordination of the Ca2+-binding site II 2(E/D)XCXG(X)4C(X)2(D/N) [2+ ions in sites II and III are deeply buried and tightly coordinated and cannot be stripped from ADAM22, even using EDTA [2+ ions are likely to remain permanently in place once the D domain is folded.Both the Dequences ,87. In t site II C. On theubdomain D. These X)2(D/N) ,87. Bothing EDTA . Therefow subdomain, and a disulfide bond (Cys468/Cys499) and bound Ca2+ ion at site III further stabilize the continuous rigid Da/Cw structure. Therefore, in ADAMs and P-III SVMPs, the disintegrin-like loop is inaccessible for protein–protein interactions due to steric hindrance. Disintegrins (40–100 amino acids) are typically generated by proteolytic processing of larger precursor P-II SVMPs [s subdomain than P-III SVMPs, and thus one or two fewer disulfide bonds. In addition, there are substitutions of the key residues constituting the Ca2+-binding site II and III in most P-II SVMPs [X-ray crystallography [2+-binding has been identified in these structures and the Ds subdomain region of disintegrins is generally shorter and less ordered than the corresponding regions of ADAMs and P-III SVMPs. Because of the lack of structural Ca2+ ions, disintegrin structures are more flexible throughout the molecule, than the corresponding region of ADAMs and P-III SVMPs. The flexibility of RGD-containing disintegrin loops is probably important for the binding of integrins. As previously mentioned, P-II SVMPs may have evolved from ancestral P-III SVMP genes after losing the genetic information encoding the protein regions downstream of the D domain [2+-binding sites), imposed both on the disintegrin loop and the Ds subdomain in the ancestral P-II SVMPs, has been postulated as the key event that permitted the subsequent evolution of both integrin-binding activity and the proteolytic release mechanism.The overall structures of the D domain of P-III SVMPs and ADAM22 are similar to that of trimestatin, an RGD (Arg-Gly-Asp sequence)-containing classic disintegrin  Figure E. The inII SVMPs ,99,100, II SVMPs . Most P-II SVMPs . AlthougD domain ,59,60. Rh subdomain of ADAM17 revealed that PDI can act on this subdomain and convert it from the inactive to the active conformation by disulfide-bond isomerization [While the pattern of disulfide-bond pairing in the D domain determined thus far is strictly conserved among ADAMs and P-III SVMPs, with the exception of K-like proteinase, it may be possible that multiple structural isoforms of the same SVMPs exist in the venom, perhaps as the result of alternative disulfide-bond pairing . For exarization .w, residues 437–503) and the “hand“  subdomains [w subdomain tightly associates with the D domain, and the two are integrated into one continuous structure. On the other hand, the Ch subdomain constitutes a separate unit and has a unique structure consisting of irregularly folded loops with a core α/β-fold and four to five disulfide bonds. The Ch subdomain has a novel fold with no structural similarity to any currently known proteins, with the exception of the corresponding segments of M12B proteinases. The whole C domain of P-III SVMPs and ADAMs has been deposited in the Conserved Domain Database  and the Pfam database (http://pfam.xfam.org/) as the ADAM_CR domain . Here, we define the Ch subdomain of ADAMs and SVMPs and corresponding regions of ADAMTSs  as the ADAM_CR domain in a more restricted sense.The C domain of ADAMs and P-III SVMPs, typically about 80–150 amino acid residues, can be structurally subdivided into the “wrist”  also possesses essentially the same fold as the Ch subdomain, even though the two share no apparent sequence similarity [Crystallographic studies on the D* domain-containing fragments of ADAMTS1, 4, 5 and 13 revealed that the D* domain of ADAMTSs has no structural similarity to classic snake disintegrins, but is very similar in structure to the CII SVMPs ,53,66,71h subdomain of catrocollastatin/VAP2B and the D* domain of ADAMTS5, respectively, two typical ADAM_CR domain structures. Although there is negligible sequence identity between these two protein portions (~16%), they clearly show similar topologies. The topology diagram of these two protein portions is shown in N-terminal strands, S1 and S4, shown in gray. A short connecting loop of six amino acids in ADAMTS5 is replaced by a 27 amino acid residue insertion forming a central α-helix and two consecutive hairpin loops protruding out the top of the molecule in the case of catrocollastatin/VAP2B. This segment is named variable loop (V-loop) [h subdomains of SVMPs determined thus far and ADAM22 show a catrocollastatin/VAP2B type long V-loop structure  ,53. Currructures D, comparh subdomain of catrocollastatin/VAP2B is very similar to that of six other SVMPs and that of ADAM22, with variability occurring mostly in loop regions. Of note, aside from the V-loop, the loop encompassing residues 561–582  and in amino acid composition. Therefore, this region has been designated as the hypervariable region (HVR) [The overall structure of the Con (HVR) ,44. The on (HVR) . In ADAMon (HVR) ,44. DiffE. coli for functional assays. It should be noted that short peptides or E. coli expressed cysteine-rich proteins do not always mimic their counterparts in the intact molecule. The whole C domain or DC domains of ADAMs are suggested to be involved in protein–protein interactions [h subdomain in this text) of ADAM17, was shown to be responsible for recognition of two type-I transmembrane substrates, the IL-6R and the IL-1RII, but not for the interaction with the type-II transmembrane molecule TNF-α [in vitro, is also involved both in substrate recognition and in regulating the shedding activity of ADAM17 [A domains of ADAMTS13 have actually been shown to constitute VWF-binding exosites (see below).Several reports suggest that the HVR region directly contributes to the substrate recognition of ADAMs and SVMPs. Most of these studies, however, used synthetic peptides derived from the HVR region or the isolated domains expressed in ractions ,107,108.ractions . Recentlle TNF-α . Furtherf ADAM17 ,111, as f ADAM17 . Most ofProteins with multimer and/or heterogeneous complex structures are frequently observed in snake venoms. Such multimers or protein complexes generally exhibit markedly enhanced pharmacological activities compared to the individual components and thus may play significant roles in snake venom toxicity . Some SVC-terminal region of this segment (residues 322–324) forms an antiparallel β-sheet with its counterpart. In addition, water molecules are bound to the side-chain oxygen atoms of His322 and Ser323 and form a hydrogen-bond network that further stabilizes the interface between the monomers. Lys324 plays a pivotal role in the key-to-keyhole recognition between the monomers. The Nε amino group of Lys324 is coordinated by six oxygen atoms, which belong to the opposite chain and are located at the corners of a pentagonal pyramid. The six atoms include the side-chain oxygen atoms of Asn295 and Gln320, the carbonyl oxygen atoms of Phe296, Gly298 and Thr300, and a water molecule (molecule C. The inmolecule D. TherefDaboia russelli) has been recognized for its potent coagulation activity. Two major components, RVV-X and RVV-V, of this venom can collaboratively accelerate formation of the prothrombinase complex (Factor Xa (FXa)/Factor Va (FVa) complex) that converts prothrombin to thrombin, resulting in a disseminated intravascular coagulation in the body of the prey [X (FX) by cleaving the Arg194-Ile195 bond in FX, which is also cleaved by factors IXa and VIIa during physiological coagulation [Vipera lebetina venom [A few P-III SVMP members exist as heterocomplexes due to the existence of an extra subunit that interacts through covalent or non-covalent interactions. The venom of Russell’s viper  of the light chain-A (LA). In addition, the residues in the HVR and the surrounding regions in the heavy chain form multiple aromatic and hydrophobic interactions and hydrogen bonds with the N- and C-terminal residues in LA, further stabilizing the continuous C/LA structure. The RVV-X structure provides the first direct observation of a protein–protein interaction mediated by HVR.RVV-X is a heterotrimeric complex consisting of an MDC-containing heavy and two light chains ,125. Twosnaclecs ). Insteasnaclecs ,127. The2+-dependent primary capture site for circulating FX. The docking model indicates that the Ch/snaclec domains act as a scaffold to accommodate the elongated FX model. The relatively large separation (~65 Å) between the catalytic site and the exosite explains the high specificity of RVV-X for FX. This is in sharp contrast to thrombin-like RVV-V which cleaves the Arg1545-Ser1546 bond specifically by recognizing the side-chains of Ile1539 (P7)-Arg1545 (P1) located in close proximity to the scissile bond of FV [The structure of the snaclec domain of RVV-X is quite similar to that of the FX-binding protein (X-Bp) whose crystal structure was solved in complex with the γ-carboxyglutamic acid (Gla) domain of FX . This stnd of FV . The RVVEchis carinatus and Echis multisquamatus, respectively [S. Takeda and T. Morita, unpublished work). Each subdomain in multactivase is similar in structure to the corresponding one in RVV-X. However, the interactions between the heavy chain and the snaclec domain are remarkably different. The snaclec domain interacts with the Ds subdomain in multactivase but the Ch subdomain in RVV-X, resulting in a different overall shape and configuration of the catalytic site and the exosite between these two P-IIId SVMPs. The multactivase structure represents the first crystallographic observation of the interaction between an ADAM D domain and another polypeptide chain, providing additional insights into protein–protein interactions by the M12B clan of proteinases.Carinactivase-1 and multactivase are potent prothrombin activators isolated from the venom of ectively ,131. TheA and S domains, which are connected by two elongated structural modules, T1 and CB. Unlike ADAMs, ADAMTSs lack the Ds/Da/Cw arm structure, and the D* domain with an ADAM_CR domain fold is directly connected to the M domain by a connector loop (16–20 residues) that wraps around the opposite surface of the catalytic site [tic site ,71. The tic site  althoughtic site . On the tic site . These rA, are separated by about 45Å along T1. T1 has a very similar structure to the prototypical TSR, TSR2 in TSP-1 [C-terminal β-sheet in the CA subdomain, forming a mini β-sandwich structure with a hydrophobic core that strengthens the interactions between T1 and CA, thus fixing the CA domain position relative to T1. On the other hand, there are few specific interactions between the D* and T1 domains in the crystal structure of ADAMTS13-DTCS, suggesting that the relative orientation between the D* and T1 domains may be fixed by crystal packing and would be variable in solution. The flexibility of the molecule between the D* and T1 domains is reflected by the low isomorphism of the ADAMTS13-DTCS crystals [B subdomain has no apparent secondary structure but comprises a series of turns stabilized by a pair of disulfide bonds and forms a rod shape with its N and C termini about 25Å apart  is a plasma glycoprotein that plays an essential role in platelet dependent hemostasis ,138. VWFin vitro ,147,148.in vitro . Recent The segment corresponding to the HVR runs across the middle of the D* domain of ADAMTSs in close proximity to the active site, suggesting that the HVR might be ideally positioned to directly influence cleavage of the substrate . In the A subdomain adopts an ADAM_CR domain fold and thus potentially functions as a protein–protein interaction site. As expected, ΔV-loop, a triple alanine substitution (H476A/S477A/Q478A) in the V-loop and R488E in the HVR mutants had significantly reduced proteolytic activity, suggesting that these hydrophilic or charged residues play a pivotal role in VWF recognition and constitute exosite-2 [et al., reported the results of a comprehensive analysis of the C domain in ADAMTS13 that identified its functional importance for interacting with VWF [A subdomain) had no major effect on ADAMTS13 function, and five out of six engineered glycans on the C domain also had no effect on ADAMTS13 function. However, glycans attached at position 476 appreciably reduced both VWF binding and proteolysis.The Cxosite-2 . Recentlwith VWF . They foA subdomain form a hydrophobic pocket that appears to be involved in binding hydrophobic residues Ile1642, Trp1644, Ile1649, Leu1650 and Ile1651 in VWF. The east Asian-specific P475S polymorphism in the ADAMTS13 gene causes approximately 16% reduction in plasma ADAMTS13 activity [A subdomain of this mutant was significantly different from that of the wild type [By substituting the segments of the C domain with the corresponding regions in ADAMTS1, they identified that residues Gly471-Val474 at the base of the V-loop within the Cactivity . The cryild type .C-terminal deletion mutants of the VWF115 (VWF residues 1554–1668) and VWF73 fragments demonstrated that VWF A2 domain residues Glu1660-Arg1668 appreciably contribute to the cleavage of the Try1605-Met1606 scissile bond [The S domain in ADAMTS13 has the highest binding affinity for the A2 site of VWF. ile bond  and thatile bond . Mutantsile bond . In ADAMile bond , and furile bond . It was ile bond . The ADAile bond . These gile bond . Both thile bond . Similarile bond . An antiTremendous progress has been made in the past decade towards our understanding of the structure–function relationship of the M12B clan of proteinases. Crystallographic studies have revealed the structures and spatial relationships of the functionally important domains of both ADAM and ADAMTS family proteinases. Most of the structural information of the overall MDC domains of ADAMs has come from SVMPs. The higher abundance, stability and resistance to proteolysis of SVMPs compared to mammalian ADAMs have made them attractive models for structural studies. The key message from these findings is that the MDC domains adopt a C-shaped configuration, whereby the HVR in the ADAM_CR domain faces toward the catalytic site. This raises the intriguing possibility that the HVR creates an exosite for capturing substrates directly or via binding to an associated protein. The RVV-X structure is consistent with this hypothesis. The multactivase structure suggests a potential function of the D domain in protein–protein interactions. These structural studies on SVMPs have provided radical new insights into the structure–function relationship of ADAMs. Some molecules have been shown to work as cofactors in the process of ectodomain shedding by membrane-bound ADAMs ,162,163;


Understanding the evolution of a protein, including both close and distant relationships, often reveals insight into its structure and function. Fast and easy access to such up-to-date information facilitates research. We have developed a hierarchical evolutionary classification of all proteins with experimentally determined spatial structures, and presented it as an interactive and updatable online database. ECOD (Evolutionary Classification of protein Domains) is distinct from other structural classifications in that it groups domains primarily by evolutionary relationships (homology), rather than topology (or “fold”). This distinction highlights cases of homology between domains of differing topology to aid in understanding of protein structure evolution. ECOD uniquely emphasizes distantly related homologs that are difficult to detect, and thus catalogs the largest number of evolutionary links among structural domain classifications. Placing distant homologs together underscores the ancestral similarities of these proteins and draws attention to the most important regions of sequence and structure, as well as conserved functional sites. ECOD also recognizes closer sequence-based relationships between protein domains. Currently, approximately 100,000 protein structures are classified in ECOD into 9,000 sequence families clustered into close to 2,000 evolutionary groups. The classification is assisted by an automated pipeline that quickly and consistently classifies weekly releases of PDB structures and allows for continual updates. This synchronization with PDB uniquely distinguishes ECOD among all protein classifications. Finally, we present several case studies of homologous proteins not recorded in other classifications, illustrating the potential of how ECOD can be used to further biological and evolutionary studies. Protein structural domain databases offer a vital resource for structural bioinformatics. These databases provide functional inference for homologous structures, supply templates for structural prediction experiments, and differentiate between homologs and analogs. The rate of structure determination and deposition has increased dramatically over recent years, overwhelming the ability of current classifications to incorporate all new structures. We have developed a fast and reliable methodology for updating domain databases automatically, and created a revised hierarchy for domain classification that emphasizes evolutionary relationships. By classifying all known structures in our database with continuing automatic updates, we provide an up-to-date alternative to current resources. We illustrate several concepts that guided our classification scheme with examples of homology between domains in ECOD that are not observed in other resources. The billions of proteins in extant species constitute a bewilderingly diverse protein world. To understand this world, systematic classifications are needed to reduce its complexity and to bring order to its relationships. As proteins are the products of evolution, their phylogeny provides a natural foundation for a meaningful hierarchical classification. As in the classification of species, a phylogenetic classification of proteins identifies evolutionary relationships between proteins and groups homologs (proteins that are descendants of a common ancestor) together. Because homologs generally share similar three-dimensional (3D) structures and functional properties, such a classification provides a valuable platform for studying the laws of protein evolution by comparative analysis as well as for predicting structure and function by homology-based inference.http://www.cathdb.info) is largely automatic with added manual curation and emphasizes more on geometry, while SCOP is mainly manual and focuses on function and evolution.Many protein classifications are currently available. Comprehensive sequence-based classifications such as Pfam http://scop.mrc-lmb.cam.ac.uk/scop/index.html) hierarchical classification, closely related domains are grouped into families; families with structural and/or functional similarities supporting common ancestry are grouped into superfamilies; superfamilies with similar 3D architectures and topologies are grouped into folds; and folds with similar secondary structure compositions are grouped into classes. Cataloging remote homologies identified by a combination of visual inspection, sequence and structure similarity search, and expert knowledge, the SCOP superfamily is the broadest level indicating homology and offers invaluable insights in protein evolution. However, SCOP tends to be conservative in assessing evolutionary relationships, and many homologous links reported in literature are not currently reflected http://www.pdb.org) hinders careful manual curation in SCOP. Recently, a new version of SCOP (SCOP2) In the SCOP Here we introduce the ECOD (Evolutionary Classification Of protein Domains) database. Our goal is threefold: (1) to construct a comprehensive domain classification based on evolutionary connections, (2) to extend the realm of connections to include remote homology, and (3) to maintain concurrent updates with the PDB. Because experimental data is very sparse compared to sequence data, establishing an evolutionary-based classification scheme of structures allows for biological insight into related proteins that otherwise lack functional information. In such a scheme, close homologs admittedly represent the most relevant source of functional inference. However for most proteins, only distant homologs have been studied in detail. Fortunately, many examples have shown that analysis of proteins in the context of their distant homologs provides functional clues that advance biological research http://prodata.swmed.edu/ecod/). By focusing on remote homology and weekly updates, ECOD strives to provide a more simplified and up-to-date view of the protein world than is currently available in existing classifications. As such, ECOD is unique in combining the following features: 1) the aforementioned weekly updates, following new releases from the PDB; 2) a hierarchy that specifically incorporates sequence-based relationships in a family level of close homology; 3) a classification that reflects more distant evolutionary connections; 4) a hierarchy that lacks a SCOP-like fold level, as the definition of “fold” is often subjective ECOD is a publicly available database  or physical/chemical restrictions  We examined the distribution of domains mapped to SCOP folds and CATH topologies among ECOD X-groups. Of 1,799 ECOD X-groups, 598 include domains from only one SCOP fold and 564 include domains from only one CATH topology, reflecting agreement between classifications for these groups. 89 ECOD X-groups contain domains from multiple SCOP folds and 315 X-groups include domains from multiple CATH topologies. For example, the SCOP folds c.1-TIM beta/alpha-barrel and c.6-7-stranded beta/alpha barrel both contain domains mapped to the ECOD TIM beta/alpha barrel X-group. ECOD unifies such groups due to their shared structural similarity (7- versus 8- stranded) and similar locations of functional sites, but with insufficient evidence of homology to belong to the same H-group. 935 ECOD X-groups are not mapped to any SCOP fold, whereas 1,014 ECOD X-groups are not mapped to any CATH topology. The majority of these unmapped X-groups are simply due to proteins that are not classified by SCOP or CATH ; the remainder are shared proteins that are partitioned differently. Taken together, these results suggest that ECOD tends to merge both SCOP folds and CATH topologies into X-groups.An ECOD H-group can contain more distant homologous links than the equivalent SCOP superfamily or CATH homologous superfamily. Although the majority of ECOD H-groups contain only a single SCOP superfamily (88%) or CATH homologous superfamily (81%), some H-groups contain many more . For exaIn total, 53 ECOD H-groups contain domains from two or more SCOP folds, and these H-groups contain domains from 151 unique SCOP folds, indicating that fold change in evolution of protein structures is not a very uncommon phenomenon. Similarly, 169 ECOD H-groups contain domains from two or more CATH topologies, and these H-groups contain domains from 357 unique CATH topologies. Additionally, 36 H-groups contain domains mapped to more than one CATH class, indicating homologous domains that nonetheless contain fairly different topologies.To readily incorporate the observation that homologs can adopt different folds, ECOD has a topology (T-) level below the homology (H-) level. As a result, homologs with different topologies that SCOP necessarily separates into different folds (and thus different superfamilies) are unified in the same H-group but different T-groups in ECOD. For example, β-propellers are comprised of differing numbers of repeated β-meanders, all of which are evolutionarily related. The five different beta-propeller folds outlined in SCOP are organized in ECOD into a single H-group, with child T-groups for domains with differing number of blades Within T-groups, ECOD organizes domains into families based on sequence similarity. We employ Pfam as the standard for family definition. ECOD domains were attributed to Pfam families by HMMER3 th, 2013 (version 22b) are presented in Summary statistics for the ECOD database as of July 31stWe also compare ECOD to the most recent releases of SCOP and CATH. ECOD, SCOP, and CATH differ in domain partition strategy, classification hierarchy, and simply in the number of structures considered. At the time of writing, ECOD classifies 93,663 PDB depositions containing 239,303 protein chains, SCOP 1.75 contains 38,221 PDBs and 85,141 chains, and CATH v3.5 contains 51,334 PDBs and 118,792 chains. Of those chains classified in ECOD that are not in SCOP , 137,794 were automatically classified and 2,484 were classified manually. Of those chains classified in ECOD, but not in CATH , 106,474 were automatically classified and 2,521 were classified manually. The growth of the PDB over time is compared to the number of structures classified in ECOD, CATH, and SCOP . The difSince the July 2013 version, whose statistics are presented here, the subsequent 25 weekly releases by the PDB have been automatically classified . Each weWe analyzed the distribution of domains in hierarchical levels in ECOD. The most populated homologous groups (H-groups) are placed in context with their architecture in ECOD  and are We compared our H-groups to SCOP superfamilies and folds by considering sequence and structure similarity of domain pairs within each level. ECOD manual representatives and ASTRAL40 domains were evaluated by HHsearch to reflect sequence similarity and TMalign to reflect structure similarity We compared the domain partition observed in ECOD, SCOP, and CATH. Domain partition strategy can differ markedly between classifications, depending generally on whether the presence of compact structural units or overall sequence similarity is emphasized. The number of domains per chain observed in the domain classifications is presented in The difference in homologous links among equivalent domains was analyzed in ECOD, SCOP, and CATH. We define equivalent domains as those that share 80% residue coverage in all classifications. This subset of domains contains those domains whose partition is similar among classifications, but whose classification and homologous cluster size differ. We then analyze whether those domains that share a homologous link within one classification also share that link in other classifications. For the purposes of this analysis, only SCOP domains from canonical SCOP classes [a–d] are considered. Of the total domains in ECOD, 67,559 are defined equivalently (by 80% residue coverage) in SCOP and CATH. As many of these domains are identical or near identical in sequence, only domains with less than 95% sequence identity are used. There are 9,523 equivalent, non-redundant domains, shared among SCOP, CATH, and ECOD. Any pair of those equivalent domains belonging to the same H-group is considered to be homologous, 1,030,085 of these homologous domain pairs were observed in ECOD. Similar analysis was performed on SCOP superfamilies and CATH homologous superfamilies, where 711,894 and 680,726 homologous domain pairs were observed respectively. On average, 49.5% of domain pairs were shared between classifications, 36.6% of domain pairs were only observed in ECOD, 11.4% of domain pairs were observed only between ECOD and CATH . NegligiSCOP recently diverged into two separate projects: SCOPe SCOPe (v2.03-stable) uses a conservative automated method to add domains to the SCOP v1.75 hierarchy. Since both ECOD and SCOPe were derived from SCOP v1.75, we were particularly interested in classification of recent chains. ECOD v49 and SCOPe v2.03 (stable) contain 261,704 and 163,351 domains from shared protein chains, respectively. Of those SCOP-mapped ECOD domains, 94,292 were derived from SCOP v1.75 domains and 57,929 were independently classified. 27,142 ECOD domains derived from SCOPe shared chains do not map to any SCOPe domain, reflecting direct differences in domain partition strategy between SCOPe and ECOD. 1,493 SCOPe domains arise from structures classified only by SCOPe, but these structures are dominated by peptides and coiled-coils, regions that are not classified as domains by ECOD. 9,164 ECOD domains were derived from SCOP v1.75 domains, but are not mapped to SCOPe. These domains were generally the result of subdivision of a larger SCOP domain. There is a core set of domains that are shared by SCOPe and ECOD, both arising due to their shared origin and also due to independent classification of more recent domains. The differences in domain partition likely arise from differences in treatment of domain duplication and subdomains and are a potential target for further study.We consider the growth-over-time analysis of ECOD in the context of the domain mapping between ECOD, SCOP and CATH . Where aIn addition to comparison of broad statistics of ECOD, we also present three examples of homologous relationships recorded in ECOD but not observed in other classifications. We consider any two homologous domains to have a “homologous link.” Firstly, we demonstrate the homologous link between SAM-dependent methyltransferases and NAD(P)-binding Rossmann-fold domains. These domains share topological connections, but a strand invasion causes them to bear distinct topologies, nonetheless, they share strong sequence similarity. Secondly, we show how members of the cysteine-rich domains of Frizzled share homology with other domain families that can primarily be detected by conserved patterns of cysteines. Finally, we describe a novel homologous link between Duf371 and the GutA-like PTSIIA component domain families within the topologically diverse cradle-loop barrel X-group. Each of these distinct examples demonstrates how the particular focus of distant homology in ECOD can reveal previously unknown relationships.ECOD contains many homologous links that are not recorded in other classification databases. One example is the relationship between S-adenosyl-L-methionine-dependent methyltransferases (SAM MTases) and NAD(P)-binding Rossmann-fold domains (Rossmann domains). SAM MTases methylate a wide range of substrates using the methyl group donated by the cofactor SAM, which is comprised of an adenosine nucleoside and a methionine amino acid joined together. Rossmann domains are found in many oxidoreductases that transfer electrons between substrates and the cofactor NAD(P), which is comprised of a nicotinamide nucleotide and an adenine nucleotide joined together. Thus, SAM and NAD(P) share the adenosine part but differ in the other half, and the two enzyme superfamilies exploit the dissimilar parts of the cofactors to catalyze different reactions In SCOP, SAM MTases and Rossmann domains are classified in different folds , while in CATH, they are in the same topology group but different homology groups . Although both SCOP and CATH indicate by their classification that SAM MTases and Rossmann domains are not homologous, literature suggests that they are actually related Frizzled receptors possess an extracellular cysteine-rich domain (FZ-CRD) for binding the Wnt ligands. FZ-CRD, as a mobile evolutionary module, has been found in other proteins such as the Smoothened receptor in Hedgehog signaling, secreted Frizzled-related proteins (SFRPs), and receptor tyrosine kinases MuSK and ROR. Sequence similarity searches and structural comparisons revealed distant similarities among FZ-CRD, Niemann-Pick type C1 protein (NPC1) that functions in cholesterol transportation, folate receptors and riboflavin-binding proteins (FRBPs) 8CX6C*CX3CX6,7C*C*C’   . The disesidues) . The homX6C*C*C’  similar X6C*C*C’  in glypiX6C*C*C’  and a riX6C*C*C’ . TogetheX6C*C*C’ , was obsMethanocaldococcus fervens sequence Mefer0473 . PTSIIA adopts a similar β-barrel topology as Duf371 and is noted in SCOP as consisting of two intertwined structural repeats  and the bacterial GutA-like PTS system glucitol/sorbitol-specific IIA component . While Duf371 is absent in SCOP, CATH classifies its fold (2.60.120.630) separately from that of PTSIIA (2.40.33.40). Duf371 forms an 8-stranded β-barrel from the intertwined β-strands of a tandem duplication . The dup repeats . The oveA similar overside connection occupies the N-terminal half of pyruvate kinase (PK) β-barrel domain-like folds (ECOD/SCOP domain e1pklA1/d1pkla1). The PK barrel adopts a duplicated topology like PTSIIA and Duf371, although it lacks the C-terminal overside connection. The absence of this structural element in PK results in a 7-stranded β-barrel . The PK Structural similarity between Duf371 and PTSIIA is evidenced by their being reciprocal top Dali hits of each other , with the next best hits being to various RIFT-related homologs such as the PH barrel. The resulting structural alignment of the PTSIIA C-terminal sequence with both Duf371 sequence repeats is shown in The ECOD database summarizes our views about partitioning of protein structures into domains and this evolutionary classification is a comprehensive resource for the research community. Data about a protein can be retrieved by PDB ID, keyword(s), or protein sequence search. Protein domains of interest are placed close to their close homologs, facilitating analysis of closely related protein structures. Information about more distant homologs is available by browsing representatives of this protein's homology group. ECOD database emphasizes distant evolutionary relationships that otherwise cannot be found. Finally, it is the only classification of protein domain structures that is kept current with the PDB, and every structure is classified with a week delay from its release by the PDB. This feature is significant because other classifications lag behind in updates and researchers are frequently interested in the newest protein structures. Future developments of ECOD will include incorporation of protein sequences without experimentally determined structures to cover as much of the protein world as possible.


The origin of the eukaryotic cell is one of the most important transitions in the history of life. However, the emergence and early evolution of eukaryotes remains poorly understood. Recent data have shown that the last eukaryotic common ancestor (LECA) was much more complex than previously thought. The LECA already had the genetic machinery encoding the endomembrane apparatus, spliceosome, nuclear pore, and myosin and kinesin cytoskeletal motors. It is unclear, however, when the functional regulation of these cellular components evolved. Here, we address this question by analyzing the origin and evolution of the ubiquitin (Ub) signaling system, one of the most important regulatory layers in eukaryotes. We delineated the evolution of the whole Ub, Small-Ub-related MOdifier (SUMO), and Ub-fold modifier 1 (Ufm1) signaling networks by analyzing representatives from all major eukaryotic, bacterial, and archaeal lineages. We found that the Ub toolkit had a pre-eukaryotic origin and is present in three extant archaeal groups. The pre-eukaryotic Ub toolkit greatly expanded during eukaryogenesis, through massive gene innovation and diversification of protein domain architectures. This resulted in a LECA with essentially all of the Ub-related genes, including the SUMO and Ufm1 Ub-like systems. Ub and SUMO signaling further expanded during eukaryotic evolution, especially labeling and delabeling enzymes responsible for substrate selection. Additionally, we analyzed protein domain architecture evolution and found that multicellular lineages have the most complex Ub systems in terms of domain architectures. Together, we demonstrate that the Ub system predates the origin of eukaryotes and that a burst of innovation during eukaryogenesis led to a LECA with complex posttranslational regulation. Of the three domains of life, eukaryotes have the most complex forms of cell organization. Understanding the emergence and early evolution of the eukaryotic cell is a major challenge for evolutionary biology. Recent findings have profoundly changed our long-held view of a simple last eukaryotic common ancestor (LECA) , 1991, pWe know that signaling systems are crucial in complex cells, as they provide the basis for finely tuned regulation of processes such as transcription , the celUbiquitination consists of the posttranslational modification of proteins by the covalent attachment of Ub, a 76-residue peptide . Ub can Since the discovery of Ub, other posttranslational signaling pathways, collectively known as Ub-like systems, have been characterized. These systems use different labeling peptides, which often do not have significant sequence similarity with Ub but nonetheless have the same tertiary structure (a β-grasp fold ). Ub-likThe three systems share the same E1 and E2 enzymes, both of which belong to ancient protein families present in Eukaryota, Bacteria, and Archaea. The prokaryotic E1s and E2s are involved in other signaling systems and were co-opted into new functions with the emergence of the early Ub system . Unlike The ligase activity of E3s can be reversed by DUBs, isopeptidase enzymes that cleave Ub chains after the C-terminus of the peptide label . Some DUIn this work, we use comparative genomics to decipher the origin and evolution of three Ub-like systems: Ub itself, SUMO, and Ufm1. Our reconstruction shows that the ubiquitination toolkit of the LECA was as complex as that of most modern eukaryotes, in terms of diversity of gene families. Furthermore, various species of Archaea belonging to three different lineages  already had a minimal but complete ubiquitination toolkit. Thus, Ub signaling existed prior to the origin of eukaryotes and underwent a profound process of innovation during eukaryogenesis, resulting in a complex Ub system in the LECA. Analysis of the subsequent evolution of the Ub-like posttranslational systems in eukaryotes shows that E1 and E2 predate the LECA and underwent little innovation during early eukaryotic evolution, whereas most E3 families appeared concomitantly with eukaryotes and underwent multiple lineage-specific expansions and diversifications of protein domain architectures. We also describe two independent expansions of the Ub signaling system at the origins of multicellularity in animals and plants. Overall, we show that the complexity of the LECA involved the capacity to perform posttranslational regulation of different cell processes by Ub and Ub-like systems. This suggests that Ub signaling was key to the origin of eukaryotes and was later expanded in some specific, mostly multicellular, lineages.supplementary table S1, Supplementary Material online, and Materials and Methods). Specifically, we surveyed the generalist E1 and E2 enzyme protein families, 27 specific components of the Ub system , 7 families related to SUMO, and 4 related to Ufm1 . Our survey revealed that 38 of these 40 protein families are widespread among eukaryotic groups  events . Although none of the analyzed bacterial genomes contained a complete Ub toolkit, many bacteria were found to possess signaling systems that employ JAB peptidases, and E1 and E2 enzymes akin to the ones acting in ubiquitination . These b) events . Despite) events . OverallCaldiarchaeum subterraneum. In our survey, we found evidence of eukaryotic-like Ub toolkits in three independent Archaea lineages: Crenarchaeota , Euryarchaeota , and Aigarchaeota  .Unlike in bacteria, Ub-specific protein families were observed in many Archaea. Previous work by toolkit) . Interessupplementary figs. S6 and S7 and file S3, Supplementary Material online).To determine whether HGT of eukaryotic sequences into prokaryotic genomes could have occurred, we conducted Basic Local Alignment Search Tool (BLAST) similarity searches for all the protein families present in Archaea and phylogenetic analyses of Ub, UQ_con, and UCH . None of the prokaryotic genes were found to be unexpectedly similar to eukaryotic sequences according to these methods. Thus, under the current taxon sampling, we can rule out a HGT origin for the archaeal toolkit , and two different DUBs (JAB and USP) , whereasTo better understand the evolution of the Ub system in eukaryotes, we examined the counts of two generalist gene families (E1 and E2 enzymes) and 38 protein families that are specific to a particular Ub-like system  . We thenAsupplementary fig. S1, Supplementary Material online). Finally, using likelihood-based gain/loss reconstruction , we obtained a similar result compared with the parsimony-based analysis . This shows that the recruitment of novel machinery in Ub-like systems is a relatively exceptional event during eukaryotic evolution, especially when compared with the frequent losses of individual system-specific gene families.Our analysis indicates that the LECA already had most of the surveyed gene families, independently of whether we root eukaryotes between unikonts/amorpheans and bikonts  or betwesupplementary fig. S2, Supplementary Material online). Indeed, the proportion of Ub-related genes can add up to approximately 5% in some plant genomes , thereby implying a functionally diversifying gene expansion process.To further analyze the diversification of Ub-like systems in eukaryotes, we used the array of domain architectures of each protein family as a proxy to assess the diversity and versatility of the Ub, SUMO, and Ufm1 toolkits. In particular, we compared the number of different protein domains that co-occur alongside the core protein domain of each protein family . The most abundant families  are also the most diverse in terms of architectures (A). In particular, we found that the principal component 1 identified a group of genomes rich in genes related to Ub-like signaling systems, including embryophytes, many animals  and ichthyosporeans . Furthermore, PC2 differentiated most holozoans from embryophytes, which both clustered separately from the rest of the eukaryotes due to the loadings of many protein families that appeared or expanded in holozoans  and plants , respectively. The distinction between plants and holozoans  was also recovered by the PCA based on protein architectures (fig. 4B): Plants and animals, while sharing all the surveyed protein families, had specific sets of protein architectures that distinguished them from the rest of the eukaryotes.To test whether there are phylogenetic patterns in the profiles of gene counts and architectural diversity of the Ub-like systems, we performed principal component analyses  . The PCAfig. 4A–G, see To gain insight into the complexity of Ub-like signaling during eukaryotic evolution, we used the protein domain architectures of extant species to reconstruct ancestral domain networks at various ancestral nodes of the eukaryotic tree  (see Matfig. 5D), although less than in most extant eukaryotes. For example, the LECA’s Ub toolkit used highly promiscuous domains such as Ankyrin repeats (linked to C3HC4 RINGs), UBA , and LRR (linked to F-box). Architectural diversification during eukaryogenesis also led to specific domain combinations in E1 and E2 protein families, which use exclusive sets of accessory domains  and have little interconnection with other nodes. These E1 and E2 types are conserved in all the other ancestral nodes and characterize the eukaryotic Ub network. Also, the usage of multidomain proteins in the early eukaryote appeared as an important difference compared with archaeal systems, in which all genes encode single-domain proteins.We inferred that many Ub-related genes already employed multiple accessory protein domains (in black) in several Ub-related genes in the LECA D, althouA–C and E–G), confirmed by the PCA based on domain architectures (B). Nevertheless, we found that the network structure of the deep ancestors influenced later ancestors and extant organisms. For example, the urembryophyte’s less extensively connected domain network could be traced back to the urbikont (fig. 5E–G). This phylogenetic inertia constrained the Ub and SUMO systems of plants, whose expansion was not accompanied by a significant increase of protein architectures. Conversely, the diversified toolkits of animals were recapitulated in the denser domain networks of the urmetazoan, the urholozoan, and the uramorphean (fig. 5A–C).Since the origin of eukaryotes, the connectivity and network density of Ub and SUMO toolkits independently increased in Amorphea and Bikonta, although to a lesser extent in Bikonta. This led to rich signaling systems in multicellular animals and plants (fig. 5tectures B. NevertA–C and E–G). The most abundant catalytic machinery of Ub signaling employed a similar core of highly connected nodes in all the post-LECA ancestors. This included the C3HC4 variants (which shared most of their accessory domains and often co-occurred themselves), C3H2C3/zf-RING_2 (highly connected but not directly linked to other RINGs), IBR, or U-box. The CRL substrate recognition subunits BTB and F-box were both highly connected, particularly to protein-binding domains. In contrast, BTB and F-box shared few nodes, thus suggesting independent diversifications. For example, F-box often co-occurred with Kelch (in plants), LRR, and WD40, whereas BTB used Ankyrin, Kelch, BACK, and NPH3 .Despite these differences in network density, patterns common to all the ancestral networks emerged  reveals that eukaryotic Ub-like systems switched to the use of multidomain protein families during their early evolution, whereas archaeal toolkits consist only of the catalytic protein domains. The presence of accessory domains within protein families reflect their ability to physically interact with other cellular components  and CRLs’ substrate selector subunits (BTB and F-box), HECTs, and USPs. Likewise, HECTs are also rich in motifs that bind to lipids, complex sugars, and poly-A tails of RNA . The preArabidopsis thaliana entailed sub- and neofunctionalization events, whereas its E3s are often redundant . This might be explained by the fact that HECTs’ tertiary structure is intrinsically constrained, as they require their catalytic site to be at the C-terminus to be active . This may be due to the complex multicellularity of plants and animals, which requires fine-tuned regulation of cellular functions. Indeed, parallel to this complexification of posttranslational regulation, animals and plants are known to have a rich transcriptional regulation machinery, probably related to their complex development  and diversified its toolkit with different accessory domains , two RING E3 types , and two peptidases (USP and JAB). This early Ub system underwent an important process of innovation during the eukaryogenic phase that led to the LECA.We propose that three processes shaped Ub signaling during early eukaryotic evolution. First, almost all the Ub-related gene families seen in extant eukaryotes emerged at that time. This includes new catalytic mechanisms  and, most importantly, two eukaryote-specific signaling systems (SUMO and Ufm1). Second, some gene families underwent massive expansions . Finally, new and diverse protein domain architectures were acquired in both ancient and new enzyme families . Altogether, these events identify the stem phase of eukaryotic evolution as a period of rapid and intense innovation in posttranslational signaling.After the initial eukaryotic radiation, the Ub and Ub-like systems further evolved by protein family expansion and domain architectural diversification, in a largely lineage-specific manner. There was, however, little protein family innovation, with only IR1-M  and SOCS-box selectors (holozoan CRLs) evolving later on. These diversification processes particularly affected E3s ligases (in the case of the Ub system) and delabeling peptidases (in the case of the SUMO system) probably because they are in charge of the target selection specificity. In this sense, the diversification of domain architectures in these families is related to the substrate specificity, with new accompanying domains allowing selective interaction with other proteins, complex sugars, lipids or nucleic acids. This process of architectural innovation was especially intense at the origin of animals and plants, coinciding with their need for a precise regulation of multicellularity-related protein products and processes. Thus, alongside the eukaryogenic phase of Ub expansion, the origins of multicellular animals and plants represent the main bursts of innovation in Ub systems in eukaryotes.Overall, our investigation into the diversity of early eukaryotic Ub signaling clearly points to an important burst of evolutionary innovation at the origin of eukaryotes. This suggests that the LECA was much more complex than previously thought, not only in terms of cellular machineries but also in terms of elaborate regulation systems such as Ub signaling.supplementary table S1, Supplementary Material online). The selection of eukaryotic taxa includes 14 animals, 10 unicellular holozoans, 16 fungi, 1 apusozoan, 4 amoebozoans, 7 embryophytes, 7 unicellular algae , 6 heterokonts/stramenopiles, 5 alveolates, 1 rhizarian, 1 haptophyte, 1 cryptophyte, and 5 excavates. We obtained the proteomes from publicly available databases, with the exception of Oscarella carmela and Mnemiopsis leidyi, kindly provided by Scott A. Nichols (University of Denver) and Andy Baxevanis , respectively. We also used RNA-Seq data generated in-house   against the NCBI Bacteria and Archaea databases and the Microbial Dark Matter project database , and genomic data from the Microbial Dark Matter project  (supplemisporum) . We perfisporum) . The ideisporum) , searchidatabase .supplementary table S2, Supplementary Material online, for a complete list of protein families, associated Pfam domains, and examples of specific genes in model organisms). The ThiF, zf-MIZ, and DCAF protein families were identified, refining the domain search with specific amino acid motifs. Specifically, proteins with ThiF and Moez/MoeB catalytic motifs do not have E1 activity and were discarded  to a certain Pfam domain, referred to as the core defining domains of each protein family . Statistical analyses were performed using R to detect enrichments or depletions in gene content in different lineages, using the Wilcoxon rank sum tests with a significance threshold of P < 0.01.Using R , we builsupplementary fig. S7 and table S3, Supplementary Material online). We searched all the archaeal sequences  with a cut-off value of 10−5 and against a combined database including the full NCBI nonredundant protein database, the Microbial Dark Matter database, and the full genomes and transcriptomes included in this study. We took the top 50 hits and searched them back to the same combined database, with a cut-off value of 10−10. The network visualizations of this reciprocal BLAST analyses were generated using Cytoscape 3.1.1 . For these analyses, we used 1) all the Pfamscan-identified proteins from our selection of eukaryotes, 2) the identified archaeal sequences from NCBI and the Microbial Dark Matter databases, and 3) the top 100 hits from the BLAST searches in these databases. The alignments were performed using the Mafft L-INS-i algorithm, optimized for local sequence homology (http://www.csiro.au/Homo last accessed 1 October 2014), to determine whether the aligned sequences of amino acids are consistent with evolved under time-reversible conditions (assumed by most model-based phylogenetic programs). Based on the PP plots shown in Asupplementary figure S6, Supplementary Material online, it was concluded that the data did not violate this assumption. The phylogenetic trees of UQ_con, UCH, and Ub were estimated using the Le and Gascuel  evolutionary model with a discrete gamma (Γ) distribution of among-site variation rates (four categories), according to the respective analyses performed with ProtTest 3.4  BTest 3.4 , using thttp://mesquiteproject.org, last accessed 1 October 2014). We assumed two scenarios for the root of eukaryotes: 1) the modified “unikont-bikont” hypothesis . In this case, ProtTest showed that the best evolutionary model for our data set was LG with a Γ distribution of four discrete categories and a proportion of invariable sites (LG+Γ+I). Using this model (PROTGMMAILG), we used RAxML with a fixed topology .The reconstruction of ancestral states of each core element was inferred with Mesquite 2.75 using both a parsimony criterion and the AsymmMk likelihood model (pothesis  but renapothesis  and 2) tpothesis . For thepothesis  and “thrpothesis . The Asypothesis , which wprcomp function, using scaling  and a covariance matrix, and plotted using bpca R package. We used scaling because our data, although presenting the same units (counts of number of genes), show very different ranges of values (with some families having hundreds of genes and others just one or two). The PCA of the protein counts (A) was based on the number of genes of each family in each species. In the PCA of protein domain architectures (B), instead, the species were clustered based on the number of proteins with a particular domain architecture. To this end, we first created a list of all the existing protein domain architectures  and then counted how many proteins (with each particular architecture) each species has. These raw counts can be visualized in supplementary figure S5, Supplementary Material online.A PCA was performed using built-in R n counts A was bastectures B, insteaB and supplementary fig. S5, Supplementary Material online) within the corresponding clades. We represented these reconstructions as networks of co-occurring domains using Cytoscape 3.1.1  to accessory domains (other protein domains that co-occur with a core domain in the same protein) if such co-occurrence existed in at least the earliest-branching lineage of a clade and another internal taxon. We used a nested approach, first reconstructing the most external nodes and proceeding inward . The abundance of each core domain (represented by the size of the node) at the reconstructed ancestors of particular clades was estimated with the median gene count of all the analyzed species in that clade . The frequency of each domain co-occurrence (represented by the thickness of the edge between nodes) was estimated analogously. We calculated the network density index of each reconstructed ancestor using the Cytoscape NetworkAnalyzer module .


S.cerevisiae, D. melanogaster, C.elegans, M.musculus, T.rubripes and H.sapiens–and discusses their functions. The presence of such kinases necessitates a revisiting of the classification scheme of the protein kinase family using full length sequences apart from classical classification using solely the sequences of kinase catalytic domains. The study of these kinases provides a good insight in engineering signalling pathways for a desired output. Lastly, identification of hybrids and rogues in pathogenic protozoa such as P.falciparum sheds light on possible strategies in host-pathogen interactions.The highly modular nature of protein kinases generates diverse functional roles mediated by evolutionary events such as domain recombination, insertion and deletion of domains. Usually domain architecture of a kinase is related to the subfamily to which the kinase catalytic domain belongs. However outlier kinases with unusual domain architectures serve in the expansion of the functional space of the protein kinase family. For example, Src kinases are made-up of SH2 and SH3 domains in addition to the kinase catalytic domain. A kinase which lacks these two domains but retains sequence characteristics within the kinase catalytic domain is an outlier that is likely to have modes of regulation different from classical src kinases. This study defines two types of outlier kinases: hybrids and rogues depending on the nature of domain recombination. Hybrid kinases are those where the catalytic kinase domain belongs to a kinase subfamily but the domain architecture is typical of another kinase subfamily. Rogue kinases are those with kinase catalytic domain characteristic of a kinase subfamily but the domain architecture is typical of neither that subfamily nor any other kinase subfamily. This report provides a consolidated set of such hybrid and rogue kinases gleaned from six eukaryotic genomes– Living cells constantly respond to both internal and external stimuli with the help of signalling systems. As the complexity of the organism increases, the complexity of signalling systems also increases Many of the signalling domains function in the process of cellular localization, provide interacting partners and regulate the activity of the protein Protein kinases are a group of enzymes that play important roles in almost all signalling pathways. In this work, we consider Ser/Thr and Tyr kinases only. About 280 different subfamilies of protein kinases have been identified so far, and these are involved in regulating different parts of signalling pathways in various organisms T. rubripes. Therefore, this kinase is localized to the membrane due to the property of the domain associated with the STE11 kinase catalytic domain. Rogue kinases are those where the domain architecture of the kinase is not usually observed among currently known Ser/Thr/Tyr kinases. For example, the association of the DAXX domain with the TTBK subfamily of kinases indicates potential association of this kinase with transcriptional machinery. Such domain combinations may determine some of the properties of the protein and also introduce cross-talks in the pathway, thereby leading to more complex networks However, in nature, sometimes such subfamily-characteristic domain architectures may not be strictly followed Kinases are classified into their respective subfamilies by means of clustering of sequences of solely kinase catalytic domains Plasmodium falciparum) has been probed for existence of such sequences with hybrid or rogue features to probe if this feature is specific to higher eukaryotes alone or is also present in eukaryotic pathogens that attack them.Protozoans are known to exhibit various non-canonical features in their protein sequences H. sapiens, S. cerevisiae, M. musculus, T. rubripes, C. elegans, and D. melanogaster) have been recognized by remote homology detection protocols adopted in previous publications from this laboratory Sequences of protein kinases from six model organisms  . The DAP2+ ions at the C2 and C1 domains, respectively The NDR kinase plays a role in regulating the MAPK pathways. An example of a hybrid is an NDR kinase associated with C1 and C2 domains (Q8MPZ6) . ClassicCertain domains mediate protein-protein interactions. One such example is the SAM domain which induces dimerization in the proteins containing them. This domain is specifically seen to be associated with the Eph receptor family Domain recombination leading to multi-domain proteins is instrumental in the evolution of signalling pathways. Certain domain architectures are more commonly observed involving certain domain families. Although there are such preferences, deviations do occur where domains are recruited in such a way so as to result in proteins with uncommon domain combinations leading to new functional features. From the dataset, 18 such rogue kinases have been identified, and they impart a wide range of functions to protein kinases.The domain architectures of the rogue kinases identified in the current study are shown in Calcium/Calmodulin dependent kinases are of various types, some of which are involved in glucose metabolism by phosphorylation of Glycogen-synthase Similarly, functions of such proteins could be extrapolated depending on the domains associated with the kinase domain and experimental studies that may indicate the rogue nature. An example of this is the Q13237 , with thThe set of 92 outliers presented as hybrids and rogues in the sections above have been identified on the basis of comparison of domain architecture of the sequence with the cognate architectures of the classified subfamily. These hybrids/rogues are likely to be functionally different from the corresponding classical subfamily and hence need to be resolved and differentiated from them. In other words, these hybrid and rogue kinases are new emergent subfamilies that may be evolutionary offsprings of two subfamilies displaying hybrid function. The ClaP method developed earlier in this group P. falciparum is an obligate parasite and shows a lot of variations in kinase distribution, with certain subfamilies being absent altogether like the MAP2K kinases and many members of the STE group 2+ binding and regulation. This protozoan organism not only shows variation in its distribution but also at the level of the sequence. Fifty-seven P. falciparum kinases were analyzed for their hybridnature. Forty-two of these kinases are either hybrids or rogues and these are listed in Plasmodium genome is very well studied, although their evolutionary significance and function is largely debated. Structural studies on these proteins indicate the presence of zinc fingers in such regions In addition to sequences containing such inserts, many kinases belong to the single kinase domain hybrid category that may be extrinsically regulated by the host proteins as well. Another hybrid is the MLK and Dicty4 subfamily kinase with the SAM domain tethered to it , which iPlasmodium kinome . Such chimeric proteins could be introduced into tumour cells to induce programmed cell death. The examples of hybrids and rogues discussed in this study could help in widening the prospects for designing more synthetic cell circuits.The protein kinase family is crucial in regulating important cellular pathways in the cell. Kinases are promiscuous in nature and occur with many associated domains that help in its localization, regulation and interaction with other proteins so as to relay the signal in a specific and time dependent manner. These kinases have been classified into groups and subfamilies that give an indication on the function based on specific motifs within the kinase catalytic domain. Although this has proven to be useful in a large number of cases, there exists a sub-population of kinases that have “inconsistencies” in the subfamily classification and associated domain combinations. We refer to them as hybrid and rogue kinases.This study provides a consolidated list of such kinases from 6 eukaryotes and a eukaryotic pathogen. The AGC group is largely represented in the list of hybrid and rogue kinases identified. This is specifically interesting because the AGC kinase group comprises of proteins involved in core intracellular signalling and are subject to various modes of regulation including phosphorylation, binding to small molecules and forming higher order oligomers These hybrid kinases, due to their dual functional properties, may be eventually classified into separate subfamilies that constitute such outliers although their kinase catalytic domain shows significant similarity to one of the currently known subfamilies. The method presented to identify such novel and rare kinases on the basis of their local matching score identifies specific clusters that have high population of hybrid kinases, thereby re-iterating the fact that such kinases can be classified as a new subfamily.Some of the domain architectures represented in these hybrids and rogues are more commonly noted across organisms while others are more organism-specific or may be referred to as orphan kinases. The objective of this study was to identify all deviant kinases. The deviant architectures seen only once so far have been marked as orphans in The presence of hybrid and rogue kinases indicates an elegant evolutionary mechanism that causes variations in the signal transduction pathways that are important for the adaptation of an organism especially in case of pathogens. Study of such hybrid kinases also provides an understanding for engineering signal transduction pathways for a desired output. Such a mechanism of domain recombination leading to evolution/rewiring of signal transduction pathways has been described in a review by Bhattacharya et al. http://kinase.com−4 on sequences greater than 200 residues (the length cut-off of 200 residues has been chosen since the kinase domain is about 200–300 residues long) in the genome, an initial set of kinase-like sequences were identified. These were then filtered out using a profile coverage criteria of ≥70% to weed out false positives. A sequence is assigned to a particular subfamily of Ser/Thr/Tyr kinase only if it shares at least 30% sequence identity with the profile of that subfamily.Protein kinase sequences encoded in the genomes of these organisms have been identified using a well-established protocol involving profile search methods adopted previously DLKXXN. The most crucial residue is the Asp, which is usually identified by conservation of Asn four residues further. Substitutions in other residues  have been observed in certain sub-families which are likely to be still functional.The catalytic Asp is the most crucial residue for the kinase to be active since it mediates the phosphate transfer Homo sapiens 2. Saccharomyces cerevisiae 3. Caenorhabditis elegans 4. Drosophila melanogaster 5. Takifugu rubripes 6. Mus musculus. Only the sequences belonging to the well-characterized protein kinase subfamilies for which all level of function are well established were considered for this analysis. The outlier group also known as the “Other” group has been excluded from this analysis due to debated gross level function annotation for these subfamilies. The complete list of kinases identified from these 6 genomes and their sub-families obtained by considering solely the kinase catalytic domain are provided in the Plasmodium falciparum  is the fraction of sequences belonging to a subfamily i in a particular cluster. Details of the scoring schemes have been described in greater detail by Bhaskara et al. The dendrogram is parsed at 0.25 cut off to obtain clusters by hierarchical clustering using Wards method as employed in R package. The individual clusters give an estimate of the possible subfamilies that the dataset can be divided into. Since subfamily information based solely on the sequence of kinase catalytic domain is well known, the variation of subfamilies within each cluster was estimated as a function similar to the Shannon entropy score.Figure S1Maximum likelihood trees showing various subfamilies that contain hybrid/rogue kinases with the canonical cases in black and hybrids highlighted in red and rogues are highlighted in green. Scale bars indicate distances as number of amino acid substitutions per site. A) Eph, B) Focal adhesion kinase, C) Met, D) Ror, E) Fer, F) PKC, G) Src H) PDGFR I) MAST and J) NDR.(PDF)Click here for additional data file.Table S1Canonical domain architectures for each of the 91 subfamilies used in the study based on literature survey.(DOCX)Click here for additional data file.Table S2S. cerevisiae, C.elegans, D.melanogaster, T.rubripes, M.musmusculus, H.sapiens.List of hybrid and rogue kinases from the six model eukaryotes (DOCX)Click here for additional data file.Table S3Clustering of the 1498 sequences using full length alignment free method. Number of sequences, entropy score and subfamily variation for each cluster are also provided.(DOCX)Click here for additional data file.Table S4P.falciparum.List of hybrid and rogue kinases from (DOCX)Click here for additional data file.Table S5P.falciparum.Domain architectures, subfamily information and the length of 1498 sequences from 6 eukaryotes and 57 sequences from (XLSX)Click here for additional data file.Text S1Indirect method to ascertain reliability to dendrograms generated using ClaP method.(DOCX)Click here for additional data file.


TNF-receptor associated factor (TRAF) proteins are key adaptor molecules containing E3 ubiquitin ligase activity that play a critical role in immune cell signaling. TRAF1 is a unique family of TRAF lacking the N-terminal RING finger domain. TRAF1 is an important scaffold protein that participates in TNFR2 signaling in T cells as a negative or positive regulator via direct interaction with TRAF2, which has recently been identified as a pro-apoptotic regulator in neuronal cell death. Here, we report the first crystal structure of the TRAF1 TRAF domain containing both the TRAF-N coiled-coil domain and the TRAF-C domain. Our structure reveals both similarities and differences with other TRAF family members, which may be functionally relevant to TRAFs. We also found that the TRAF-N coiled-coil domain of TRAF1 is critical for the trimer formation and stability of the protein. Finally, we found that conserved surface residues on the TRAF1 TRAF domain that might be binding hot spots that are critical for interaction with signaling molecules. TNF-receptor associated factor (TRAF) proteins are key intracellular signaling molecules in the tumor necrosis receptor (TNFR) and toll like receptor (TLR) family signaling pathways that play critical roles in the immune system124569Seven TRAF proteins, TRAF1–TRAF7, have been identified in mammalsTRAF1 is a well-known adaptor molecule that regulates the activation of NF-kappaB and JNK151819One of the main functions of TRAF1 is suppression of TNF-α or T cell receptor (TCR)-mediated apoptosis, which is known to be an anti-apoptotic function of TRAF1Because of the importance of TRAF proteins in signal transductions, all structural information regarding the TRAF domain of TRAF family, except the TRAF1 TRAF domain, have been elucidated. Despite the emerging roles of TRAF1 in human disease states, including recently reported hepatic and cerebral ischemia/reperfusion injury22Seven TRAF proteins have been identified in mammals, TRAF1–TRAF7, all of which except TRAF7 contain the TRAF domain. TRAF1 is considered a unique family of TRAFs in that it does not contain the N-terminal RING finger domain, which is another common feature in the TRAF family that may be critical for the E3 ubiquitin ligase function . The TRAn) was detected at the N-terminus. The overall fold of the TRAF1 TRAF domain was similar to that of other TRAF domains. Specifically, they resembled the shape of a mushroom, in which the TRAF-N coiled-coil domain forms the stalk and the TRAF-C domain forms the cap.The 2.8 Å crystal structure of the TRAF domain (corresponding to amino acids 220–416) consisting of the TRAF-N coiled-coil domain and the TRAF-C anti-parallel β-sandwich domain was solved using the molecular replacement (MR) method and refined to Rwork = 21.0% and Rfree = 26.2%. The crystallographic and refinement statistics are summarized at There was one trimer (three monomers) in the asymmetric unit, chain A, chain B, and chain C, and three molecules related by near perfect three-fold symmetry . A model2 and 3264 Å2 and were buried upon complex formation, which corresponds to an average of 1088 Å2 per molecule.The structure of the trimeric TRAF1 TRAF domain exhibited a typical mushroom shape, in which the TRAF domain forms the cap and the coiled-coil domain forms the stalk. The trimeric TRAF-C domains were loosely packed with the narrow portion of the binding interface, while the TRAF-N coiled-coil domain was tightly packed with massive hydrophobic interactions and salt bridges. The trimeric interface of the TRAF1 TARF-C domain is formed by packing one end of the (β2)-β3 connecting loop, the β4-β5 connecting loop, against β1 of the neighboring monomer . The majThe TRAF1 TRAF domain, which contains both the TRAF-N coiled-coil domain and TRAF-C domain and was used for the current structural study, was over-expressed in bacteria and purified by affinity chromatography followed by size-exclusion chromatography. Upon size-exclusion chromatography, the TRAF1 TRAF domain was eluted at around 16 ml, indicating that it forms a stable trimer in solution similar with other TRAF family members . InteresUnlike other TRAFs, the TRAF1 TRAF domain was insoluble under physiological conditions, 20 mM Tris-HCl pH 8.0 and 150 mM NaCl. To make the TRAF1 TRAF domain soluble, we attempted solubility tests using different pH values and salt concentrations. We were finally able to solubilize TRAF1 in buffer containing 20 mM sodium citrate and 1 M NaCl, which is an extremely low pH and high salt level. During the solubility test, we realized that the TRAF1 TRAF-C domain alone precipitated readily about 1 hour after size-exclusion chromatography. Due to the insoluble nature of the TRAF-C domain, the solubility difference between the TRAF domain and TRAF-C domain was analyzed by different time points . Our anan helix of the coiled-coil domain also differed in position. Especially, the αn helix of the coiled-coil domain of TRAF4 is out-layered when compared with the αn helix of the coiled-coil domain of TRAF1  of 13.6 uM. This affinity of TANK peptide is somewhat higher than affinity reported for TRAF3 interaction (KD of 23.9 uM), indicating that TANK can interact to TRAF1 more tightly. Finally, we produced F325D, S368R, and R308D mutations to analyse the interaction sites on TRAF1 and performed ITC. TRAFs are important adaptor molecules that mediate cellular signaling events. These compounds can interact with various receptors and intra-cellular proteins including CD40, TRADD, LMP1, TNFR2, RANK, and TANK during the particular signaling events. Structural and biochemical studies of TRAFs and their interaction proteins showed that TRAFs use three regions , β3 and the loop connecting β3 and β4 , β6 and the loop connecting β6 and β7 , and many dispersed areas including β4, β5, β6 and β7 , to accommodate their various partner molecules28293234E. coli BL21 (DE 3) by overnight induction at 20 °C. The protein contained a carboxyl terminal His-tag and was purified by nickel affinity and gel filtration chromatography. A superdex 200 gel filtration column 10/30  that had been pre-equilibrated with a solution of 20 mM sodium citrate at pH 5.0 and 1 M NaCl was used for gel-filtration chromatography. The protein eluted at around 16 ml upon gel-filtration chromatography was collected and concentrated to 5–6 mg/ml for crystallization.The expression and purification methods used in this study have been described elsewhere in detail. Briefly, human TRAF1 TRAF domain corresponding to amino acids 220–416 was cloned into pET24a vector and expressed in The molar mass of the TRAF1 TRAF domain was determined by multi angle light scattering (MALS). The target protein was loaded onto a Superdex 200 HR 10/30 gel-filtration column  that had been pre-equilibrated in buffer containing 20 mM sodium citrate at pH 5.0 and 1 M NaCl. The acta chromatography system was coupled to a MALS detector (mini-DAWM treos) and a refractive index detector (Optilab DSP) (Wyatt Technology).Crystallization was conducted at 20 °C by the hanging drop vapor-diffusion method using various screening kits. The crystals used for the x-ray diffraction study were grown on plates by equilibrating a mixture containing 1 μl of protein solution (5–6 mg ml-1 protein in 20 mM sodium citrate at pH 5.0 and 1 M NaCl) and 1 μl of a reservoir solution containing 1.26 M ammonium sulfate, 0.1 M CHES pH 9.5, and 200 mM NaCl against 0.3 ml of reservoir solution. A 2.8 Å native dataset was collected at the BL-4A beamline of the Pohang Accelerator Laboratory (PAL), Republic of Korea. Data processing and scaling was carried out using HKL2000The structure was determined by the molecular replacement phasing method using PhaserThe general strategy of the solubility assay was based on the method introduced by Bondos and Bicknellhttp://www.ebi.ac.uk/Tools/msa/clustalo/).The amino acid sequence of TRAFs was analyzed using Clustal Omega (Site-directed mutagenesis was performed using the Quickchange kit (Stratagene) following the manufacturer’s protocols. Mutagenesis was then confirmed by sequencing. Mutant proteins were prepared using the same method described above.Isothermal titration calorimetry experiments were performed by NanoITC (TA Instruments). The wildtype and mutants proteins were dialyzed intensively against PBS buffer, and the TANK peptide (SVPIQCTDKT) was dissolved in the same buffer to minimize heats of dilution. TANK peptide was synthesized and purified by Peptron . Prior to titration, the protein samples and the peptide were contrifugated at 13,000 rpm at 4 °C for 5 min to remove any debris. For each titration, a concentrated peptide solution (1 mM) was injected into a cell containing wildtype or each mutant of TRAF1 at a concentration of ~20 uM. All the titrations were carried out 15 °C with 25 injections at 160 sec intervals. Binding isotherms were analysed by using the software provided by TA instruments. Baseline controls were acquired with buffer and pure peptide solutions.Data availability: Coordinates and structural factors were deposited in the Protein Data Bank under PDB ID code 5E1T.How to cite this article: Kim, C. M. et al. Crystal structure of TRAF1 TRAF domain and its implications in the TRAF1-mediated intracellular signaling pathway. Sci. Rep.6, 25526; doi: 10.1038/srep25526 (2016).


Periplasmic Adaptor Proteins (PAPs) – relatively understudied. Here we review the current state of knowledge of these versatile proteins which, far from being passive linkers between the OMF and the transporter, emerge as active architects of tripartite assemblies, and play diverse roles in the transport process. Recognition between the PAPs and OMFs is essential for pump assembly and function, and targeting this interaction may provide a novel avenue for combating multidrug resistance. With the recent advances elucidating the drug eﬄux and energetics of the tripartite assemblies, the understanding of the interaction between the OMFs and PAPs is the last piece remaining in the complete structure of the tripartite pump assembly puzzle.Recent years have seen major advances in the structural understanding of the different components of tripartite eﬄux assemblies, which encompass the multidrug eﬄux (MDR) pumps and type I secretion systems. The majority of these investigations have focused on the role played by the inner membrane transporters and the outer membrane factor (OMF), leaving the third component of the system – the  Gram-negative bacteria have to export a number of cargoes across their double membrane, which presents a formidable barrier for free diffusion of molecules. Amongst a number of secretion systems , tripartmultidrug eﬄux and type I secretion systems which draws from several different families of primary and secondary inner-membrane transporters . With the help of the so-called periplasmic adaptor proteins (PAPs), the inner-membrane transporters are linked to the outer membrane factors (OMFs) of the TolC family to create continuous conduits from the cytoplasm to the extracellular space, shown in Figure Tripartite assemblies are a heterogeneous group of Multidrug eﬄux-pumps are grouped into a number of families including the primary transporters of the ABC-family , and seWhile the roles of the OMFs and transporters have been subject of much scrutiny , the rolThe OMFs, which are the outer membrane components of tripartite pumps, are trimeric integral membrane proteins. Although TolC was identified as a colicin-susceptibility factor in the early 1970s , its assThe structure of the prototypical member of the family, TolC, was solved by Outer membrane factors have a β-barrel domain resembling the porin fold, which, unlike the canonical porins is formed by all three subunits, each of which contributes four β-strands to form a pseudo-continuous barrel. In addition, OMFs possess a unique periplasmic domain, which, like the β-barrel, is a pseudo-continuous structure built with the participation of all three protomers. Unlike the β-barrel domain, the periplasmic part is almost entirely α-helical , 2000. TIn vitro studies of the TolC channel in isolation showed that it is predominantly closed with only very short stochastic opening sequences, and exhibiting strong cation selectivity  based on perceived sequence similarity to ascribed . The latascribed . We showPseudomonas aeruginosa became the first member of the family to be crystallized (1VF7.pdb and 1T5E.pdb), followed by the structure of AcrA  and Actinobacillus actinomycetemcomitans (4DK0.pdb) were solved  has become available from a structural genomics effort. Last year saw the report of the first MFS-transporter associated PAP – EmrA from Aquifex aeolicus  it is dispensed with entirely . The coien lieu of the loop that harbors it. While the exact functional role of this domain is still to be established, analysis of mutations targeting it suggest that it has a role in stabilizing the complex assembly. This may be achieved either by interaction with the transporter, as indicated by cross-linking of the AcrA lipoyl domain to AcrB  following processing by signal peptidase 2.Cupriavidus metallidurans CH34 (E. coli) and possess a unique metal-binding β-barrel fold  family of RND transporters may also present additional N- and C-terminal domains. Involvement of the latter in metal-chaperoning function has been demonstrated in the SilB adaptor protein from ans CH34 . These drel fold . The domThe possible conformational transitions associated with ion binding in CusB have recently been revealed by modeling of the N-terminal domains based on extensive homology modeling combined with molecular dynamics and NMR spectroscopy data . DespiteFigures 4A,B show the comparison of the detailed topology of the β-barrel and the MPDs from MexA. The key conserved elements in these domains is the combination of a β strand with a helix or helical turn (shown in green) followed by a β-meander . The subsequent β-hairpin strands  and an N-terminal strand (blue) are associated with this β-meander in the complete barrel domain. In contrast the MPD has a split in the barrel giving a β-roll structure. There is a characteristic folding over of the β-hairpin  family, both of which have unique PAPs.RND HAE1 transporters are trimeric assemblies, with each protomer consisting of a transmembrane domain containing 12 transmembrane α-helices and characteristic two large hydrophilic loops that comprise the substrate-binding porter (or pore) domain and the OMF-coupling docking domain . The HMEin vivo crosslinking by As soon as the AcrB structure became available it was speculated that TolC and AcrB might come into direct contact , based oTaking into account the then-available MexA structures and this suggestion, The 3:3:3 model of Desulfitobacterium hafniense  and pull-down assays . Furthersitivity .These results, combined with the similar cross-linking studies of AcrAB , served in vivo functional and cross-linking assays, the main support for the tip-to-tip model came from in vitro structural studies of isolated components. While CusBA crystallographic complex is sometimes considered as supportive of tip-to-tip assembly due to the narrow aperture of the ring of the PAPs which may imply that there is no direct contact between the transporter and the OMF, the organization of the CusB hexamer is rather different from that in the MacA structure  MacA on which the tip region was replaced by the tip regions of the TolC α-barrel have been analyzed for structural formation with wild-type E. coli MacA by electron microscopy, and showed dumbbell-shaped structures with a central bulge  when a leaky E434K OMF mutant is used as a partner .E. coli do not seem to possess identifiable RLS sequence altogether – e.g., CvaA  and/or non-hydrolysable ATP-analogs. Given this, it has been anticipated that energy is also required for the formation of the complex, and likely conformational changes in the transporter are relayed to the OMF channel, omplexes , ITC /Ag(I) eﬄux pump CusCFBA which is composed of the OMF CusC, the RND-transporter CusA, metallochaperone CusF, and the PAP CusB. CusF and CusB have been shown by NMR spectroscopy to freely exchange Ag(I) and Cu(I) toward equilibrium in highly specific protein–protein interactions  pumps have been instrumental for establishing the active role of the PAPs in the transport process. ractions . Similar to CusF .in vivo. It has also been shown that the N-terminal domain acquires the metal from the metallochaperone (CusF) and is able to pass it on to the transporter  as identified by X-ray crystallography and NMR and by mass-spectrometry and X-ray absorption spectroscopy , creatinnsporter . In thatThe involvement of the PAPs in the cargo selectivity in the RND multidrug eﬄux pumps is less studied, but some indication of their role could be found from studies of non-cognate PAP complementation. Change of the substrate profile brought by the PAP change was clearly demonstrated by the complementation analysis of AcrA interactions with MexB . In thisAs mentioned previously, some adaptor proteins contain N-terminal membrane spanning domains, and these have been suggested to interact within the membrane with their cognate transporters . This isIt is notable, that MPDs appear exclusively in PAPs associated with RND- and ABC-transporters that feature prominent periplasmic domains. As these classes of transporters are also known to acquire their eﬄux substrates from the periplasmic space or the outer leaflet of the cytoplasmic membrane, we propose that the role of the MPDs in these systems may be associated with active cargo presentation and regulation of energy-coupling of the transport cycling.in vivo functional binding to cargo and/or transporter. Furthermore, it has been reported that mutations in the PAP HlyD affected folding of the substrate , PlnH from Lactobacillus plantarum (WP_015379778.1); SppE from Lactobacillus sakei (CAA86947.1). The list could be expanded by the related Streptococcal competence protein ComB, as well as the Enteroccocal iron ABC transporter (WP_025481776.1), and Carnobacterium maltaromaticum CbaC (AAF18149.1).While in some cases it is difficult to establish functionality of these genes, which may have been acquired via a lateral gene transfer and are dormant in the genome – e.g., in the case of nsporter . The genVirgibacillus halodenitrificans showing high similarity to the PAPs associated with MFS transporters  is more difficult to explain and requires further studies.These PAPs possess extremely large hairpin domains, which is difficult to rationalize if the only function of the hairpin is OMF-transporter bridging. However, as mentioned above it may have a role in folding the nascent polypeptide chain of the cargo. Preservation of the large hairpins in PAPs such as MdtN in Bacillus subtilis YknX (BSU14350), which seems to function with a multi-component ABC-transporter, YknZYW, involved in the resistance to antimicrobial killing factor SdpC (A rather unusual case is presented by the tor SdpC . This sytor SdpC . All thetor SdpC , which anX BSU1430, which Our structural analysis of the available PAP-transporter pairs in combination with the examination of the available biochemical evidence, leads us to believe that there is a very clear pattern of structural matching of specific PAP domain combinations to certain transporter types, summarized in Figure This pairing is far from random and most likely underlies a functional connection between the domains in question. We have identified that MPDs occur without exception in PAPs paired with transporters possessing large periplasmic domains and which are suggested to load their cargo either exclusively or preferentially from the periplasm or the outer leaflet of the inner membrane, such as RND-transporters and MacB-family of ABC transporters. There are two likely explanations for this – one is that due to purely spatial requirements the MPDs are required as “spacers” to prevent displacement of the PAP by the large transporter, which would prevent the PAP from reaching from the inner membrane to the OMF. An alternative and, in light of the increasing amount of functional data, more likely explanation is that the MPDs critically complement the cargo selection and presentation to the transporters.Similarly, the requirement for effective sealing of the transport conduit in PAP-transporter combinations, such as MFS and ABC-transporters of the Type I Secretion System where the transporter lacks a pronounced periplasmic domain, necessitates an introduction of the recognizable “hairpin extension” subdomain . The preUltimately, none of the evidence provided above proves conclusively either of the models of OMF-PAP association. However, while structural biology seemingly gives us a hint toward the tip-to-tip interaction taking place, the ability of such interactions to provide sufficient energy for stabilization of a large multi-protein assembly, which also undergoes significant conformational changes during its transport cycle, is still called into question, especially in light of the wealth of functional data.Recent data have shown the PAPs to be a versatile group of proteins which, far from being passive linkers between the OMF and the energized inner-membrane transporters actively control the tripartite complex assembly. They play an active role in assembly energetics and cargo selection and presentation, while at the same time providing highly specific differentiation between a number of homologous partners to provide high fidelity and specificity of transport.Our analysis has expanded the current understanding of structural relations of the multiple domains of these highly modular proteins, and revealed some unexpected connections linking them to other secretion systems and transporters. Finally, we have identified a pattern in the domain organization of the PAP families, which underlies their functional association with their cognate transporters.Summing up the available data also shows that despite these recent advances, the ultimate answer of the complete pump architecture remains elusive.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.