A new study uses genomics to show that fusions of plant immune receptors and other protein domains occur in significant numbers. This finding will generate many new research hypotheses and provide new opportunities for breeding resistant plant varieties.http://dx.doi.org/10.1186/s12915-016-0228-7Please see related Research article:  BMC Biology, Sarris et al. [Plants are constantly challenged by potential and actual pathogenic microbes, including viruses, bacteria and fungi, and so have evolved sophisticated molecular defense mechanisms that recognize, stop or slow down potential pathogens. Damage to host plants caused by microbial organisms is of particular importance in agriculture where plant diseases are a major cause of crop losses. Upon infection, microbes inject into host cells a set of virulence effectors tailored to target protein domains that are involved in the host plant’s basal immunity, thus disarming or disabling the plant’s immune response. When fused to intracellular immune receptors, however, the same protein domains become ‘baits’ that plants use to detect effector molecules and to activate defense responses. In a recent article in s et al.  expand oArabidopsis is a master regulator of basal defense and it is the target of several bacterial effector proteins [The immune system of plants is composed of two tiers of receptors . The firproteins . The secEffector perception by NLR immune receptors occurs either directly or indirectly. Indirect recognition can be achieved when NLR receptors monitor the activity of effectors on other host proteins involved in basal plant immunity. This is referred to as the ‘guard’ model, with the host proteins being known as ‘guardees’. A sophisticated advancement of the ‘guard’ model involves ‘decoy’ proteins. In contrast to the guardees, decoy proteins do not have a function in plant immunity; their sole role is to mimic effector targets and to trap virulence effectors. If a decoy is monitored by an NLR receptor, effector binding to the decoy will trigger a defense response Fig. .Fig. 1CoArabidopsis NLR protein RRS1, which contains an additional WRKY domain [Magnaporthe oryzae [Interestingly, decoys and the decoy-monitoring NLR proteins are sometimes encoded by the same gene; these are known as ‘integrated domain’ NLRs (NLR-IDs). Two independent studies recently described the functional analysis of the Y domain , 5. WRKYe oryzae , 7. ThesBMC Biology article, Sarris et al. [In their s et al.  made uses et al. . Of thesSome of the domain fusions have been conserved during evolution, but many probably evolved through convergent evolution, resulting in similar NLR-IDs across different plant lineages. For example, there were at least five independent events in which WRKY domains were fused to NLRs in different plant families. The observation that recruitment of certain domains into NLRs occurred independently in different plant families suggests that these fusions have a selective advantage for the plant. The fusion of integrated domains and NLRs ensures that the decoy and the corresponding immune receptor are inherited together; separate coding of the decoy and NLR bears the risk that the two genes are separated during meiosis, resulting in susceptible offspring. In addition, integrating the decoy domain into the respective NLR reduces the risk of hybrid incompatibility between different alleles of NLRs and decoys.Since some integrated domains  have previously been shown to be targets of effector proteins, the newly identified integrated domains represent new candidate effector targets. The study identified several integrated domains that have not yet been reported as effector targets. These protein domains most probably play an as yet unidentified role in plant immunity.The study of Sarris et al.  identifiAnother interesting aspect and perspective of the Sarris et al. article relates to the observation that NLR-IDs seem to work in pairs of closely linked NLRs, at least in the cases studied in detail to date . Thus, wAvr-Pia result in virulence to RGA5, indicating that these modifications in Avr-Pia disrupt recognition by RGA5 [Pi21 results in durable resistance [Pi21 gene is a susceptibility factor. This suggests that the HMA domain is a target of a pathogen effector. Therefore, integrated domains could provide information about susceptibility factors that might serve as targets to generate knock-out mutations in the host that have the potential to provide durable resistance. Novel gene-editing technologies in crops provide the tools necessary to achieve this relatively easily.As fusion domains have been shown to act as targets for pathogen effectors, each newly identified integrated domain provides a promising lead for studying how pathogens manipulate host metabolism. Thus, the work by Sarris et al. represents a rich resource for new research projects. Initial experiments might aim to identify effectors that target these newly identified domains. An important question pertaining to all resistance mechanisms relates to their durability in a crop cultivar grown on a large area and over several years. NLR-mediated immunity is often not durable because effector modification can disrupt recognition by the respective immune receptor, resulting in the emergence of new, virulent pathogen races. Because pathogens can modify their effector repertoire very rapidly, NLR genes are frequently overcome in the field, sometimes only a few years after the release of a new crop cultivar. There is no evidence that NLR-IDs provide a more durable resistance than normal NLRs. For example, the Lr10–RGA2 pair, in which an additional nucleotide-binding domain is integrated in RGA2, does not provide durable resistance; virulent races that overcame Lr10–RGA2 resistance are commonly found , 9. Simi by RGA5 . Neverth by RGA5 . Loss of


The extracellular matrix (ECM) is a defining characteristic of metazoans and consists of a meshwork of self-assembling, fibrous proteins, and their functionally related neighbours. Previous studies, focusing on a limited number of gene families, suggest that vertebrate complexity predominantly arose through the duplication and subsequent modification of retained, preexisting ECM genes. These genes provided the structural underpinnings to support a variety of specialized tissues, as well as a platform for the organization of spatio-temporal signaling and cell migration. However, the relative contributions of ancient versus novel domains to ECM evolution have not been quantified across the full range of ECM proteins. Here, utilizing a high quality list comprising 324 ECM genes, we reveal general and clade-specific domain combinations, identifying domains of eukaryotic and metazoan origin recruited into new roles in approximately two-third of the ECM proteins in humans representing novel vertebrate proteins. We show that, rather than acquiring new domains, sampling of new domain combinations has been key to the innovation of paralogous ECM genes during vertebrate evolution. Applying a novel framework for identifying potentially important, noncontiguous, conserved arrangements of domains, we find that the distinct biological characteristics of the ECM have arisen through unique evolutionary processes. These include the preferential recruitment of novel domains to existing architectures and the utilization of high promiscuity domains in organizing the ECM network around a connected array of structural hubs. Our focus on ECM proteins reveals that distinct types of proteins and/or the biological systems in which they operate have influenced the types of evolutionary forces that drive protein innovation. This emphasizes the need for rigorously defined systems to address questions of evolution that focus on specific systems of interacting proteins. The emergence of complex, multicellular organisms required innovation of biological processes facilitating a variety of new structures and functions. In vertebrates, distinguishing features include specialized matrices such as cartilage, tendons, bones and teeth, and a pressurized vascular system. Technological advances in genetics and proteomics, begun over the past decade, continue to provide insights into the composition and organization of these processes . At the The human ECM comprises approximately 324 proteins that self-organize into a complex array of fibers and ancillary proteins, providing essential scaffolding for arranging cells into tissues . The ECMCiona, the authors concluded that the increased complexity observed in vertebrate ECM proteins was largely driven by large-scale duplications of preexisting genes, , few stg genes, . Subsequg genes, . Indeed,g genes, . To dateg genes, . IncreasAt the whole-proteome level, recent studies suggest that domain gain and loss events, occurring most commonly at the C-terminus, dominate and that transposition, exon shuffling, and recombination appear to play only minor roles . HoweverCharacterized by large, multidomain proteins with distinct physical and functional characteristics, the ECM represents an ideal system to explore how patterns of domain evolution may vary for a given class of functionally related proteins. Here, we present a systematic study of domain gain, loss, and rearrangement events that have contributed to human ECM innovation and highlight how the evolution of domain architectures has resulted in the emergence of vertebrate innovations.supplementary spreadsheet S1, Supplementary Material online). To identify orthologs of these components, protein sequences for 131 fully sequenced eukaryotic genomes  were obtained as previously described  which would otherwise inappropriately skew domain frequencies. This was done by iteratively removing duplicated domains until only a single (A-A) pair remained. Proteins were then classified as either single or multidomain depending on their domain content. Domains were classified as appearing in single or multidomain proteins or both. Domain enrichment was determined using the hypergeometric test with false discovery rate.Domains found in human ECM reference sequences were compared against the full proteome of each species. As above, domain architectures were preprocessed to remove tandem duplications which would otherwise inappropriately weight domain frequencies. Domain pairs refer to immediately adjacent domains. Domain pairs were defined in the N- to C-terminal direction and reverse orientations were considered to be unique . A domain or domain pair was considered to be conserved if it appeared in at least one of the proteins in a given species. Patterns of domains and domain-pair conservation were hierarchically clustered using Cluster 3.0 grouping them into blocks representing similar conservation profiles.The domain arrangement for each reference sequence was compared with each of its corresponding orthologs . Tandem duplications were included in this analysis. Domain gains, losses, gains and losses of domain repeats, and complex rearrangements, were identified based on the most parsimonious change . Complex rearrangements were those involving combinations of gain and loss events. Domain architecture conservation profiles were hierarchically clustered using Cluster 3.0.supplementary spreadsheet S1, Supplementary Material online). Enrichment for late domain gains was assessed via hypergeometric test with Bonferroni correction.We assessed the total number of Pfam-A domains in each human ECM protein and the relative contribution of “first instance domains” (FIDs) and “non-FIDs” (NFIDs) using a simple counting method. The first occurrence of a domain was counted as an FID and any subsequent occurrence of the same domain as a NFID. By definition, the occurrence of NFIDs is highly correlated with tandem repeats and for our purposes we assume they are equivalent with the caveat that some NFIDs could be the result of two or more occurrences of the same domain separated by one or more other domains. For example, an alternating pattern such as ABABA would be recorded as having three NFIDs and algorithmically we would not distinguish this from AAABB—which has bona fide tandem repeats. To mitigate this source of error, we only considered proteins with a larger number of NFIDs than FIDs to contain tandem domain repeats. To quantify the association between late domain gains and NFIDs, we classified proteins according to their domain architecture conservation patterns as having undergone prevertebrate (“early”) or postvertebrate (“late”) domain gains and whether or not those gains were associated with tandem repeats (NFIDs > FIDs) . The resulting short strings of letters were rapidly aligned using a custom Perl script implementing the BioPerl SimpleAlign module with ClustalW as the alignment method. The ktuple parameter was set to 2 (“ktuple” ≥ 2) and we used an identity substitution matrix (“matrix” ≥ “ID”).P value represents the number of times out of 10,000 simulations that a given pair was found as frequently as or more frequently than in the real proteome by chance alone. The corresponding z-scores were used to weight the edges of the subnetwork of all human pairs and clustered using the Markov Cluster Algorithm (MCL)  arrangement of domains occurring in our reference proteins, we constructed a list of domain pairs occurring in the human ECM and their orthologs from each species and used them to construct a network of domain adjacency. The statistical significance of domain pairs was determined by comparing the frequency of each pair in the real human proteome with that of 10,000 simulated proteomes (“Bootstrap resampling”—see below). The reported hm (MCL)  with a dmappings  were usemappings . Overlapmappings .We adopted the weighted bigram frequency used by Basu  as a meaThe bigram frequencyt is the number of distinct domain types. i and i in the genome, calculated as i and N is the total number of domains detected in the given genome:where Note ated set . To deteated set , we defiated set .n proteins (support = n). For example, the sequential pattern  can be found in proteins with domain architectures: , , , . It should be noted that the pattern can be discontinuous as long as the ordering is preserved . We used PrefixSpan  representing the presumed orthologs of the reference sequence . Because the presence of highly related sequences would tend to inflate the occurrences of patterns found in, for example, similar splice variants, the sequences were prefiltered to remove redundant sequences (above 90% similarity) prior to pattern analysis. Thresholds of 90%, 95%, and 97% are commonly used to filter out redundant sequences in taxonomic studies  in the real proteome. To populate each simulated proteome, we constructed a set of “pseudo-proteins” by randomly selecting domains (without replacement) from a pool reflecting the domain frequencies of the real human proteome. As domain pairs were created in the growing pseudo-proteins, the pair was propagated across eligible pseudo-proteins a random number of times before individual domain selection resumed. Individual domains propagated as pairs continued to be removed from the domain pool during this process. If the availability of either domain in the pair was exhausted in the domain pool or if the random propagation limit for that pair was reached, the propagation of that pair ceased and individual domain selection was resumed. This process was continued until all domains in the pool were exhausted. For domain pairs, simulated proteomes were constructed using domain frequencies corresponding to the preprocessed domain architectures of human ECM proteins , and the random placement of domains was constrained so as to prevent the random creation of tandem domain repeats. Random domain pairs resulting from these simulations therefore reflected the conditions used to evaluate domain pairs in the real proteome. As sequential pattern mining was performed on unprocessed domain architectures, simulated proteomes created for assessment of HOODs were not constrained in this way.P < 0.05, Hypergeometric test with false discovery rate correction). Among the enriched domains, 35 appear exclusive to ECM proteins (A) and supplementary spreadsheets S3–S6, Supplementary Material online). To assess the origins of these 144 domains, we examined their occurrence within the genomes of 131 fully sequenced eukaryotes. Three distinct groups are apparent, corresponding to domains with eukaryotic, metazoan, and vertebrate origins (B) and supplementary spreadsheet S7, Supplementary Material online). Approximately four out of every five ECM domains (79.2%) are conserved but specific to choanoflagellates and metazoans, suggesting that the emergence and subsequent evolution of the ECM involved the recruitment of both a limited number of ancestral, premetazoan domains (20.8%), together with the innovation of a larger number of novel domains, prior to the branching of the various metazoan lineages.In this section, we establish the domain repertoire of ECM proteins and, examine their origins and the contribution of novel domains to novel ECM functions. Using domain definitions as provided by the curated Pfam-A resource , we find that of 4,243 domains in the human proteome, 144 ( ∼ 3.4%) are associated with ECM proteins, of which 101 are significantly ECM-enriched (proteins A and sup origins B and supB) are several that are ECM-enriched as well as one apparently exclusive to the ECM in humans; MFAP1_C (PF06991) is a conserved C-terminal domain of human MFAP1 (microfibrillar-associated protein), an important component of elastin-associated microfibrils . The latter are enriched in ECM-exclusive domains associated with proteins involved in biomineralization   . Correspfig. 1PF07175) , enamel PF07175) , and bonPF07175) . Given tPF07175) .Having established a list of ECM-associated domains, their origins and the role of novel domains in establishing new functions, in the next section we consider the tendency for domains in the ECM to form stable combinations with other domains (a trait referred to as “promiscuity”), and its potential role in the context of a previously defined network of ECM protein interactions .supplementary spreadsheet S8, Supplementary Material online). Domains were grouped into three age categories based on their conservation across the Eukarya (E), Metazoa  or, Vertebrata (V). Of the three age categories, vertebrate-derived domains were characterized by domains of low frequency and promiscuity while metazoan and eukaryotic domains, included domains with higher frequency and/or higher promiscuity .Previous studies have found extracellular proteins possess a relatively high incidence of promiscuous domains relative to most other proteins , 2009. Tsupplementary fig. S3 and spreadsheet S10, Supplementary Material online). Of the 124 ECM-associated domains that appear in multidomain proteins, we found 38 (30.6%) could be defined as highly promiscuous which represents a significant enrichment compared with non-ECM associated domains at 224/2,282 (9.8%) . Overall, as the remaining 86/124 ECM-associated domains  were of low promiscuity, we did not find a statistically significant association between promiscuity and domain age . Given the sigmoidal distribution of promiscuity scores , we conclude that promiscuity is not a general characteristic of ECM domains, but rather, restricted to a small subset of ECM domains in which high promiscuity likely plays some functionally important role.On the basis of the top ten percentile of promiscuity scores , we defined a weighted bigram frequency of 0.0021 as a cutoff for “high promiscuity” . We found that hubs (defined as having a node degree ≥5) are significantly enriched in highly promiscuous domains . More intriguingly, network hubs are significantly enriched in structural proteins  and these structural proteins, which accounted for 60/173 proteins, were significantly enriched in promiscuous domains .Domains, via protein binding, play a fundamental role in the organization of protein-interaction networks with multidomain proteins often representing “hubs” , b. To eConsistent with the proposed role of domains in network connectivity, the three highest promiscuity domains in humans . Where we observed changes in human ECM protein domain architecture, domain gain was found to be more common than domain loss (28.9% vs. 5.2% of orthologs) .To obtain a global overview of domain gain and loss events, we generated domain architectures for orthologs of ECM proteins identified in 131 fully sequenced eukaryotic genomes. We found that across 33 deuterostome genomes, 62.8% of ECM orthologs had identical domain architectures, suggesting selective pressure to maintain architecture . Thus, as for other systems , whereas more complex changes (combinations of gain and loss events) were relatively rare (2.8% of orthologs), as consistent with previous global domain studies . As expe systems , domain P < 0.05, hypergeometric test with Bonferroni correction), whereas prevertebrate domain gains corresponded with low numbers of domain repeats . Interestingly, repeats were enriched within structural proteins . Despite an appearance that recent domain gains occur solely at the divergence of primates from other mammals as might be inferred for groups 4, 5, 7, 8, and 9 in supplementary spreadsheets S13–S17, Supplementary Material online), revealed that gains (as well as losses) occur throughout the deuterostome lineage. For example, perlecan (HSPG2) is composed of a conserved core of Laminin B and Laminin EGF domains, supplemented with increasing numbers of I-set domains. Fibulin-2 (FBLN2) has acquired an ANATO domain (PF01821) initially detected in fish and subsequently duplicated in mammals. Finally, hepatocyte growth factor activator (HGFAC) demonstrates a mosaic of domain gains and losses throughout the deuterostome lineage.Proteins comprised of a majority of NFIDs, associated with tandem domain repeats , were enriched for recent (vertebrate) domain gains  , we findP < 0.01 χ2 test; supplementary fig. S5, Supplementary Material online). Of the 144 ECM domains, 62 (43%) are found exclusively in multidomain proteins, whereas an additional 67 (46.5%) are found both in single and multidomain proteins. This compares with 1,665 (39.2%) and 775 (18.3%), respectively, for non-ECM domains. In general, ECM domains rarely occur only in single-domain architectures (15/144 [10.4%]) though when they do, tend to be of recent origin. For example, 5/10 of the highlighted, tetrapod-specific domains are found exclusively in singleton architectures (B) though the other five occur exclusively in multidomain proteins. Together these findings suggest that when new ECM domains evolve, they tend to be rapidly integrated into multidomain architectures.The recruitment of additional domains to the ECM, in addition to providing intrinsic functionality, offers the potential to derive new functions through combining with other domains. Compared with other human domains, ECM domains are more often associated with multidomain proteins . Although 28 of 144 domains precede the emergence of metazoans, with a single exception involving SMC3, all domain pairs appear unique to metazoans. SMC3, a component of the cohesin complex, is involved in spindle pole assembly. However, posttranslational addition of chondroitin sulfate gives rise to the secreted proteoglycan bamacan, an abundant basement membrane protein . Of the 90 vertebrate-specific domain pairs 34 (37.8%) involved at least one vertebrate domain of which, only 7 (7.8%) were comprised of two vertebrate domains (V:V). In contrast, 75% (67/90) of vertebrate-specific pairs involve at least one domain of metazoan origin . Noteworthy, eukaryotic domain pair combinations (E:E) were significantly enriched , highlighting the capacity of even ancient  domains to contribute to new functional contexts. These findings suggest that emergence of novel ECM domain combinations, which help drive lineage specific innovations, are not dependent on domains of recent origin. Rather, they arise through sampling existing domains irrespective of their age. We next explore whether related domain architectures comprise functionally relevant modules within the ECM network.We next examined the source of domains driving new combinations, categorizing domains as eukaryotic (E), metazoan (M) or vertebrate (V) in origin . In geneTo examine functional relationships between domain pairs and how these relationships have changed across different lineages, we constructed a domain adjacency network comprising 117 nodes (domains) connected by 201 edges  represenP < 0.005—see Materials and Methods, Clustering the network , revealed 15 putative domain modules consisting of three or more domains. Exploiting a previously generated set of PPI-based module annotations  togetherWithin these modules we observe expansion of vertebrate-specific pairs, extending the variety of ECM domain architectures conserved with protostomes. For example, module 3 (cell adhesion) transitioned from a core comprised of EGF and laminin domains to a larger module in which the EGF domain serves as a central hub for a variety of ECM-based architectures. Vertebrate-specific domains such as FN1 and FN2 form further connections  associated with matrix remodeling and protein binding. Vertebrate-specific domain pairings are also responsible for the emergence of new modules .In addition to novel links, the network displays rewiring of domain relationships across evolution. For example, modules 2, 7, 8, 11, and 14 are generally well-conserved with arthropods but many domain relationships are not present in basal metazoans or nematodes. Conversely, several domain pair relationships appear to have been lost in arthropods  indicating either sequence divergence, loss of function provided by the domain combination or recruitment of additional proteins to replace the function. Apparent losses include a number of domain combinations involving Kazal, Laminin, and CUB domains. Importantly, it is the unique combination of these domains that has been lost rather than the domains themselves. Serine protease inhibitors (Serpins) in which kazal domains are found are involved in protection against autophagy in metazoan digestive systems . Althougsupplementary fig. S7 and spreadsheet S21, Supplementary Material online). Further, since a diversity of compatible domain architectures exist for many proteins, individual species represent only a fraction of the possible inventory of domain pairs consistent with a particular function.Among orthologs of human ECM proteins are a large number of poorly conserved, species-specific domain pairs (305/510), suggesting the recruitment or shuffling of domains  . In the P < 0.05, Bootstrap resampling; supplementary spreadsheet S22, Supplementary Material online). Among 490 patterns which were most significant (P < 0.005), 256 were comprised of four domains while 150 were comprised of three domains . For each pattern, we examined its conservation across ten representative metazoans (A) and supplementary spreadsheet S24, Supplementary Material online).To identify recurring HOOD architectures within the human ECM we used PrefixSpan , a sequeetazoans A and supD. rerio ortholog.Although there were a number of lineage-specific pattern losses in worm, only a single fly specific loss was observed. The former include patterns involving domains TIL, C8, and VWD, which occur in the human proteins: VWF, TECTA, OTOG and two mucins, MUC5A and MUC6. Consistent with the missing domain patterns, despite worm possessing homologs for at least some of these proteins, the C8 domain appears to have been lost within the nematode lineage . InteresB) and supplementary spreadsheets S25–S29, Supplementary Material online). We identified 13 pattern groups (PGs) whose members share similar domain composition. The relative frequency of specific domains within each PG is shown in figure 6C. Rather than PGs being composed of a mosaic of all conservation groups, each PG reflected a limited set of conservation groups. For example, PGs 1, 5, and 12 are composed of patterns associated with vertebrates, whereas patterns in PG 8 are associated with protostomes and deuterostomes. The four largest PGs are associated with a larger range of conservation groups, suggesting expansion of patterns for these groups within distinct lineages. For example, PG 2 includes patterns associated with basal metazoans, together with those that later emerged in the vertebrate lineage, highlighting the ability of apparently fixed domain architectures to acquire new domains that may help drive lineage-specific adaptations.To examine potential overlap in patterns arising from similar domain architectures, we constructed an enrichment map in which nodes represent discrete patterns and links indicate sharing of common domains B and supsupplementary fig. S8, Supplementary Material online). This correspondence suggests that HOODs, like their component pairs, cluster around common functional themes, validating the inherent assumptions underlying prior studies of domain pairs. Our study of domain patterns has revealed that HOOD architectures exist and are conserved across multidomain ECM proteins.Mapping HOODs onto the network of domain adjacency revealed a correspondence between HOODs and modules defined on the basis of domain pairs  of all human domains were considered to be vertebrate in origin . At the Although we acknowledge that these findings may be impacted by our reliance on domain detection algorithms that may fail to identify divergent members  , there iRecent studies probing the mechanisms of de novo domain creation and recombination suggest that the majority of vertebrate-specific  domains first emerged as single-domain proteins . HoweverProteins involved in extracellular structures were previously found to be enriched in promiscuous domains . HoweverP < 0.0003). Such preferential recruitment might be explained through the potential impact of novel and consequently disordered domains disrupting existing biological functions; established domains, by their very nature, being less likely to cause such disruptions  vertebrate-specific domain pairs involved at least one domain of more ancient origin, suggesting that ECM innovation in vertebrates was largely driven through the generation of novel domain combinations. Furthermore, while the ECM is enriched in vertebrate-specific domains, pairwise patterns of domain age were consistent with a random model of domain propagation: New domains participate in a continuous process of random domain assortment and new functions arise as much from novel domain combinations as from new domains themselves . As an aruptions .The dependency on new domains to provide novel innovations in vertebrates is further minimized through the reuse of domains either through tandem duplications or reordering resulting in new functions . Domain supplementary fig. S7 and spreadsheet S21, Supplementary Material online).To further elucidate origins of domain-pairs, we applied a network-based approach to identify domains of eukaryotic and metazoan origin recruited into vertebrate-specific combinations. This network revealed a surge of vertebrate innovation, driven by the acquisition of novel domain arrangements. Although many aspects of vertebrate skeletal evolution remain unclear , the ideAn important contribution of this study is the application of sequential pattern mining as a method to investigate HOOD architectures and their conservation. Vogel et al. previously suggested that contiguous two and three domain combinations can result in evolutionary conserved three-dimensional structures, termed “supra-domains” . SubsequThe emergence of metazoan life involved the innovation of a large number of novel ECM domains. Vertebrates subsequently exploited these domains through the generation of novel domain combinations to yield systems such as a biomineralized skeleton, a network of elastic fibers and a variety of organ systems supported by an array of specialized matrices.Consistent with the current consensus model of domain evolution in which the accretion of domains and domain combinations and selective losses lead to increasingly complex, multidomain architectures, our study has shown that the major driving force for human ECM evolution has been the innovation of novel domain combinations, rather than novel domains. These domain combinations, which we have extended to include HOODs, have evolved to support the unique, dual roles of the ECM in structure and signaling. We identified specific domains of eukaryotic, metazoan, and vertebrate origin which, independent of their age, gave rise to clade-specific domain combinations. However, the prevalence of older domain pairs among the large number of vertebrate-specific pairs suggests the mechanism of novel domain acquisition by ECM proteins may be different than other proteins; dominated by domain fusion and the recruitment of additional domains to existing architectures rather than the de novo creation of independent domains. This study reveals that the organization of the matrix is mediated by a relatively small number of highly promiscuous domains which are enriched in structural proteins that have emerged as the basis for network hubs. Together these results emphasize the importance of validating models derived from global domain analyses, through focusing on specific biological processes and/or specific classes of proteins.Supplementary figures S1–S8 and excel spreadsheets S1–S29 are available at Genome Biology and Evolution online .


The α + β barrel superfamily of the ferredoxin-like fold consists of a functionally diverse group of evolutionarily related proteins. The barrel architecture of these proteins is formed by either homo-/hetero-dimerization or duplication and fusion of ferredoxin-like domains. Several members of this superfamily bind heme in order to carry out their functions.We analyze the heme-binding sites in these proteins as well as their barrel topologies. Our comparative structural analysis of these heme-binding barrels reveals two distinct modes of packing of the ferredoxin-like domains to constitute the α + β barrel, which is typified by the Type-1/IsdG-like and Type-2/OxdA-like proteins, respectively. We examine the heme-binding pockets and explore the versatility of the α + β barrels ability to accommodate heme or heme-related moieties, such as siroheme, in at least three different sites, namely, the mode seen in IsdG/OxdA, Cld/DyP/EfeB/HemQ and siroheme decarboxylase barrels.Our study offers insights into the plausible evolutionary relationships between the two distinct barrel packing topologies and relate the observed heme-binding sites to these topologies.The online version of this article (doi:10.1186/s12859-016-1033-6) contains supplementary material, which is available to authorized users. The “dimeric α + β barrel superfamily”  is an evolutionarily conserved group of protein families found in all three kingdoms of life –4. ProteHeme, a metal-porphyrin compound, is associated with at least six different families of this superfamily , 12, 13.The proteins of this superfamily are made up of two domains that pack together as a closed barrel (SCOP identifier 54909) Fig. . Each doWhile many studies describing the structural and biochemical characterization of these proteins are available, an evolutionary basis for the emergence of different heme-binding sites in the ferredoxin α + β barrel and rationale for the apparently different barrel packing is not available. Here, we present a comparative view of the different heme-binding ferredoxin α + β barrel families based on evolutionary considerations and identify two different barrel packing modes of the ferredoxin-like domains. The evolutionary scenarios that likely resulted in the topologically different packing modes are discussed. Further, we provide a comparative topological- and structural view of the various heme-binding sites in the versatile ferredoxin α + β barrel. It is particularly interesting to observe how the ferredoxin-like protein domain has evolved to utilize different interfaces for performing varied functions around the heme moiety. This work could be potentially useful for future protein structural and evolutionary studies and in understanding the spatial packing of protein domains.Hydrogenobacter thermophilus TK-6 and Desulfovibrio desulfuricans have been determined recently [H. thermophilus TK-6 siroheme decarboxylase (PDB identifier 4CZC) and two ferredoxin-like domains from different chains in D. desulfuricans siroheme decarboxylase (PDB identifier 4UN1). However, in the D. desulfuricans siroheme decarboxylase the monomeric ferredoxin-like domains are products of evolutionarily related genes and thus, the barrel is a result of heterodimerization of ferredoxin-like domains, unlike the homodimeric IsdG barrel.The structure of the ferredoxin α + β barrel superfamily contains two ferredoxin-like domains that may be present on a single polypeptide either as tandemly duplicated and fused domains or as independent domains on two different polypeptides. Further, the barrel may involve packing of identical domains i.e. same gene products (homodimerization) or non-identical domains i.e. duplicated but not fused gene products (heterodimerization) or of tandemly duplicated and fused domains. The arrangement of the ferredoxin-like domains in the heme-binding families of this α + β barrel superfamily is summarized in Table recently , 24. Theviz., members of the ABM and Cld/DyP/EfeB/HemQ families. Type-2-packing is mostly confined to the OxdA family members and a group of non-heme-binding ferredoxin α + β barrel proteins typified by the hypothetical protein YqjZ (PDB identifier 2GO8)  Fig. . Interes0, which would result in a Type-2 packing , and the C-terminal domains of the OxdA (PDB identifier 3A16_A) and the Cld/DyP/EfeB/HemQ (PDB identifier 3NN1_A) barrels  could be manual superimposed with an RMSD of 1.16 Å over 50 pairs of Cα-atoms, while the C-terminal domain of OxdA (PDB identifier 3A16) and Cld (PDB identifier 3NN1) could be superimposed with an RMSD of 2.63 Å over 53 pairs of Cα-atoms.The second heme-binding site, structurally distinct from IsdG and OxdA , is seenels Fig. . The IsdHomologs of IsdG, OxdA, and Cld/DyP/EfeB/HemQ were obtained by sequence similarity searches initiated using JackHMMER . A multiB. subtilis (PDB identifier 4FVC_A), heme is seen to bind to a nearby Tyr residue . Further, duplication and divergence would have likely resulted in non-identical ferredoxin-like domains that could heterodimerize. The heterodimerization could also be of either type, but the structure for only the Type-1 packing mode is known currently . Likewise, a tandem duplication and fusion of the ferredoxin-like domains would have resulted in a barrel formed from two domains present on a single polypeptide. However, these could again pack in the two aforementioned modes dictated possibly by the flexibility of the linker region between the domains . The presence of tandemly duplicated ferredoxin-like domains is likely to have eased the evolutionary pressure for heme binding on both the domains and thus, only one domain retained the heme-binding histidine . The N-terminal domain, thereafter, likely served a mere structural role in barrel packing and is even seen to have lost some of the secondary structural elements of the ferredoxin α + β barrel scaffold, for example in the HmoB protein. The structure of HmoB  reveals the degradation of the α-helix, equivalent to the heme-binding α-helix of the ferredoxin-like domain of evolutionary-related IsdG proteins, in its N-terminal domain  finds matches to uncharacterized HTH-type transcriptional regulator PH1519  and uncharacterized HTH-type transcriptional regulator PYRAB06490 .A vivid example of the independent emergence of heme-like-moiety-binding is that of siroheme decarboxylase, an enzyme of the alternative heme synthesis pathway which catalyzes the conversion of siroheme to 12, 18 didecarboxysiroheme –25. EvidD. desulfuricans (PDB identifier 4UN1) and from β6 of the barrel of H. thermophilus (PDB identifier 4CZC). H. thermophilus siroheme decarboxylase (PDB identifier 4CZC) as mentioned above has duplicated and fused ferredoxin-like domain (single polypeptide) and thus, the β6 of H. thermophilus (PDB identifier 4CZC) is equivalent to β2 of an individual ferredoxin-like domain of D. desulfuricans (PDB identifier 4UN1). We observe that in each of these proteins, both the ferredoxin-like domains of the barrel possess a histidine residue on the β2 strand. A superimposition of the siroheme decarboxylase structures from D. desulfuricans and H. thermophilus, reveals that the bound siroheme molecules are located at spatially distinct locations and conformation changes to a few side-chains of the siroheme moiety could allow the ferredoxin α + β barrel to accommodate two siroheme molecules simultaneously, without steric clashes.We refer to the mode of siroheme-binding in siroheme decarboxylase  as the third mode of heme binding observed in ferredoxin α + β barrel families, as unlike the aforementioned families which bind heme in the cleft of the ferredoxin-like domain, siroheme decarboxylase binds siroheme in the barrel cavity formed by the two ferredoxin-like domains Fig. . The hisWe compared the various modes in which the ferredoxin-like domains may pack in the heme-binding ferredoxin α + β barrel families. We find that the ferredoxin-like domains may pack in two modes to form the barrel, which differ in the orientation of the constituent domains. Of the 18 families of ferredoxin α + β barrel superfamily (as classified by Pfam), Type-1 packing mode is seen in proteins belonging to 17 families of which proteins from the ABM, Cld, DyP and AsnC families are known bind heme and Type-2 is observed only in the heme-binding OxdA family proteins and the non-heme YqjZ-like proteins of the ABM family. Our analysis helps rationalize the underlying relationships between the different heme-binding sites and barrels of IsdG, OxdA and Cld/DyP/EfeB/HemQ proteins.Representatives of the ferredoxin α + β barrel superfamily were selected from the SCOP and PDB databases after considering the following criteria. The presence of bound heme/heme-like ligand molecules to ferredoxin-like domain(s) was the primary criteria followed by a preference for structures without disordered regions and/or non-natural mutation(s) with maximum sequence length coverage. X-ray diffraction structures were preferred over NMR structures and the one with the highest resolution was selected. Representative structures that were selected included IsdG (PDB identifier 2ZDP_A), MhuD 3HX9_A), chlorite dismutase (PDB identifier 3NN1_A), DyP-type peroxidase (PDB identifier 3VXJ_A), EfeB (PDB identifier 3O72_A), HemQ (PDB identifier 1VDH_A) and aldoxime dehydratase (PDB identifier 3A16_A). Siroheme decarboxylase, which binds siroheme and whose structure was characterized recently, was also included in the representative dataset  along with the representative of AsnC transcription regulator family (PDB identifier 2E7X_A). Other related proteins were obtained by sequence and structural similarity searches as outlined below  and the sequences from the UniProtKB resource (http://www.uniprot.org/).All the structural data used for this study is freely available from the Protein Data Bank (


To this end, the electric-field-induced and stress-induced strain curves of KNN-based ceramics over a wide compositional range across PPT were characterized. It was found that the coercive field of the virgin samples was highest in PPT region, which was related to the inhibited domain wall motion due to the presence of nanodomains. However, the coercive field was found to be the lowest in the PPT region after electrical poling. This was related to the irreversible transformation of the nanodomains into micron-sized domains during the poling process. With the similar micron-sized domain configuration for all poled ceramics, the domains in the PPT region move more easily due to the additional polarization vectors. The results demonstrate that the poling process can give rise to the irreversible domain configuration transformation and then account for the inverted macroscopic piezoelectricity in the PPT region of KNN-based ceramics.Miniaturization of domains to the nanometer scale has been previously reported in many piezoelectrics with two-phase coexistence. Despite the observation of nanoscale domain configuration near the polymorphic phase transition (PPT) regionin virgin (K In particular, -modified textured KNN (KNN-LTS) ceramics have been reported to have piezoelectric properties (d33 ≈ 416 pC/N) comparable to those of some commercial PZT ceramics3578101112Recently, (Ks d33 ≈ 46 pC/N co1−xTix)O310141/3Nb2/3)O3-PbTiO31/3Nb2/3)O3-PbTiO31/2Na1/2)TiO3-xBaTiO3171920213- and KNN-based ceramics. In (1−x)Ba(Zr0.2Ti0.8)O3-x(Ba0.7Ca0.3)TiO3 (BZT-xBCT) ceramics, the ferroelectric phases converge near the line of Curie temperatures. The anisotropic energy contribution in the phase convergence region reduces sharply and even vanishes at a certain composition, as estimated by phenomenological theory23253-KNbO3 phase diagramAs compared to the micron-sized domains in the single-phase region, the nanodomains have different response mechanisms to the external loads. A lower-symmetry monoclinic structure was detected in the lead-based and lead-free materials with MPB composition, such as Pb(Zrin situ transmission electron microscopy (TEM) on bulk KNN-based ceramics revealed that the domain morphology changed significantly during poling process, leading to the noticeable variation of macroscopic piezoelectricityA miniaturized nanoscale domain morphology was previously observed in the virgin state of KNN-based ceramics with two-phase coexistence and was believed to be responsible for the enhanced quasistatic piezoelectric coefficient of the poled ceramics80.52K0.4425Li0.0375)(Nb0.92-xTaxSb0.08)O3  ceramics measured at a frequency of 1 kHz during heating with a rate of 2 °C/min is shown in TO–T), while the one at higher temperature to the tetragonal-cubic transition . The compositional dependences of TC and TO–T are summarized in the inset of TC and TO–T decrease almost linearly with increasing Ta content and TC lies above room temperature for all investigated compositions. While the ceramics with x < 0.07 have an orthorhombic structure at room temperature, the crystal structure changes to tetragonal for the specimens with x > 0.07. A two-phase coexistence zone composed of orthorhombic and tetragonal ferroelectric phases is formed at room temperature for the composition with x = 0.07. The x = 0.03 specimen is found to have an orthorhombic structure at room temperature, a two-phase coexistence region at about 50 °C, and a tetragonal structure above 50 °C up to 150 °C, as determined by in-situ X-ray diffraction (XRD) measurement  images of the KNN-LTS ceramics shown in The relative density, quasistatic piezoelectric coefficient rend of  . The shoTC, while the rapid decrease of the hysteresis above 200 °C indicates vanishing of the ferroelectric properties. The The bipolar electric-field-induced polarization and strain hysteresis loops, and unipolar electric-field-induced strain curves at 4 kV/mm and 1 Hz for three representative poled KNN-LTS compositions were measured in a broad temperature range, as displayed in Room temperature TEM bright field images of the virgin KNN-LTS ceramics are shown in 318The electric-field-and stress-induced strain curves from the virgin state were measured in order to study the domain behaviour of the virgin KNN-LTS ceramics under the external loads. The coercive field is directly related to the domain switching possibility during the application of an external load33 samples . Both pa−3%∙°C−1 from 25 °C to 50 °C and 0.9∙10−3%∙°C−1 from 50 °C to 100 °C, respectively. The sharp reduction of stress-induced On the other hand, the electric-field- and stress-induced This domain behaviour in the virgin samples can be elucidated from the view of the domain configuration. The evolution of the domain configuration for two-phase coexistence ceramics and the pure orthorhombic or pure tetragonal ceramics is displayed in the schematic diagrams of Fig. 11a2,b2−3 kV/mm∙°C−1 from 25 °C to 50 °C and 1.8∙10−3 kV/mm∙°C−1 from 50 °C to 75 °C, respectively. These results illustrate that the The electrical properties and domain behaviour of the poled samples are different from that of the virgin samples. The T region . As evidTO–T indicates a short range order with random nanodomains, corresponding to the TEM images in 1−xTix)O3 based1/3Nb2/3)O3-PbTiO3 based411/2Na1/2)TiO3-BaTiO3 based3 based44The electro-mechanical properties can be elucidated from the domain behaviour in the poled samples, which is closely related to the domain configuration. In many lead-free and lead-based piezoelectric ceramics, it is verified that regular ferroelectric domains correspond to minimum frequency dispersions in dielectric permittivity while nanodomains lead to strong frequency dispersions133738The Li, Ta, Sb-modified KNN ceramic samples with different Ta contents were prepared using the conventional mixed-oxide method and the electro-mechanical properties were investigated. A significant variation in the electrical and mechanical properties was observed before and after the poling process. The highest coercive field and lowest field-induced strain were obtained in the virgin samples with PPT composition, while the opposite trend was detected after the ceramics were poled. The electro-mechanical properties could be elucidated from the differences in the domain configuration. In the virgin samples, the micron-sized domain structures are dominant in the pure tetragonal or pure orthorhombic ceramics, while nanoscale domain structures exist in the two-phase coexistence ceramics. The miniaturized domain induced high domain wall density leads to a larger domain wall energy barrier in the PPT region, making the domain wall mobility difficult. Therefore, the coercive field of the virgin samples reaches the maximum in the proximity of the PPT region. During the poling process, the nanodomains transform irreversibly into micron-sized domain structure and thus all the poled KNN-LTS ceramics have similar micron-sized domain configuration. The domains in the PPT region move more easily due to the additional polarization vectors. Therefore, the coercive field of the poled ceramics has a minimum value for the PPT composition.0.52K0.4425Li0.0375)(Nb0.92−xTaxSb0.08)O3  powders were prepared using the conventional mix-oxide method. Analytically pure powders of Nb2O5 , Ta2O5 , Sb2O3 , Na2CO3 , K2CO3 , and Li2CO3  were utilized as starting materials. The alkali carbonates were first dried at 80 °C and then homogenized with other oxides in a planetary mill for 8 h according to the nominal stoichiometric composition using ethyl alcohol as medium. The powder mixtures were calcined twice at 850 °C for 5 h with intermediate milling. Subsequently, they were milled, dried, and sieved. The powders were then compacted into pellets with a diameter of 10 mm and a thickness of 1 mm by uniaxial pressing in a stainless-steel die using Polyvinyl Butyral as binder. The specimens were sintered at 1090 °C for 2 h with a heating/cooling rate of 3 °C/min in a sealed crucible to minimize the evaporation of alkaline metals. The density of the samples was determined by the Archimedes method. Scanning electron microscopy  was used to examine the microstructure of the sintered ceramics. The crystalline structure of the crushed sintered ceramics was determined by XRD  with Cu Kα radiation. After mechanical polishing and ion milling of ceramics, domain structure was studied using the TEM, carried out on electron microscope TECNAI G2 20  at 200 kV.. Dielectric permittivity δ at 1 kHz and the planar electromechanical coupling factor kp at room temperature were measured using a capacitance meter . The temperature-dependent dielectric properties of the poled and virgin samples were measured from −50 °C to 300 °C with a heating rate of 2 °C/min . The electric field-polarization and electric field-strain curves were measured from room temperature to 250 °C by using the TF ANALYZER 2000E ferroelectric measuring system .For electric measurement, the two main surfaces of the sintered disk samples were coated with silver paste and then heat-treated at 550 °C for 30 min with a cooling rate lower than 1 °C/min to reduce internal stress. During measurement a unipolar triangular electrical load was applied to the sample at a frequency of 1 Hz using a high voltage amplifier  and a function generator . The strain and polarization were measured during testing with a LVDT and a Sawyer-Tower circuit, respectively. The disk samples were poled in silicon oil under a direct current electric field of 4 kV/mm at 70 °C for 30 min. The quasistatic piezoelectric coefficient How to cite this article: Huan, Y. et al. Inverted electro-mechanical behaviour induced by the irreversible domain configuration transformation in NbO3-based ceramics. Sci. Rep. 6, 22053; doi: 10.1038/srep22053 (2016).


Journal of Research of the National Institute of Standards and Technology of the following, based on Refs. ..Journal 2H/1H relative ratios of all substances be expressed relative to VSMOW (Vienna Standard Mean Ocean Water) on a scale such that 2H/1H of SLAP (Standard Light Antarctic Precipitation) is 0.572 times that of VSMOW; (ii) 13C/12C relative ratios of all substances be expressed relative to VPDB (Vienna Peedee belemnite) on a scale such that 13C/12C of NBS19-limestone (RM 8544) is 1.00195 times that of VPDB; and (iii) 18O/16O ratios of all substances be expressed relative to either VSMOW or VPDB on scales such that 18O/16O of SLAP is 0.9445 times that of VSMOW.To eliminate possible confusion in the reporting of isotopic abundances on noncorresponding scales, the Commission on Atomic Weights and Isotopic Abundances of the International Union of Pure and Applied Chemistry (IUPAC) recommended at the IUPAC 37th General Assembly in August 1993 at Lisbon, Portugal, that (i) discontinue reporting isotopic abundances relative to SMOW (Standard Mean Ocean Water) and PDB (Peedee belemnite);2H of SLAP  relative to VS-MOW is −0.428;express hydrogen isotopic ratios of all substances relative to VSMOW  on a normalized scale such that the δ13C value of +0.001 95 for NBS19-limestone (RM 8544) relative to VPDB;express carbon isotopic ratios of all substances relative to VPDB (Vienna Peedee belemnite) on a scale defined by adopting a δ18O of SLAP (RM 8537) is −0.0555 relative to VSMOW (RM 8535); andexpress oxygen isotopic ratios relative to either VSMOW or VPDB on a normalized scale such that the δindicate values of isotopic fractionation factors if isotopic abundance measurements of minerals or compounds depend upon such factors.As a consequence, the Commission recommends that researchers should:


The SLC4 The solute carrier SLC4 family consists of 10 members, nine of which are  SLC4A11 encoding BTR1 which likely mediates electrogenic Na+-coupled borate transport  encode +-independent or Na+-dependent  AE1 (SLC4A1), AE2 (SLC4A2), and AE3 (SLC4A3) conducting electroneutral transmembrane exchange of Cl− and +-dependent members, commonly known as Na+-coupled +/+/+-driven Cl−/+-dependence of “AE4” (SLC4A9) remains controversial. Several groups have shown evidences that the product of SLC4A9 performs Cl−/+-dependent +-independent Cl−/SLC4A9 is more similar to those of SLC4A4 and SLC4A5 than to those of the three AEs  family represents a major group of bicarbonate transporters. In mammals, the SLC4 family consists of 10 genes. Except for + and Cl−) in diverse tissues. The pH regulation and transepithelial ion transport mediated by these transporters are critical for a diverse range of biological processes and functions in the body, such as modulation of neuronal excitability . The apparent molecular weights of the native SLC4 transporters, mostly with oligosaccharide moieties, range from about 90 to 200 kDa  of the membrane proteins from erythrocytes. AE1 is extremely abundant in erythrocytes, accounting for 25-30% of total proteins in the plasma membrane  or specific chemical degradation . The intracellular localization of the Nt end was determined by a series of comprehensive biochemical analyses with the fragments of AE1.In the 1970s, considerable number of studies were carried out with AE1 by classical biochemical approaches to establish the intracellular localization and the gross size of the Nt domain of AE1 in erythrocytes , but not on entact red blood cells, suggesting that the Ct domain of AE1 is located in the intracellular side  studies . However, there is a major discrepancy among different models regarding the number of transmembrane alpha helices in the TMDs. Some models propose that the TMD contains 13 transmembrane helices  between TM9 and TM10 in model A is proposed to form a transmebrane helix (TM10) and an intracellular loop (IL5) in model B. Therefore, TM10 and TM11 in model A would correspond to, but be in the opposite direction with, TM11 and TM12 in model B, respectively. Second, the re-entrant loop RL2 (TM12) which is proposed to be non-helical in model A is mostly assigned to the 6th intracellular loop (IL6) in model B.858 in NBCe1-A is accessible to chemical labeling by BM and MTS-TAMRA  can be slightly N-glycosylated, consistent with extracellular localization of this position , but are not accessible to membrane impermeant MTS-TAMRA [2-((5(6)-tetramethylrhodamine) carboxylamino) ethyl methanethiosulfonate], consistent with the idea that this region is localized in the intracellular aquaeous environment  between TM11 and TM13 in model A is highly hydrophilic containing 7-8 charged residues depending on specific SLC4 members. Different studies have shown contradictory data regarding the topological localization of residues in a specific region (888-905 in NBCe1-A which corresponds to 815-829 in AE1) in RL2. There are both evidences favoring and against the intracellular localization of this region.Evidences favoring the intracellular localization of this region include: (1) N-glycosylation motifs introduced at 820, 821, 828 of AE1 are not subjected to N-glycosylation modification in a cell-free translation system supplemented with microsomal membranes  The binding of BM to cysteine residues introduced in region 815-829 of human AE1 is highly sensitive to inhibition by monobromo (trimethylammonio) bimane bromide (qBBr), which is believed to be membrane impermeant  in the cryo-EM structure of AE1 appears to favor topological model B which has 14 transmembrane helices  that is indispensable for the transport activity of the proteins?Xenopus oocytes . Studies have shown that the transport activity of the anion exchangers is largely retained even with the entire Nt domain removed .Consistent with the dimerization of the Nt domain observed in crystal structures (see discussion above in “Role of Nt domain in SLC4 transporters”), it is generally believed that the SLC4 transporters primarily exist as dimers in the plasma membrane  has no effect on the activity of AE1  in human  or Phe471 (corresponding to Phe491 in NBCe1-A) in TM3 substituted by cysteine are highly sensitive to inhibition by pCMBS, suggesting that these two residues are lining along the ion pathway of AE1  in TM5 likely plays a role in determining the ion selectivity of the transporter , 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS)  for stilbene disulfonate derivatives, such as 4,4′-diisothiocyanodihydrostilbene-2,2′-disulfonic acid  of AE1-3 expressed in Xenopus oocytes  is highly sensitive to inhibition by pCMBS and MTSEA  in IL4 immediately following the intracellular end of TM8 are also important for ion transport. AE1 mutants with Ile684 or Ile688 replaced by Cys are sensitive to inhibition by sulfhydryl reagents. In some models, it is proposed that the region beyond Ile688 (corresponding to Ile761 in NBCe1-A) forms an extended portion of the helix of the putative TM8 , is of particular interest. This Glu residue is conserved in all three established AEs but is replaced by Asp in all five NCBTs plus AE4. This Glu residue plays a role in determining the substrate selectivity of AE1 and AE2 as well as the pH sensitivity of AE2  are basically consistent between model A and model B, except for some minor differences in the sequence boundary Figure . These t849 and Val850 (corresponding to Leu923 in NBCe1-A) at the extracellular end of the putative TM13, Ser852 and Thr853 in the putative EL7, and Leu857 (corresponding to Ile930 in NBCe1-A), Ala858, Phe861, Val862, and Ile863 in the putative TM14. The observations suggest that these redidues in TM13 and TM14 is important for ion translocation. The residues accessible to sulfhydral reagents are likely involved in the formation of the ion translocation pore. The inactivation of Cl−/833, His834, Phe836, Thr837, Gln840 in the putative TM13, and Lys851 in the putative EL7 also support the functional and/or structural significance of TM13 in AE1  and Lys851 in human AE1 (corresponding to Lys559 in TM5 and Lys924 in TM13 of NBCe1-A) react with the same DIDS molecule  contains ~130 residues. The initial portion of the Ct domain (Ct-CR1) right following the TMD is highly conserved among all SLC4 family members, whereas the last portion (Ct-VR1) of the Ct domain is highly variable and has little similarity among different members.Compared to the large Nt domain and the TMD, the Ct domain of the SLC4 Xenopus oocytes, suggesting that the Ct domain is important for the membrane trafficking of NBCe1  contains OSEs that could affect the intrinsic activity of the transporters.+/Xenopus oocytes  which is likely the binding site of carbonic anhydrase. Studies have provided evidences showing that the Ct of AE1 interacts with carbonic anhydrase CAII  transcription using distinct promoters, e.g., the generation of alternative Nts of NBCe1-A vs. NBCe1-B  in the present article. These OSEs play critical roles in the functional modulation of the transporters. Functionally, the OSEs could contain structural determinants establishing the biophysical properties, e.g., the intrinsic activity and pH sensitivity, of the transporters. Moreover, the OSEs could regulate the transport function by interacting with protein partners.Firstly, the intrinsic transport activity (or single molecular activity) of different variants of some SLC4 members can vary greatly, e.g., NBCe1-A vs. NBCe1-B/C  of NBCn1 contains binding site for calcineurin Aβ (CnAβ). Finally, as just discussed in the previous section, the Ct ends of variants of several SLC4 members contain binding motif for PDZ-domain containing scalfold proteins. Some of these protein interactions have great effect on the activities of the transporters.Thirdly, the OSEs in the Nt and Ct domains of SLC4 transporters often contain binding sites for interacting partners. The Nt-VR1 of eAE1 contains structural determinants for the binding of cytoskeletal proteins. Nt-VR1 of NBCe1-B/C, NBCn1, and NBCn2 contains binding site for IRBIT, which is an inositol trisphosphate (IP3)-receptor (IP3R) binding protein released with IP3 , was the first to be cloned in the SLC4 family, originally from murine spleen  have been identified, whereas in human, only the first three have been identified. The five mouse AE2 variants are derived from three different promoters, designated as a, b, and c  is expressed under the control of the proximal promoter  in the Nt domain, and the alternative Cts. As shown in Figure SLC4A4  in the Nt domain and cassette ζ (aka cassette III) in the Ct domain. Cassette β of NBCn1 is the largest (123 aa in rodent and 124 aa in human) among all reported optional cassettes in the SLC4 family In addition to the differences in the initial Nt ends, NBCn1 contains four optional cassettes: cassettes α, β, and γ  and as many as seven cassette exons  as well as a specific variant rb7NCBE containing just the Nt domain. These variants have three different initial Nt ends starting with MEIK- (NBCn2-A through -D plus rb3NCBE and rb7NCBE), MCDL- (NBCn2-E through -J), and MHAN-NBCn2 (NBCn2-K through -N), respectively. MEIK-NBCn2 is expressed under the control of the most distal promoter P1. MCDL-NBCn2 could be expressed from either the middle promoter P2 or the proximal promoter P3. Finally, MHAN-NBCn2 is expressed under the control of the middle promoter P2 . Cassette γ is present in NBCn2-B/D/F/H/J/L/N and “rb3NCBE.” The functional consequence of the presence vs. absence of cassette γ in NBCn2 remains unclear. In addition, the Nt domain of NBCn2 contains one minor variation, i.e., the optional inclusion of a single residue Ala256 (numbering according to MEIK-NBCn2) due to alternative usage of a cryptic splicing acceptor site. The optional inclusion of this single Ala residue appears to be random and presumably has no significant effect on the structure and function of NBCn2.The Nt domain of NBCn2 contains one major optional cassettes, i.e., cassette γ  The last 66 residues of the full-length NDCBE-A/C is unique compared to NDCBE-B/D/E; (2) The last 17 residues of NDCBE-B/D/E is unique compared to NDCBE-A/C; (3) The last 15 residues of THYMU3021755 with the isolated Nt domain is unique compared to the full-length variants. The differnce in the Ct ends of the full-length NDCBE arises from alternative splicing of exon 25 of Since the identification of the erythrocytic AE1 more than four decades ago, our knowledge about the SLC4 family In future studies on the structure and function of the SLC4 transporters, the following issues are of particular interest to investigators. Firstly, although the crystal structure of the soluble Nt domain of some members have been reported, some major issues remain to be addressed regarding the role of the large Nt domain in the ion translocation. Why the Nt domains of AEs are not necessary, whereas those of NCBTs are indispensible for the ion transport activities? Except for their regulatory roles for the transporters, are the Nt domains also directly involved in the ion translocation process, e.g., being a part of the ion translocation pathway as suggested by Chang et al. ?Secondly, the fine 3D structure of the TMD of the SLC4 transporters remains mystic. Cryo-EM could be a promising strategy to resolve this issue, given the great progresses in the development of cryo-EM technology and its application in determining the near-atomic level structures of highly complex macromolecules including integral membrane proteins during the past years  depending on the expression systems or in response to specific stimulus  between the AEs and NCBTs? Is NDCBE (SLC4A8) a hybrid of anion exchanger and Na+/Fourthly, what is the structural basis for the differences in the NaAnswering these questions will require studies involving structural, biophysical, and functional approaches. It is no doubt that the knowledge about the structure and function of the SLC4 family transporters derived from these studies will greatly enhance our understanding about the physiology of these proteins. The knowledge about structure and function will also be of high relevance to clinical and pharmaceutical studies.YL and LC wrote the manuscript. JY contributed critical discussions for the preparation of the manuscript.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.


Calotropis procera R. Br., a traditional medicinal plant in India, is a promising source of commercial proteases, because the cysteine proteases from the plant exhibit high thermo-stability, broad pH optima, and plasma-clotting activity. Though several proteases such as Procerain, Procerain B, CpCp-1, CpCp-2, and CpCp-3 have been isolated and characterized, the information of their transcripts is limited to cDNAs encoding their mature peptides. Due to this limitation, in this study, to determine the cDNA sequences encoding full open reading frame of these cysteine proteases, transcripts were sequenced with an Illumina Hiseq2000 sequencer. A total of 171,253,393 clean reads were assembled into 106,093 contigs with an average length of 1,614 bp and an N50 of 2,703 bp, and 70,797 contigs with an average length of 1,565 bp and N50 of 2,082 bp using Trinity and Velvet-Oases software, respectively. Among these contigs, we found 20 unigenes related to papain-like cysteine proteases by BLASTX analysis against a non-redundant NCBI protein database. Our expression analysis revealed that the cysteine protease contains an N-terminal pro-peptide domain (inhibitor region), which is necessary for correct folding and proteolytic activity. It was evident that expression yields using an inducible T7 expression system in Escherichia coli were considerably higher with the pro-peptide domain than without the domain, which could contribute to molecular cloning of the Calotropis procera protease as an active form with correct folding. Proteases are a class of enzymes that occupies a crucial position with respect to physiological roles as well as various industrial and therapeutic applications. Latex from plants has been considered an important source of papain-like cysteine proteases, which are promising candidates with high proteolytic activity and unique characteristics as biocatalysts.Calotropis procera R. Br. (Asclepiadaceae family) is a tropical plant that has been widely used in Indian traditional medicinal system. The latex from this plant has various therapeutic uses, including as hepatoprotective, anti-arthritic, anti-inflammatory, antipyretic, and anticancer treatments ,22A. fruEXXXRXXXFXXNXXXIXXXN) motif, which is a highly conserved motif, this motif is present in the long α-helix which contributes a major part in the scaffold of the cathepsin L like pro-peptide. Another highly conserved GNFD (GXNXFXD) motif is also found for this family. This motif is located at the kink of the β-sheet ahead of the short α-helix (α3) capping the opening of the interdomain cleft. The detection of ERFNIN-GNFD motif in the pro-region of cysteine proteases clearly implies its relationship to the cathepsin L group and sets it apart from cathepsin B subfamily [These proteases contain the ERFNIN , namely Cys25, His159, and Asn175 (papain numbering) as well as the six cysteine residues known to be involved in the formation of the disulfide bond. The Cys25 is embedded within a highly conserved peptide sequence, CGSCWAFS. The His159 is adjacent to small amino acid residues, such as glycine or alanine, and Asn175 is a portion of the Asn-Ser-Trp motif.Calotropis procera, we selected SnuCalCp03 that had been previously investigated as a purified mature protease. In order to express and characterize the cysteine proteases of Carica papaya whereas the identity of pept-SnuCalCp03 was 53% with GPII (PDB ID:1CQD) from Zingiber officinale. Thus, two aforementioned structures were selected as templates to construct the three-dimensional structure of pro-SnuCalCp03 and pept-SnuCalCp03, respectively. The quality of final model was validated by Ramachandran plot analysis with PROCHECK and ERRAT plot. PROCHECK analysis showed that residues of pro-SnuCalCp03 and pept-SnuCalCp03 in the most favorable region were 82.8% and 86.2%, and in the additional allowed region were 14.2% and 13.7%, respectively. The overall quality factor of the model from ERRAT plot for pro-SnuCalCp03 and pept-SnuCalCp03 were 93.7 and 86.4, respectively, indicating that the modeled structure had low steric hindrance . Furthermore, structural comparison between pro-SnuCalCp03 and pept-SnuCalCp03 showed that both the proteins were superimposed completely with RMSD of 1.04 A° over 208 residues. The mature part is subdivided into two typical domains referred to as the left domain (L-domain) and the right domain (R-domain) of papain-like cysteine proteases sharing “V” shaped active site comprised of Cys27, His157, and Asn177. Modeled structure of pept-SnuCalCp03 is structurally similar to the mature part of pro-SnuCalCp03 and both the proteins are almost superimposable, revealing that inhibitor poses steric hindrance to substrate binding at the catalytic cleft. The structural analysis demonstrated that the pro-peptide of the zymogen undergoes a rearrangement in the form of a structural loosening at acidic pH which triggers the proteolytic activation cascade [Structural analysis of pro-SnuCalCp03 and pept-SnuCalCp03 was performed, which helps us to understand the zymogen structure and the activation mechanism of SnuCalCp03 itself . Sequenc cascade ,27. We tE. coli with and without a propetide sequence, we obtained a 1,079 bp cDNA and a 660 bp cDNA fragment containing a zymogen domain and protease C1A domain, respectively (E. coli BL21 (DE3) Star and the cultures were sonicated to release the recombinant proteins. SDS-PAGE  with and without pelB leader sequence to induce proper folding were also tested for protease expression, but the protein was not expressed without pro-peptide domain. Most of the recombinant zymogen was expressed in form of inclusion bodies present in pellet and those were dissolved with 8 M urea and purified by Ni-affinity chromatography. Attempts to oxidative refolding of enzyme were made using reduced and oxidized glutathione. Autocatalytic activation of zymogen was processed at pH 4.0, but enzyme was still found in non-active form. The lack of proteolytic activity may be attributed to misfolding of protein due to the absence of proper folding conditions during in vitro refolding.In order to confirm the expression level of recombinant proteins encoding partial sequences of SnuCalCp03 in ectively . The cDNSDS-PAGE  and WestSDS-PAGE  analysisThe genetic information and the expression pattern of recombinant protease obtained from this study could help to design biologically active cysteine proteases with inhibitors, which is essential for the correct folding, transport, maturation, and regulation of activity, although further studies to elucidate the exact roles of domains for these proteases should be conducted.S1 FigThe deduced amino acid sequences of (A) pro-peptide domain and (B) mature domain were aligned by ClustalW. Identical and conserved amino acid residues are darkly shaded, and the amino acids numbers are shown on the left. Conserved signatures (ERFNIN and GNFD) and catalytic triad residues  are highlighted in bold and indicated above the alignment.(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


The winter wheat variety Kitahonami shows a superior flour yield in comparison to other Japanese soft wheat varieties. To map the quantitative trait loci (QTL) associated with this trait, association mapping was performed using a panel of lines from Kitahonami’s pedigree, along with leading Japanese varieties and advanced breeding lines. Using a mixed linear model corrected for kernel types and familial relatedness, 62 marker-trait associations for flour yield were identified and classified into 21 QTLs. In eighteen of these, Kitahonami alleles showed positive effects. Pedigree analysis demonstrated that a continuous pyramiding of QTLs had occurred throughout the breeding history of Kitahonami. Linkage analyses using three sets of doubled haploid populations from crosses in which Kitahonami was used as a parent were performed, leading to the validation of five of the eight QTLs tested. Among these, QTLs on chromosomes 3B and 7A showed highly significant and consistent effects across the three populations. This study shows that pedigree-based association mapping using breeding materials can be a useful method for QTL identification at the early stages of breeding programs. Flour yield, or the percentage of flour from a given quantity of grain, is of great importance to flour milling companies. Flour yield can be increased by the enhancement of techniques in the milling process, or through the development of varieties with higher flour yields. In 2006, the soft winter wheat variety Kitahonami was released in the Hokkaido prefecture of Japan Pinb on 5D chromosome had a strong influence on flour yield However, flour yield is a complex trait that appears to be strongly influenced by genetic background. QTL studies using bi-parental populations have been conducted within hard varieties or within populations of interclass hybridizations between hard and soft varieties. These studies have indicated that QTLs for flour yield are located on 16 out of 21 chromosomes: 1B, 1D, 2A, 2B, 3A, 3B, 4A, 4B, 4D, 5A, 5B, 5D, 6B, 6D, 7A and 7D Phytophthora infestans in potato. Such association mapping techniques might be useful in rapid marker development for MAS.Because developing mapping populations and performing mapping studies are time consuming processes, breeders often have already introgressed target QTL into breeding lines using traditional selection methods before markers are available, especially for highly desirable traits. Thus, the most effective stage for using marker-assisted selection (MAS) to introduce a new trait into breeding programs is often missed. One solution to this could be to take advantage of populations developed within a breeding program to identify QTLs. Jannink et al. The objective of this study was to dissect the genetic factors contributing to the high flour-yield trait of Kitahonami by an association mapping approach. After identification of QTLs related to flour yield, the pedigree record of Kitahonami was used to trace the origin of QTLs which had been inadvertently accumulated through selection for high flour yield. To confirm the utility of this approach, QTLs identified by association mapping were validated using our own bi-parental populations.T. spelta var. duhamelianum.Plant materials: One hundred eighty-five accessions were used in this study (Table S1 in PstI (TaqI) v.3]  and 164 accessions were genotyped by SNP (PrivKSU_WheatCons_9k) http://wheat.pw.usda.gov/GG2/index.shtml/) and established diagnostic markers, such as Pina-D1, Pinb-D1, Wx-A1, Wx-B1, Ppo-A1, Ppo-D1, Psy-A1 and Psy-B1  according to the manufacturer’s instructions. For the diversity analysis, 151 accessions were genotyped by DArT , during the three successive cropping seasons from 2008/2009 to 2010/2011. The plot size was 3.0 m×0.7 m, and each plot consisted of 40–50 plants separated from one another by 10–15 cm. Two replications were conducted in each season except 2008/2009.Trait analyses: Grain samples were tempered to 14.5% moisture and 100 g of each sample was milled on a Quadrumat Junior mill . The mill was preheated to prevent expansion of the rolls during operation. Ground grain samples were sifted with an 8XX silk reel sieve and a 94-mesh (180 µm) screen. Flour yield (FlYd) was expressed as the percentage of total flour weight to initial sample weight. Measurements were also taken for the following 14 traits: flour efficiency (FlEf), median size of flour particles (x50), specific surface area of flour particles (Sv), flour protein content (FPC), flour ash content (Fash), flour color L* (FlL), flour color a* (Fla), flour color b* (Flb), grain protein content (GPC), grain ash content (Gash), test weight (TestW), 1000-kernel weight (TKW), heading date (HD) and maturity date (MD). Detailed explanations for each trait are described in Table S3 in Statistical analysis: Principal component analysis (PCA) and cladogram construction were performed with TASSEL 3.0 1 plants from crosses between Kitahonami and three other varieties, namely Kinuhime, Tohoku224 and Shunyou. At least 151 lines from each population were field-grown without replication during the 2010/2011 season and subjected to validation. FlYd values for these lines were obtained as described above. To reduce genotyping costs, markers showing significant association with this trait in the association mapping analysis were converted from array-based SNP markers into PCR-based markers. To do this, probe sequences of the SNPs of interest were identified; since most of these sequences have been mapped, the chromosome number to which they have been assigned is known. These sequences were used as queries in BLASTN searches  against the wheat survey sequences  2, 0.2 mM dNTP (each), 1×Ex Taq buffer, and 0.5 U of TaKaRa Ex Taq . The concentrations of GSPs and ASPs in PCR mixtures are provided in the Table S4 in QTL validation: QTLs obtained as described above were validated using three doubled haploid (DH) populations, which were developed from FArray-based marker analyses allowed the identification of 2,933 polymorphic DArT  analyses of the associated loci were performed. Obvious LD blocks were observed in 11 of 18 QTLs ; Fig. S2For validation of the QTLs detected by association mapping, we performed linkage analysis using three sets of DH populations  rather than population structure (Q) as a covariant. Based on a plot of expected versus observed p values, this correction achieved a reduction in the false positive rate (data not shown). Kernel type is known to have a great impact on milling yield traits enotypes . TherefoIt was not possible to precisely compare the positions of QTLs detected in this study to those identified in previous reports, since few identical markers were used. However, based on the microsatellite consensus map The level of LD can be affected by various factors including linkage, selection, and admixture Segregation analysis confirmed that five of the eight QTLs tested had significant effects on the FlYd . PreviouThe effects of the markers on FlYd were not identical between single and multiple regression models . These dhttp://www.wheatgenome.org/) In this study, PCR-based markers linked to eight QTLs were developed . RecentlMilling tests require a substantial quantity of grain, meaning that selection for flour yield cannot be performed at the early stages of wheat breeding programs. The identification of markers linked to the flour yield trait can circumvent this problem. Using association mapping with a mixed model, we identified 21 QTLs influencing flour yield. The role of these QTLs was supported by pedigree information and results of linkage analysis. Notably, we identified several QTLs which were consistently associated with high flour yield across different genetic backgrounds. The introduction of these QTLs from Kitahonami into other lines by MAS is a promising method of improving flour yield in Japanese soft wheat varieties.Figure S1Record of Kitahonami's pedigree.(PDF)Click here for additional data file.Figure S2LD charts produced by TASSEL 3.0.(PDF)Click here for additional data file.Figure S3Distribution of FlYd in the three DH populations.(PDF)Click here for additional data file.File S1Supporting tables. Table S1, List of accessions used in this study. Table S2, Distribution of the markers used for association mapping across the wheat genome. Table S3, Description of traits investigated. Table S4, Details of PCR-based markers developed in this study. Table S5, Variance components and heritabilities of traits investigated. Table S6, Correlation coefficients among traits. Table S7, List of marker-trait associations (MTAs) detected by genome-wide association mapping. Table S8, Full matrix produced by similarity analysis in LD blocks. Table S9, Results of multiple regression analysis using the three DH populations.(XLSX)Click here for additional data file.File S2Datasets. Dataset S1, DArT genotyping data for diversity analyses. Dataset S2, SNP genotyping data for diversity analyses. Dataset S3, Phenotyping data for association mapping. Dataset S4, Genotyping data for association mapping. Dataset S5, Genotype and phenotype data of doubled haploid populations.(ZIP)Click here for additional data file.


Arf1 is activated at the cis-Golgi by the guanine nucleotide exchange factor (GEF) GBF1 and at the trans-Golgi by the related GEF BIG1 or its paralog, BIG2. The trans-Golgi-specific targeting of BIG1 and BIG2 depends on the Arf-like GTPase Arl1. We find that Arl1 binds to the dimerization and cyclophilin binding (DCB) domain in BIG1 and report a crystal structure of human Arl1 bound to this domain. Residues in the DCB domain that bind Arl1 are required for BIG1 to locate to the Golgi in vivo. DCB domain-binding residues in Arl1 have a distinct conformation from those in known Arl1-effector complexes, and this plasticity allows Arl1 to interact with different effectors of unrelated structure. The findings provide structural insight into how Arf1 GEFs, and hence active Arf1, achieve their correct subcellular distribution.The GTPase Arf1 is the major regulator of vesicle traffic at both the   •Arl1 binds the N-terminal DCB domain of the Arf1-GEF BIG1•BIG1/Arl1 complex is solved at 2.3 Å resolutionStructure of the human DCB•The Arl1 binding surface on the DCB domain is unrelated to known Arl1 effectors•Structure-guided mutation shows BIG1 needs Ar11 binding for Golgi targeting trans-Golgi by the BIG1 exchange factor. A second GTPase, Arl1, is required for BIG1 recruitment. Galindo et al. find that Arl1 binds to the DCB domain of BIG1 and determine the structure of human Arl1 bound to this domain.The GTPase Arf1 is essential for Golgi function and is activated on the  Membrane trafficking depends on the Arf and Rab small GTPase families . In the cis-Golgi to generate vesicles for transport within the stack and back to the ER, while on the trans-Golgi, Arf1 recruits the clathrin adaptor proteins AP-1 and AP-3 to make vesicles directed to endosomes  and the HUS (homology upstream of Sec7) domains . The asstrans-Golgi, we have mapped the Arl1 binding site on BIG1 to the N-terminal DCB domain and then determined its structure in a complex with Arl1GTP at a resolution of 2.3 Å. Mutagenesis shows that interaction between the DCB domain and Arl1 is a major determinant of BIG1’s ability to recognize the trans-Golgi. The Arl1 binding surface on the DCB domain consists of four parallel α helices, a structure unrelated to any known Arl1 effectors, and the Arl1 residues in the binding interface show a conformational plasticity that allows it to specifically recognize structurally unrelated effectors.To gain insight into how Arl1 regulates Arf1 activation at the Drosophila BIG1 homolog Sec71 binds directly to GTP-bound Arl1, and the analogous region of human BIG1 (1–559) is sufficient for Golgi targeting in mammalian cells (BIG1 [1–228]) bound to Arl1GTP, while the HUS domain did not, with a similar result obtained with the Drosophila proteins  was coexpressed in Escherichia coli with human Arl1 lacking residues 1–14. These residues form an amphipathic helix that becomes fully exposed upon GTP binding, and its removal has been found to be necessary for the crystallization of GTP-bound forms of Arf-family proteins -like repeat domain of the Drosophila melanogaster microtubule regulatory protein MAST/Orbit that was predicted to be distantly related to the DCB domain by HHpred (Δ51–71 domain with the exception of three residues (71–73) adjacent to the region that was deleted  connects helices 2 and 3, and the remaining link between these two α helices is not fully ordered in our model (residues 72 and 73). As expected, the Arl1Q71L-GTP exhibits the typical fold of small GTPases, containing a six-stranded β sheet surrounded by five α helices . Of these, those from switch 1 and the interswitch are present in many Arf family members with, for instance, all being in conserved in Arf1  and suggest a dimer of heterodimers  domain from the C terminus of golgin-245, one of several GRIP-domain-containing coiled-coil proteins that can capture endosome-derived vesicles, and Arfaptin-2, a BAR (Bin-Amphiphysin-Rvs) domain protein that is localized to the  unclear . The thr unclear A and 6B.c pocket C and S5,faptin-2 . The majKhan and Ménétrey have proposed a classification scheme to highlight properties shared by domains that bind to Arf/Rab GTPases . Many ofThe ability of Arl1 to recognize three very different effectors raises the question of how it can maintain selectivity for its own effectors over those of related GTPases while at the same time being promiscuous in the interactions formed by its effector-binding interface. Selectivity can be explained by the sequences of the switch regions themselves, and in particular switch 2, which is involved in effector binding in all three complexes and has residues conserved in few, if any, other members of the Arf family.As for the structural plasticity that underlies the binding of several distinct effectors by the same GTPase, examination of other Arf:effector complexes revealed that Arf proteins generally maintain a rigid core structure independent of their binding partners . Thus, tGTP/DCBΔ51–71 complex, the carbonyl group of Arl1 Ile49 remains buried inside the rim of the hydrophobic pocket  or DCB-HUS(1–566) and either Rab1A or Rab1B .Our work has provided structural insight into the regions that flank the central Sec7 catalytic domain of the two major classes of Golgi Arf GEF. In addition, it shows how Arl1 interacts with BIG1 via the DCB domain. Although some of the conserved residues in the BIG1 DCB domain are specific to the BIG family, this DCB region is also present in the GBF1 family and in the more distantly related MON2 and BIG3 proteins that lack GEF activity and are of unknown function . Thus, iis-Golgi C. The fiis-Golgi , but we sec7-1 allele, a thermosensitive mutant that carries the substitution S402L in the DCB domain , they are not well conserved in yeast . Thus, it appears that in yeasts Arl1 binds to a region of the protein other than the DCB domain recognized in metazoans. The second interesting feature revealed by comparing the structures is that in the DCB-HUS structure, the HUS domain covers the region on the DCB domain that forms the DCB homo-dimer interface reported here , carrying the Q71L mutation, and human BIG1 (UniProt Q9Y6D6) fragment DCBBIG1 (residues 1–228) were co-expressed from pOPTC (Q71L and His6-DCBBIG1. The same strategy was followed for the coexpression of Arl1Q71L and the BIG1 fragment DCBΔ51–71 (residues 1–228 with a deletion of 21 residues [51–71 inclusive]). Cells expressing either Arl1Q71L/DCBBIG1 or Arl1Q71L/DCBΔ51–71 were grown at 37°C until optical density 600 (OD600) ∼1 and induced with 0.2 mM IPTG at 15°C for 16 hr. Cells were pelleted, resuspended in buffer A  and lysed with an Emulsiflex-C3 at 15,000 psi at 4°C. The cell lysate was cleared by centrifugation at 16,000 × g for 30 min at 4°C and mixed with glutathione-Sepharose beads . After three washes of ten bed volumes, resin-bound material was released with 0.08 mg TEV protease per 10 mg complex for 12 hr at 4°C. The Arl1Q71L/DCB complexes were further purified by gel filtration on a Superdex 75 16/60 column equilibrated in buffer B . After elution, fractions were concentrated to 12–14 mg/ml and snap frozen in liquid nitrogen.Human Arl1 . Microseeding was used to increase crystal nucleation. Crystals were harvested and cryo-cooled in reservoir solution supplemented with 25% (w/v) PEG4000 for Arl1Q71L/DCBBIG1 or with 20% (v/v) glycerol in the case of Arl1Q71L/ DCBΔ51–71.Initial crystals of Arl1Q71L-GTP (PDB: 1UPT) (4G3A) (GTP/DCBΔ51–71 is PDB: 5EE5. Identification of key residues involved in Arl1GTP/DCBBIG1 interaction and DCBBIG1/DCBBIG1 were based on results obtained from NCONT and CONTACT from CCP4 (Diffraction data were collected at −100°C at beamlines I03 and I04 at the Diamond Light Source (STFC-UK). Crystallographic data were processed with XDS  or iMOSFB: 1UPT)  and the ) (4G3A) . Model r) (4G3A)  and PHEN) (4G3A) . Final srom CCP4 . Figuresrom CCP4 .2, 0.001% (v/v) Triton X-100, 0.1% (v/v) Tween-20, and 5 μM GTP. A control reaction with free NT-495 label showed no change in anisotropy with increasing protein concentration. Using a single-site binding model, the data were fitted to the equation:0 and F1 are the anisotropy in the absence of titrating protein and at saturation respectively, [LT] and [PT] are the total concentrations of DCBBIG1 and labeled Arl1GTP, and Kd is the dissociation constant. For all the mutant DCB proteins, except K105A/Q201V, Q200E, and L156D, the value of F1 was fixed at the value fitted for the wild-type.The binding of DCB variants to Arl1, labeled with NT-495 (NanoTemper Technologies), was measured by fluorescence anisotropy at 20°C using a PHERAstar plate-reader (BMG Labtech). The reaction was followed in 50 mM Tris-HCl (pH 8.0), 110 mM KCl, 1 mM DTT, 5 mM MgClobs is the observed CD signal at temperature T; YN and YD are the native and denatured protein CD contributions with αN and βD are the slopes for the native and denatured signals, respectively; Tm is the melting temperature; and C is a constant. To compare protein stability of DCBBIG1 variants, CD thermal denaturation data were transformed using the fitted baselines to the CD scans data, and they were plotted as fraction of unfolded protein (fD) versus temperature:Circular dichroism (CD) scans were recorded between 190 and 260 nm using a JASCO-815 CD spectrophotometer at a protein concentration of 8 μM in PBS at 20°C. Control spectra of buffer were used to subtract the baseline contribution to the signals. Thermal denaturations were preformed over the range of 4°C to 95°C at a rate of 1°C/min. Data were fitted to a Boltzmann curve with sloping baselines:BIG1 proteins were resolved on a Superdex-75 HR10/300 analytical gel filtration column  at 0.5 ml/min in PBS (pH 7.5), 150 mM NaCl, 5 mM MgCl2, and 0.001% (w/v) Triton X-100 before detection on a Wyatt Heleos II 18 angle light-scattering instrument coupled to a Wyatt Optilab rEX online refractive index detector and determination of native molecular weight  and GBF1 (UniProt Q92538) were expressed in BL21-CodonPlus (DE3)-RIPL cells and lysates prepared as described above. His6-tagged proteins were purified using Ni-NTA beads (QIAGEN) and GST-Arl1 using glutathione-Sepharose . Purified protein were eluted and desalted in buffer C . Purified GST-Arl1 was loaded with nucleotide by incubation at 37°C for 30 min in 10 mM EDTA and either 200 μM GMP-PNP or 1 mM GDP and then by increasing the MgCl2 to 20 mM and cooling to 4°C. 2 nM GST-Arl1GMP-PNP or GST-Arl1GDP was mixed with 2 nM of the His6-tagged protein in 500 μl buffer C plus 5 mM MgCl2 and rotated at 4°C for 30 min before adding 10 μl 50% glutathione-Sepharose beads. Beads were washed thrice with 1 ml buffer C plus 5 mM MgCl2 and bound material eluted with SDS buffer and analyzed by SDS-PAGE. 10 μl Ni-NTA beads preloaded with His6-tagged DCBBIG1 variants was mixed with 1 nM GST-Arl1GMP-PNP in 500 μl buffer C plus 5 mM Mg2Cl and 40 mM imidazole. Beads were washed thrice with 1 ml supplemented buffer C and bound material eluted with 20 μl of SDS buffer and analyzed by SDS-PAGE.GST-Arl1 (residues 15–181) and HisHeLa Cells were transfected using FuGene 6 (Promega) and plated onto multispot microscope slides 12 hr after transfection. Cell fixation, permeabilization, staining, and confocal imaging (Zeiss LSM 780) were performed as described previously . Live ceA.G. conceived and performed experiments and wrote the manuscript, S.M. conceived the study and experiments and wrote the manuscript, N.S. did the phasing and model refinement, M.Y. contributed to data acquisition, and S.H.M. and R.L.W. provided expertise and feedback.