Animals have a greater diversity of signalling pathways than their unicellular relatives, consistent with the evolution and expansion of these pathways occurring in parallel with the origin of animal multicellularity. However, the genomes of sponges and ctenophores – non-bilaterian basal animals – typically encode no, or far fewer, recognisable signalling ligands compared to bilaterians and cnidarians. For instance, the largest subclass of receptor tyrosine kinases (RTKs) in bilaterians, the Eph receptors (Ephs), are present in sponges and ctenophores, but their cognate ligands, the ephrins, have not yet been detected.Here, we use an iterative HMM analysis to identify for the first time membrane-bound ephrins in sponges and ctenophores. We also expand the number of Eph-receptor subtypes identified in these animals and in cnidarians. Both sequence and structural analyses are consistent with the Eph ligand binding domain (LBD) and the ephrin receptor binding domain (RBD) having evolved via the co-option of ancient galactose-binding (discoidin-domain)-like and monodomain cupredoxin domains, respectively. Although we did not detect a complete Eph-ephrin signalling pathway in closely-related unicellular holozoans or in other non-metazoan eukaryotes, truncated proteins with Eph receptor LBDs and ephrin RBDs are present in some choanoflagellates. Together, these results indicate that Eph-ephrin signalling was present in the last common ancestor of extant metazoans, and perhaps even in the last common ancestor of animals and choanoflagellates. Either scenario pushes the origin of Eph-ephrin signalling back much earlier than previously reported.We propose that the Eph-LBD and ephrin-RBD, which were ancestrally localised in the cytosol, became linked to the extracellular parts of two cell surface proteins before the divergence of sponges and ctenophores from the rest of the animal kingdom. The ephrin-RBD lost the ancestral capacity to bind copper, and the Eph-LBD became linked to an ancient RTK. The identification of divergent ephrin ligands in sponges and ctenophores suggests that these ligands evolve faster than their cognate receptors. As this may be a general phenomena, we propose that the sequence-structure approach used in this study may be usefully applied to other signalling systems where no, or a small number of, ligands have been identified.The online version of this article (10.1186/s12862-019-1418-z) contains supplementary material, which is available to authorized users. The origin of animal multicellularity and complexity appears to have required the evolution of elaborate internally-regulated intercellular signalling , 2. ConsAlthough metazoans have a number of unique intercellular signalling systems, including Wnt and TGF-β (transforming growth factor-beta) pathways, they also use signalling pathways that are more ancient, such as the receptor tyrosine kinase (RTK) pathway –12. ThesAmongst the bilaterian RTK families identified in sponges and ctenophores, the Eph-receptors are of particular interest in the context of understanding the origins of metazoan intercellular communication. The Eph-receptors comprise the largest RTK subfamily in bilaterians and, unlike the other RTK families that bind secreted diffusible ligands, they interact with cell surface-associated ephrin ligands on neighbouring cells Fig. a 3, 16,,16, 17. Despite this promiscuity, all bilaterian Eph receptors have an identical domain architecture that includes an extracellular globular ligand binding domain (LBD), a cysteine rich region (containing sushi and EGF-like motifs), a variable number of fibronectin domains, a single-pass transmembrane domain that connects to intracellular kinase, and SAM  and PDZ domains  searches of a wide range of publicly available genomes and transcriptomes, and unpublished transcriptome datasets, with a greater focus towards non-bilaterian and non-metazoan genomes. This survey of 104 unikont species included in particular a comprehensive collection of non-bilaterian metazoans  and 28 unicellular holozoans , comprising all recently published transcriptomic and genomic datasets  do contain a N-terminal domain that is not readily detectable by Pfam/CDD domain search engines. Manual inspection of an alignment with Eph-LBD of metazoan counterparts hinted at the presence of an Eph-LBD domain in these choanoflagellates, and thus each case was further investigated by searching against a database of HMM profiles constructed from individual Protein Databank (PDB) entries with the HHpred program. HHpred searches yielded significant similarity to the ephrin-binding (EPH-LBD) ectodomain of bona fide EPH receptors , 100 (15 full-length), and 9 (3 full-length), respectively Fig. . Across Mnemiopsis leidyi , and in this case the receptor lacks the intracellular Tyr-kinase domain. Thus, based on currently available genome and transcriptome data, there is no evidence for functional Eph-ephrin signalling outside of the Metazoa, even though components are present in some choanoflagellates.Taken together, our iterative HMM search results retrieved Eph-ephrin receptor-ligand pairs in most metazoans, including sponges and ctenophores, suggesting that Eph-ephrin signalling was present in the last common ancestor to all contemporary animals. Our results also raise the possibility that the origin of Eph-ephrin signalling predated the divergence of metazoan and choanoflagellate lineages, as we recovered putative Eph-like receptors with intact ligand binding domains in some choanoflagellates, and ephrins in others. Notably, however, we found only a single instance where both an Eph-like receptor and an ephrin ligand was found in the same choanoflagellate . In general, variations of this highly-conserved domain organisation are limited to the number of fibronectin repeats, differences in the length of the cysteine rich regions and absence of the intracellular SAM domains  for structure-based sequence comparisons.To further investigate the ligand-receptor recognition and binding in the newly identified Ephs and ephrins, we analysed the sequence conservation of the Eph-LBD and ephrin-RBD domains. The interaction of Eph-LBD and ephrin-RBD is the first critical step for the initiation of Eph-ephrin signalling via the formation of heterodimers and tetrameric complexes , 18, 24.Alignment of Eph-LBDs revealed a high degree of conservation, including invariant cysteine residues forming disulphide bridges  and conserved blocks of beta sheets forming the antiparallel beta-sandwich jellyroll fold of the Eph ectodomains   anchors that are characteristic of ephrin-A see Fig. . We founAs classified by the SCOP database, and examined previously , 44, 45 To examine the relationship of ephrins to cupredoxins and the SRS superfamily, we performed sequence-structure-based comparisons and phylogenetic reconstructions. Using Pfam HMM models Copper-bind PF00127), Cu_bind_like (PF02298) and Cupredoxin_1 (PF13473), we identified cupredoxin homologues in a diversity of organisms, including representative prokaryotes, non-metazoan eukaryotes and non-bilaterian metazoans. This survey recovered a range of cupredoxins, including the large multidomain enzymes comprising cupredoxin domain as one of their subunits. We restricted further analyses to the monodomain cupredoxins. Several of these were identified in sponges, ctenophores, unicellular holozoans and fungi  . Similarly, the Pfam domain Ephrin (PF00812), archetypal for ephrin-A and ephrin-B was used to search for ephrin orthologues. Novel putative Ephs and ephrins initially identified in non-bilaterian metazoans were aligned with representative full-length sequences from bilaterians to build new, distinct HMM profiles that were used in the second iteration. This iterative approach using domain specific HMM profiles was repeated for two rounds by including newly identified sequences to previous alignments. This approach maximised the chances of identifying distant homologues in basal metazoans and non-metazoans, and identified partial sequences, which may contain only one of these domains. After removal of redundant sequences using CD-HIT software, a final list of Ephs and ephrins were used in further sequence and phylogenetic analysis. For display purposes, we built heap maps using pheapmaps package in R. Absolute protein counts of Eph and ephrins, identified from each analysed species were plotted as heat maps , Cu_bind_like (PF02298) and Cupredoxin_1 (PF13473). These HMMER searches retrieved several cupredoxin domain containing sequences including monodomain and multidomain cupredoxins, however, we restricted our analysis to monodomain cupredoxins, as mutlidomain cupredoxins contain the same domain in additional copies. Our strategy to search for cupredoxins in metazoans and closest unicellular relatives was to verify if non-bilaterian metazoans containing ephrins also encode genes for monodomain cupredoxins with copper binding centres. This allowed us to examine possible scenarios of the diversification of ephrins from cupredoxins during the eukaryotic evolution. We also retrieved the SRS superfamily members, which are specific to apicomplexans, to resolve the overall phylogenetic relationships with the closely related ephrins and cupredoxins. SRS superfamily members were recovered through BLASTP searches against the NCBI NR database using representatives  from several SRS families including S48/45 that constitutes the same SRS- structural fold. Such search strategy recovered the large expansions of SRS superfamily, and thus the overall dataset was clustered based on 40% identity threshold CD-HIT  clusteriMultiple full-length sequence alignments were computed using MAFFT software (Version 7), utilising the E-INS-I algorithm, optimised for sequences with conserved motifs and carrying multiple domains, with BLOSUM62 as the scoring matrix and default parameters . Domain To analyse the potential loss of copper binding sites in ephrins, we generated a structure-based sequence alignment using the T-Coffee Expresso alignment server . With nuhttp://tree.bio.ed.ac.uk/software/figtree/).Maximum-likelihood phylogenetic trees were estimated by RAxML using PROTGAMMAAUTO to automatically determine the best protein substitution model for the given dataset . StatistTo distinguish between membrane-anchored (GPI- linked) ephrin-A or a transmembrane type ephrin-B we predicted potential TM regions and GPI anchoring sites in all newly identified ephrins from non-bilaterian metazoans. We utilised Topcons single (a reliable approach that engage a consensus predictive methodology derived from outputs of five different TM prediction tools)  for predInferred protein domain organisations were obtained using HMMER searches against Pfam database and Reverse Position-Specific BLAST searches against the pre-calculated Position-Specific Score Matrix (PSSMs) of the Conserved Domain Database (CDD) version 3.14. The cysteine rich regions of Eph-receptors, flanked between the Eph-LBD and the fibronectin domains are generally informed as either Sushi, EGF-like and other cysteine rich domains in Pfam and CDD searches. To be consistent, we uniformly denote the region between Eph-LBD and fibronectin as CRD (Cysteine rich domain). Also, as several of identified sequences lack one or more domains, we consider Eph-receptors as to be potentially full-length receptor, only if both the N-terminal ephrin-ligand binding domain, and C-terminal tyrosine kinase domain were present.Additional file 1:Figure S1. Domain architectures of Eph receptors identified across all analyzed taxa. Figure S2. Multiple sequence alignment containing selected representatives of the Eph-receptor extracellular Ligand Binding Domain (LBD) animals and choanoflagellates. Figure S3. Multiple sequence alignment of the Ephrin ligand Receptor Binding Domain (RBD) containing representatives from animals and choanoflagellates. Figure S4. Evolutionary relationships of metazoan Eph receptors inferred using the canonical intracellular Pkinase-Tyr domain. Figure S5. Evolutionary relationships of metazoan Eph receptors inferred using the extracellular ligand binding domain (LBD). Figure S6. Evolutionary relationships among metazoan ephrin ligands, highlighting multiple expansions in the phylum Cnidaria. Figure S7. Cartoon rendering of representative structures of cupredoxins, ephrins and the SRS superfamily. Figure S8. Structure based sequence alignment of ephrins, monodomain cupredoxins and the SRS superfamily from diverse taxa. Figure S9. Phylogenetic relationships between ephrins, monodomain cupredoxins and SRS superfamily. Table S1. List of analyzed taxa and database sources. Table S2. Dataset utilized to plot the heat map shown in Fig. Table S3. Distribution of Eph, ephrin and monodomain cupredoxins. Table S4. Mapping of Eph-LBD and Ephrin-RBD interaction interface residues. Table S5. Predicted TM regions and lipid anchoring sites in ephrins from non-bilaterian metazoans. Accompanying note. Genome/transcriptome datasets used for sequence search. Eph-LBD and ephrin hits in choanoflagellates. (PDF 47231 kb)Additional file 2:Complete FASTA sequences of all Eph receptors, ephrin ligands and cupredoxins identified new, or used for new analyses, in this study. (PDF 1079 kb)Additional file 3:Raw tree files of all phylogenetic trees reported in the manuscript. (PDF 271 kb)


A term rewrite system is terminating when no infinite reduction sequences are possible. Relative termination generalizes termination by permitting infinite reductions as long as some distinguished rules are not applied infinitely many times. Relative termination is thus a fundamental notion that has been used in a number of different contexts, like analyzing the confluence of rewrite systems or the termination of narrowing. In this work, we introduce a novel technique to prove relative termination by reducing it to dependency pair problems. To the best of our knowledge, this is the first significant contribution to Problem #106 of the RTA List of Open Problems. We first present a general approach that is then instantiated to provide a concrete technique for proving relative termination. The practical significance of our method is illustrated by means of an experimental evaluation. We denote by t is a subterm of s, and by proper subterm.A t. A substitution is a mapping t by We write rewrite rulel and r are called the left-hand side and the right-hand side of the rule, respectively. A term rewrite system (TRS) is a set of rewrite rules. Given a TRS defined symbols, i.e., the root symbols of the left-hand sides of the rules, and constructors; A rewrite relationp in s, a rewrite rule For a TRS R, we denote its transitive closure by Given a binary relation order pair) We say that A if A, A, and they are compatible, i.e., (The dependency pair (DP) method , 13 and t is a subterm of r with a defined root symbol, then the rule dependency pair of Let us briefly recall the fundamentals of the DP framework. Here, we consider that the signature DP problem, which is a pair finite if there is no infinite chain: A chain (à la ) reduction pairs [reduction pair is a well-founded order pair To prove DP problems finite, various techniques have been proposed and implemented in current termination tools, e.g., AProVE, on pairs  constitu, 12, 13), 1312, 1R and B, we denote the relation R / B. In particular, for two TRSs Given two relations Let us now recall the formal definition of relative termination:relative termination [relatively terminates w.r.t.t is nonterminating if it starts an infinite terminating otherwise.(mination ) Let \doIn other words, rule removal processors [monotone if Geser  proposedocessors . We say  Let proof orderingThe main goal of this paper is to introduce a counterpart of Proposition sufficient condition for relative termination can be stated as follows:Let us start with some basic conditions for relative termination in terms of DP problems. First, it is folklore that, given two TRSs For TRSs Observe that necessary condition for relative termination.On the other hand, using a proof technique from the standard DP framework, we can easily prove the following Let We prove that any infinite Now, we aim at finding more precise characterizations of relative termination in terms of DP problems. We introduce the multisets that we will use to prove our main results.t a term. The multiset maximaldefined subterms of t is defined as follows:Let It is well known that a well-founded order pair can be extended to a well-founded order pair over multisets .multiset extension) The multiset extension of an order pair A is the order pair A which is defined as follows: X and Y are written We have  We say that a TRS dominates a TRS  , and TPAer 2006) , fail onThe dominance condition and the non-duplication condition are indeed necessary for Corollary Consider the two TRSs The following example illustrates that the latter condition is also necessary:Consider the following two TRSs:When dominance and non-duplication are assumed, the condition Let The following result removes the condition mentclass2pt{minimLet Let First, observe that mentclass2pt{minimNext, observe that mentclass2pt{minimThe remaining two conditions, namely the dominance and non-duplication conditions might be still too restrictive in practice. For instance, only six out of 44 examples in the relative TRS category of TPDB 9.0 satisfy both conditions.Luckily, we can employ again Proposition Let Consider TRSs entclass1pt{minimaentclass1pt{minimaentclass1pt{minimaLet Consider the following TRSs entclass1pt{minimaduplicate an generate infinitely many To show termination of Consider the following TRSs Consider an arbitrary nonempty TRS minimal if every usable rules and the subterm criterion, have been proposed to prove absence of infinite minimal chains [A DP chain l chains .Unfortunately, the minimality property (in terms of the standard DP framework) cannot be assumed on DP problems produced by our relative termination criteria. Therefore, usable rules and subterm criterion are not generally applicable.usable from the dependency pair Consider the following TRSs entclass1pt{minimaNonetheless, we will show that both the subterm criterion and usable rules are still applicable when quasi-termination [quasi-terminating iff the set s.(mination ) We say First, we naturally extend the notion of minimality to relative termination.relative DP problem) A relative DP problem is a triple of TRSs, written chain is a possibly infinite sequenceminimal if every finite if it admits no infinite minimal chain. A simple projectionn-ary symbol ((relative subterm criterion) Let i. Using the discussion in [t is We prove the claim by contradiction. Assume that there exists a minimal infinite chain of the form:Now we prove that the quasi-termination condition also enables the usable rules technique. We roughly follow the proof by Hirokawa and Middeldorp  for the s, we define s is a variable, and for Let s. Note also that It is easy to see that A key property of Definition s is If First consider terminating if x. For an x to The following auxiliary result for substitutions is analogous to Hirokawa and Middeldorp , but theIf s. If If If Using the induction hypotheses, we obtainWe prove the claim by induction on the structure of Now we define the notion of usable rules.usable rules (usable symbols) in a relative DP problem If If Let p in a term s is usable if q of p. The following lemma is straightforward, which is also a corollary of Hirokawa and Middeldorp [Let us say that a position p is a usable position in s and If s is Let mentclasspt{minimap has a usable prefix q such that Suppose that p is a usable position and Otherwise, mentclasspt{minimaLet Finally, we present the main result of this section. Using the preceding lemmas, the main theorem is proven analogously to the standard case.(relative usable rules) Let mentclasspt{minimai. Thus, after some point . The implementation works as follows: It first checks if the rules in polynomial interpretations [etations , 26, extetations ,lexicographic path order [argument filtering [the th order  with argiltering ,weighted path order with partial status [the l status , andmatrix interpretations [ etations .As monotone reduction pairs for Proposition Afterwards, we obtain a standard dependency pair problem, on which existing reduction pair processors can be applied. In the experiments, we use the following reduction pairs for Proposition In the following, we show the significance of our technique through an experimental evaluation. The experiments were run on a server equipped with a quad-core Intel Xeon E5-3407v2 processor running at a clock rate of 2.40 GHz and 32 GB of main memory. NaTT uses z3  4.3.2termination problem database (TPDB) 9.0.Termination Competition 2015 were able to prove. For comparison, we include results for rule removal processors by matrix interpretations in the third row.The first test set consists of the 44 examples in the “TRS Relative” category of the We also prepared 44 examples by extending benchmarks by Arts and Giesl  with thementclass2pt{minimThe DP framework allows the combination of termination proving techniques. In the fourth row, we combine the rule removal processors and the technique presented in this paper. This combination indeed boosts the power of NaTT; e.g., by combining Proposition Although NaTT won the “TRS Relative” category in the Termination Competition 2015, it is clear that the mostly artificially crafted 88 examples in the category are limited as a fair benchmark set . We hope that new relative termination problems stemming from different applications will alleviate this problem in the near future.One of the most comprehensive works on relative termination is Geser’s Ph.D. thesis . Indeed,equational rewriting, a special case of relative termination where the base is symmetric (associative-commutative (AC) rewriting, a number of papers exist  [The closer approach is Iborra et al. , where tion (HC) , i.e., \This paper is a revised and extended version of . BesidesIn this paper, we have introduced a new approach to proving relative termination by reducing it to DP problems. The relevance of such a result should be clear, since it allows one to prove relative termination by reusing many existing techniques and tools for proving termination within the DP framework. Indeed, such an approach was included in the RTA List of Open Problems Problem #106). To the best of our knowledge, this work makes the first significant contribution to positively answering this problem. Moreover, as shown in Sect. 06. To thAs future work, we plan to improve the precision of our technique by extending the DP framework to be more suitable for proving relative termination. We will also continue the research on finding less restrictive conditions on


Nanobodies are a class of antigen‐binding protein derived from camelids that achieve comparable binding affinities and specificities to classical antibodies, despite comprising only a single 15 kDa variable domain. Their reduced size makes them an exciting target molecule with which we can explore the molecular code that underpins binding specificity—how is such high specificity achieved? Here, we use a novel dataset of 90 nonredundant, protein‐binding nanobodies with antigen‐bound crystal structures to address this question. To provide a baseline for comparison we construct an analogous set of classical antibodies, allowing us to probe how nanobodies achieve high specificity binding with a dramatically reduced sequence space. Our analysis reveals that nanobodies do not diversify their framework region to compensate for the loss of the VL domain. In addition to the previously reported increase in H3 loop length, we find that nanobodies create diversity by drawing their paratope regions from a significantly larger set of aligned sequence positions, and by exhibiting greater structural variation in their H1 and H2 loops. Conventional full‐length antibodies (Abs) have become essential tools in biological research, and a foundation of the biopharmaceutical industry due to their exquisite binding specificity and high affinity to target antigens. The immense diversity of binding specificity is created by sequence variation in two variable domains, the heavy chain (VH) and the light chain (VL). Together, these have been estimated to yield a diversity of at least 10A potentially more tractable system is provided by the class of heavy‐chain antibodies found in camelid species such as camels, llamas and alpacas, for which the light chain is completely absent Figure . The ≈ 1For both Nbs and Abs, the fundamental challenge is to determine the molecular code that relates amino acid sequence, and in particular choice of paratope residues, to the binding specificity of the folded molecule.To address this question, we compile a dataset of 90 nonredundant protein binding Nbs for which Nb‐antigen co‐crystal X‐ray structures are available, allowing us to identify and study a diverse set of Nb paratopes. Early studies examined sequence and structural diversity in the hypervariable loop regions of small sets of Nbs.22.1Lama glama (llama), 24 from Camelus dromedarius (camel), and 6 from Vicugna pacos . The Nbs in the dataset bind to 72 structurally unique epitopes, where a unique epitope is defined as having <50% of epitope residues in common with another epitope in the dataset. The set of 90 Ab‐antigen complex structures were chosen at random from those used in a recent study.We built two datasets of co‐crystal protein complex structures, each containing a Nb‐antigen or Ab‐antigen protein complex. Our novel dataset consists of 90 nonredundant protein binding Nbs for which Nb‐antigen co‐crystal structures are available in the PDB.2.2Sequences of Nb VHH domains were extracted from the PDB files and aligned using the online tool ANARCISimilarly, sequences of Ab VH domains were extracted from the 90 PDB files contained in our dataset and aligned using the same approach. In the resulting alignments the distinction between framework and hypervariable loop regions were clear, and concurred with visual inspection of the 3D structures, shown in Figure Throughout this work, we refer to amino acids by their aligned sequence positions. In our alignments, the variable loops H1–3 are positions 26–35, 50–59, and 98–116 for both Nbs and Ab VH domains. These loop definitions are based on those used by Sircar et al.2.3All sequence logos were generated by submitting the relevant sequence alignments to the WebLogo server Version 2.8.2.2.4The antigen‐bound Nb and Ab structures were then used to build a global structural alignment for each dataset, shown in Figure To quantify the structural variability of different sections of the Nb VHH domain and the Ab VH domain we calculated the average pairwise root mean square deviation (RMSD) among each set of structures over the residue sets indicated in Tables 3To investigate and compare the molecular code used by Nbs and Abs to generate diverse binding specificities we identified 90 co‐crystal structures for both Nb‐antigen and Ab‐antigen complexes. The set of 90 Nb complexes contains 72 distinct structural epitopes, while the set of 90 Ab‐antigen complexes contains 75 distinct structural epitopes. Using this data we analyze Nb and Ab structural variation in the antigen bound conformation.These data allow us to address the key question of how Nbs generate such a diverse range of binding specificities despite their reduced sequence length and compact single domain architecture. Much work has shown that the six hypervariable loops shown in Figure 3.1In Figure Lama glama, Vicugna pacos, and Camelus dromedaries from the abYsis database.IGHV1, IGHV2, and so forth) that give rise to Abs, compared to the smaller number of V gene segments in IGHVH that are used to produce the majority of Nbs.It is important to verify that the framework conservation shown in Figure The striking finding revealed by Figure The greater framework conservation in Nbs is surprising; as Nbs might be expected to increase their sequence variation, to compensate for the reduced potential sequence diversity due to their small size. Figure Our datasets of co‐crystal structures allow us to identify residues that lie on the interface with the protein antigen—that is, the paratope (see methods). These sequence positions play an important role in determining interaction specificity. Overall, the average number of antigen‐contacting residues is 18.76 per VHH structure, compared to 16.01 per VH structure and 24.91 per VH‐VL unit.In Figure 3.2Structural variation of the framework region is another potential source of Nb diversity. To examine this we built structural alignments of Nb VHH domains, Ab VH domains, and Ab VH‐VL complexes. Figure To measure the structural diversity present in these datasets we first extracted the C‐α atomic coordinates corresponding to the framework regions. We then used the Procrustes (Kabsch) algorithm to find the linear transformation resulting in the optimal alignment for each pair of crystal structures and built the set of all pairwise alignments for each dataset.t test, P = 3.14 E –33) reduction in framework structural variability compared to Ab VH domains, allowing us to conclude that Nbs do not increase diversity by varying their FR structure.We find that the 90 Nb structures align relatively tightly over the framework regions, with an average pairwise RMSD of 1.01 ± 0.31 Å measured over the C‐α atoms of the 87 aligned framework residues. The equivalent value for the 58 apo form Nbs is similar to this, at 0.95 ± 0.28 Å. In contrast, the 90 Ab VH domains have an average pairwise RMSD of 1.12 ± 0.50 Å measured over the 87 framework C‐α atoms. This RMSD increases to 1.39 ± 0.51 Å if the structural alignment is carried out over the FRs from both VH and VL domains. This suggests that Nbs display a slight yet significant  is 3.36 ± 1.11 Å for Nb VHH domains compared to 1.60 ± 0.92 Å for Ab VH domains. We note that aligned positions with particularly large RMSD values are concentrated at the N‐terminal end of the H1 loop. If the alignment is instead carried out across the H1 loop itself, the average pairwise RMSD values reduce to 2.06 Å for Nb VHH domains and 1.03 Å for Ab VH domains. The larger Nb H1 RMSD values may be partly explained by the fact that the conserved Phe at position 29 in Abs . In contrast, the average pairwise RMSD of 1.86 ± 0.74 Å for Nb H2 loops (1.93 ± 0.70 Å for apo Nb structures) suggests slightly greater backbone structural variation than Ab H2 loops at 1.78 ± 0.70 Å RMSD. This slight but significant  increase in Nb H2 loop average pairwise RMSD is greater if calculated across structural alignments of H2 loops for each dataset: 1.20 Å for bound Nb VHH domains compared to 1.00 Å for Ab VH domains. This is surprising, given the decrease in Nb H2 loop sequence variation, but could reflect differences in amino acid composition: Nb H2 loops feature small residues such as Gly and Ser permitting the tight backbone turn seen in a number of Nb structures, while the Ab H2 loop shows a bias toward larger residues, see Figure Our analysis finds that Nb H2 loops are less sequence‐variable than Ab VH domain H2 loops. The mean Hamming distance is 0.69 ± 0.14, corresponding to seven out of ten differences between each pair of Nb H2 loops, compared to 0.76 ± 0.15 or nearly eight out of ten differences between each pair of Ab VH domain H2 loops. A two‐tailed unpaired t test at the 0.05 significance level shows this difference is statistically significant (P = 6.28 E –7). In addition, the set of Nb H3 backbone structures are notably more structurally diverse than the Ab H3 loop superposition shown in Figure Finally, we turn to the H3 loops. Both Nb and Ab H3 loops vary significantly in length. The mean Nb H3 loop length in our dataset is 15.19 ± 4.23 residues (median length 15), while the mean Ab H3 loop length is 12.09 ± 3.81 residues (median length 12). Loop length distributions are shown in Figures cant P = .28 E –7.4Nbs provide a simple system with which to probe the question of how homologous amino acid sequences can encode a vast diversity of antigen binding specificities. These small single domain antibodies generate high affinity binding specificity against a huge range of protein antigens, despite being half the size of the antigen‐binding VH‐VL units found in classical Abs, and furthermore lacking the structural variability provided by the VH‐VL domain interface.IGHVH family of V genes, and our findings may support the hypothesis that this family evolved via duplication of a single IGHV3 gene family.IGHV4 and IGHV3 V gene origins, and so the pool of V gene sequences may not be as restricted as previously thought.We find that Nbs are notably more conserved in sequence and structure across their framework regions than classical Ab VH domain sequences. Furthermore, the Nb H1 loop is no more variable in sequence than the Ab H1 loop, and the Nb H2 loop is significantly less sequence diverse than its VH domain counterpart. Nb VHH domains are constructed from the Aside from their germline origins, the requirement for VHH domains to remain soluble in isolation without a partner light chain domain may provide a strong constraint on which framework sequence changes can be accepted. The finding that Nbs are more conserved in sequence and structure across their framework regions, suggests that the framework is not greatly exploited by Nbs to increase number of binding specificities that can be encoded.How then can Nbs generate such a diverse range of binding specificities? This work identifies three potential mechanisms. The first mechanism concerns the significantly increased structural diversity exhibited by Nb H1 and H2 loops. These loops do not display greater sequence variation than their Ab counterparts, yet our structural analysis shows that they do exhibit much greater structural variability. This structural diversity may be enabled by distinct loop sequence features compared to Abs—for example, fewer Nb H1 loops contain a stabilizing F29, and Nb H2 loops use a greater proportion of small residues such as Ser and Gly, which increase structural flexibility. Regardless of how Nbs sample a greater variety of loop backbone conformations, the fact that they do makes an important contribution to their ability to achieve high specificity antigen binding.In accordance with previous work, we find that Nb H3 loops tend to be on average longer than Ab H3 loopsThe third mechanism has not to our knowledge been previously reported in the literature. Our co‐crystal structure dataset reveals that Nb paratopes are drawn from 50 aligned sequence positions, significantly broader than the 35 positions employed by classical Ab VH domain paratopes. This is despite the fact that on average each Nb uses an average of only 2.75 additional antigen‐contacting residues compared to Ab VH domains. This suggests a novel mechanism that Nbs exploit to generate diverse binding specificities. The ability to draw paratope residues from a larger set than VH domains will promote diversity of both the shape and the physical properties of the antigen‐binding interface, enabling Nbs to use a broader range of their surface to interact with their cognate antigen through different binding modes. Perhaps surprisingly, the aligned Nb sequence positions with high contact propensity are not particularly sequence diverse. Figure In summary, we have found that Nbs do not appear to generate specificity‐enabling diversity through increased sequence or structure diversity in the framework. It is particularly surprising that, compared to Abs, Nbs do not exploit increased sequence variation in loops H1 and H2 to compensate for the loss of the VL domain. However, Nbs do exhibit increased structural variation, in particular in the H1 loop. The Nb H3 loop is the only part of the domain with greater sequence diversity than Ab VH domains; and the majority of this increased sequence diversity is achieved through the incorporation of, on average, only 3–4 additional residues. Apparently the loss of the additional diversity that would be generated by a cognate VL domain is compensated for using a very small insertion, coupled with the freedom to sample more diverse loop conformations. This indicates that the capacity for molecular specificity in a small protein domain is much higher than we might expect based on classical Abs, suggesting that there is exciting potential for generating high affinity specific binding to a diverse range of targets using short amino acid sequences that are relatively constrained.Additional Supporting Information may be found online in the supporting information tab for this article.Supporting InformationClick here for additional data file.


The folding and targeting of hydrophobic transmembrane domains poses a major challenge to the cell. Several membrane proteins have been shown to gain some degree of secondary structure within the ribosome tunnel and to retain this conformation throughout maturation. However, there is little information on one of the largest classes of eukaryotic membrane proteins; the G protein-coupled receptors (GPCRs). In this study we show that the signal anchor domain of GPR35 remains in an extended conformation whilst exiting the ribosome tunnel, the polypeptide chain then forms interactions with components of the SRP targeting pathway, and the Sec61 translocon, resulting in a compacted conformation prior to integration into the ER membrane. We conclude that transmembrane structure is most likely adopted after the domain leaves the ribosome tunnel and that the interaction of the signal anchor with SRP is dependent on the native levels of hydrophobicity within the first transmembrane domain. Therefore, we propose a mechanism by which the first transmembrane domains of multi-spanning membrane proteins adopt compacted structures following SRP targeting but before insertion into the ER membrane.The online version of this article (10.1007/s10863-019-9785-0) contains supplementary material, which is available to authorized users. The co-translational targeting of integral membrane proteins (IMPs) poses a major challenge to the cell, as they must remain insertion competent while their highly hydrophobic transmembrane domains are transferred from the ribosome, through the aqueous cytosol and into the lipid bilayer, via the Se61 machinery (SecYEG in bacteria). In the last decade, increasing amounts of research has suggested that the biogenesis of integral TM domains may begin far in advance of reaching the translocon.The ribosome tunnel itself has been highlighted as one of the major sites for the generation of both secondary structure, in the form of α-helical domains, and tertiary structure, such as small hairpin–like domains, in membrane proteins . During the initial stages of SRP mediated targeting, the ribosomal protein L17 in eukaryotes and the tunnel loop domain of L23 in the prokaryotic ribosome were proven to interact specifically with hydrophobic membrane segments. Biochemical and biophysical assays have identified that these proteins play a key role in sensing the nascent chain, and subsequently initiate downstream events that stabilise structure that may already be present in the nascent polypeptide chain   maintaining a similar trend to the previous assays.To validate the pegylation data gathered on GPR35, two subsequent membrane proteins were tested as controls; Bacterioopsin (Bop)  would require approximately 30aa to traverse the ribosome tunnel, where as a fully compacted peptide would require approximately 65aa (1.5 Å/aa) to span the same distance (Lu and Deutsch 3), we attempted to monitor interactions between the nascent chain and components of the SRP targeting pathway, and imply from these the conformational changes occurring in the signal anchor domain as it encounters SRP.To explore further the conformational changes occurring in the nascent chain of GPR35 as it becomes exposed at the exit site of the ribosome, and subsequently undergoes the process of membrane targeting, an assay was set up to utilize the prospective interaction with SRP. Using the chemical cross-linker bis(sulfosuccinimidyl)suberate  were available. As the bacterial and eukaryotic SRP pathways share common ancestry, we were confident that interactions between the GPR35 nascent polypeptide and the components of the prokaryotic system take place. Radiolabelled intermediates ranged from 25-65aa (from the PTC to K20), at increasing at intervals of 10aa. This range allowed us to explore the interactions taking place both during and after TM1 has traversed the ribosome tunnel. In the absence of the cross-linker molecule BS3, the translated nascent peptides can be seen to increase in size as they increase in length from 25-65aa , found 5aa upstream of the signal anchor, makes the GPR35 WT construct suitable for this cross-linking assay as it has a native marker lysine (MK) for interacting with BS0, found 3, translation product representative of each intermediate could be detected. Also, present in each sample at approximately 16 kDa was a double band representative of the protein haem  and an unknown band at ~28 kDa. In the presence of BS3, cross-links between the nascent chain at lengths 55 and 65aa could be detected with a protein of approximately 50 kDa  expression system, intermediates of the same length and range were generated Fig. e, producIt has been suggested that both ∝−helical structure and hydrophobicity play a key role in initiating an interaction between the nascent chain and SRP at the exit tunnel of the ribosome , C-terminus (ΔCT) or entire signal anchor domain (Δ4E) Fig. a. Substi3 was then carried out using the 55aa intermediate (K20 is 55aa from the PTC), as this peptide length showed cross-linking to uL23, Ffh and SRP54. As before, the uL23 protein was used as a cross-linking control in the S-30 expression system and was shown to interact in the same manner with all four intermediates following immunoprecipitation  were added to provide an ER membrane component and the end point for GPR35 insertion. The initial experiment to test the success of GPR35 insertion into the ER membrane was carried out using a digestion assay. This relied on the protease activity of proteinase K (PK) to assess whether or not integrated domains of mature GPR35 had gained protection from the membrane bilayer , which is capable of removing sugar group generated from a glycosylation event. In the presence of Endo H, the higher MW bands associated with glycosylation were successfully removed  long enough to interact with the OST found adjacent to the Sec61 translocon and assessed N-linked glycosylation at the native site at the N-terminus of GPR35 . At this intermediate length, for an interaction to be occurring between the nascent chain and SRP, the signal anchor would have traversed the tunnel and be located in the vestibule, with its N-terminus protruding from the exit port. Although, the GPR35 nascent chain meets with SRP in such an extended conformation, we are uncertain whether the entire TM domain remains so. Structural data analysing the interaction between a signal sequence and the M domain of SRP suggests 10 extended residues would be unlikely to fit in the hydrophobic groove  or Rabbit Reticulocyte Lysate (RRL) system, GPR35 was placed under the control of a T7 promoter. For pegylation experiments, a C8A mutation was carried out to remove a native cysteine residue. A marker cysteine (MC) residue, essential in the pegylation process, was introduced 10aa upstream of the signal anchor domain by a W15C mutation to yield pGPR35C (pTrc99a) and pcGPR35C (pcDNA3.1). Mutations that affected the properties of the first TM domain were incorporated into pGPR35 and pcGPR35; L27E, L31E, L34E and L40 resulted in pGPR35Δ4E; L27E and L31E resulted in pGPR35ΔNT; L34E and L40E resulted in pGPR35ΔCT. For experiments requiring glycosylation of pcGPR35, a single native glycosylation site was used or a second site was engineered by introducing an S residue between N6 and T7. This yielded the construct pcGPR35-gly. Mutations affecting secondary structure of the signal anchor domain were incorporated into pcGPR35-gly; L31P and L40P resulted in pGPR35-gly2P. Amplification reactions were carried out using an ExTaq PCR kit (TaKaRa), and site-directed mutagenesis was carried out using the QuikChange system (Stratagene).All plasmids used in this study are listed in Table S-30 extract was prepared from strain C41 essentially as described previously , 15 μL S-30 extract, 20 μCi [35S] methionine, and an antisense oligonucleotide to SsrA at a concentration of 200 ng/mL. Reactions were incubated at 37 °C for 30 min and chilled on ice for 5 min.35] methionine radiolabelled proteins in vitro; reactions were performed in varying volumes principally as specified by Promega Protocol and Application guide.For WG and RRL systems, in vitro transcription with T7 RNA polymerase was carried out on amplified DNA samples at 37 °C for 2 h. Purified RNA was used to generate . The average percentage pegylation is calculated from an n = 3 replicates, error bars are + SD. (PNG 1049 kb)Figure S2F0c, compacts in the ribosome. F0c, is a bacterial membrane protein, specifically subunit c of the Fo component of the ATP synthase. (a) The full 79 amino acid sequence of the F0c protein is shown with the position of the single cysteine (C5) residue, required for pegylation, labelled as amino acid 1. TM domains 1 and 2 are highlighted using boldface. Intermediate lengths of F0c, used for in vitro pegylation experiments, are underlined and the length is denoted below in amino acids (aa). (b) The first TM domain of the F0c protein was shown to compact by Robinson et al. (c) Quantification of the total pegylation in the S-30 transcription/translation system, for all intermediates. The y-axis shows percentage of intermediates successfully pegylated and was obtained by pixel densitometry (Image-J) and calculated using [pegylated band/ (unpegylated band + pegylated band)]. The average percentage pegylation is calculated from an n = 3 replicates, error bars are + SD. (PNG 917 kb)n et al. . Autoradn et al. , showing


Here, we report protein intrinsic disorder in a plant molecular system, which despite its prevalence is less studied. We present a detailed biophysical characterization of the entire cytoplasmic N-terminal domain of Brassica napus diacylglycerol acyltransferase, (DGAT1), which includes an inhibitory module and allosteric binding sites. Our results demonstrate that the monomeric N-terminal domain can be stabilized for biophysical characterization and is largely intrinsically disordered in solution. This domain interacts with allosteric modulators of DGAT1, CoA and oleoyl-CoA, at micromolar concentrations. While solution scattering studies indicate conformational heterogeneity in the N-terminal domain of DGAT1, there is a small gain of secondary structure induced by ligand binding.Proteins with multifunctional regulatory domains often demonstrate structural plasticity or protein disorder, allowing the binding of multiple regulatory factors and post-translational modifications. While the importance of protein disorder is clear, it also poses a challenge for  Disordered proteins have been identified as a unique protein tribe4, which includes entire polypeptide chains referred to as “intrinsically disordered proteins (IDPs)” or localized domains of proteins referred to as “intrinsically disordered regions (IDRs)”. Interestingly, IDRs with over 40 amino acid residues are found in more than 33% of the sequenced eukaryotic proteins6. They differ from globular proteins in having a unique signature in a charge-hydropathy plot7. The disordered regions are enriched with charged residues along with low content of hydrophobic residues. Sequence analyses show that they are abundant in disorder-promoting residues  and depleted of order-promoting counterparts 10. Prediction of disordered regions in proteins is not only based on the charge-hydropathy correlation, but also on various other physico-chemical property scales such as helix propensity, strand propensity, and aromaticity9. IDRs/IDPs, for the most part, lack single ordered structure under physiological conditions (at least in vitro)11. Hence, they exist as heterogeneous ensembles of conformers where due to temporal fluctuations, a single set of backbone Ramachandran angles cannot be used to define their conformational properties. Thus, the IDPs/IDRs are known to have large hydrodynamic radii relative to folded proteins with a similar chain length. The lack of well-defined tertiary structure allows these disordered proteins to discriminate themselves from their folded counterparts, not only based on structure but also in terms of their function12.The classic protein structure and function paradigm reveals three-dimensional structures of proteins with precisely positioned functional groups that facilitate interactions with substrates or protein partners. However, numerous studies over the past few decades have demonstrated the functional significance of protein disordered regions as an exception to this general rule14, such as a larger interaction surface that can easily overcome steric hindrances, an ability to interact with structurally diverse partners, the ability to gain a specific conformation upon interaction with partners, the ability to stay substantially disordered in bound state, an efficient regulation through post-translational modifications, and the ability to be associated with interaction cascades. Therefore, IDRs are abundant in the proteins of higher order organisms associated with recognition, regulation and signaling17. Intrinsic disorder, being crucial for various biological functions, exists in various environments. The amino-acid compositions of IDPs/IDRs are specific for their local/global environment. For instance, in transmembrane (TM) proteins, TM segments are well structured due to their low dielectric constants within the lipid bilayer18, the exterior of these domains being apolar19. IDRs being highly charged and polar are expected to localize within regions external to the membrane20. Cytoplasmic domains of TM proteins, however, contain three-fold more disorder than their extracellular counterparts21. IDRs are also known to be potential drivers of membrane curvature, an event crucial for cellular physiology. Reports suggest that 50% of TM proteins have IDRs with more than 30 amino acid residues, and these are highly phosphorylated22.The lack of rigid structure for IDRs/IDPs provides a broad functional advantage3. The formation of a versatile complex network is effective for responding to change in unavoidable environmental stress. Recently, an IDR spanning the N-terminal cytosolic domain of an intramembrane enzyme, diacylglycerol acyltransferase1 (DGAT1) from canola-type Brassica napus, has been identified23. The IDR spans amino acid residues 1–80, while residues 81–113 have a folded structure. DGAT1 (EC 2.3.1.20) catalyzes the acyl-coenzyme A (CoA)-dependent acylation of sn-1, 2-diacylglycerol (DAG) to produce triacylglycerol (TAG) and CoA25. TAG serves as an energy source for germination in plants, a component of edible oil, and a petrochemical alternative. This enzyme has a substantial effect on carbon flux into seed oil and hence a molecular understanding of the regulatory mechanism of DGAT1 is essential for its genetic manipulation to increase seed oil production in oleaginous plants26.Intrinsic disorder in plant proteins has been reported to be essential for the stress responseB. napus DGAT1 (BnaDGAT1) is a polytopic membrane protein localized on the endoplasmic reticulum (ER) with its N-terminal domain (residues 1–113) localized to the cytoplasm.  DGAT1 have been shown to interact with acyl-CoA29. Furthermore, crosslinking studies have shown that BnaDGAT1 has the ability to self-associate in membranes, but this is dependent upon the presence of the first 80 residues of the cytoplasmic N-terminal domain28. Various studies have suggested that plant DGAT1 enzymes are allosterically regulated30. In BnaDGAT1, acyl-CoA has been shown to act as both positive effector and acyl-donor whereas CoA is a negative effector. Both effectors have been shown to interact with the same non-catalytic site within the cytoplasmic N-terminal domain of the enzyme23. Lipidex-1000 binding assays have shown that CoA can displace acyl-CoA from this site28 and kinetic analysis has shown that CoA is a non-competitive inhibitor of the enzyme28. The acyl-CoA/CoA binding site has been shown to be located within amino acid residues 81–113. In contrast, an autoinhibitory domain within residues 1–80 has been identified; truncation of BnaDGAT1 to remove the first 80 amino acid residues resulted in an increase in enzyme activity23. Despite the growing biochemical evidence, there is lack of structural and biophysical understanding of the entire regulatory region of DGAT1 due to the disordered nature of this domain.1-113) of isoform BnaC.DGAT1.a23 and a truncation, the autoinhibitory domain containing region (BnaDGAT11-80), using isothermal titration calorimetry (ITC). Furthermore, we used circular dichroism (CD) and small-angle X-ray scattering (SAXS) to understand the conformational changes associated with interactions with these effectors. In addition, we provide biophysical evidence that the autoinhibitory region also interacts with CoA or acyl-CoA bringing about secondary structural changes. While both of these regions of BnaDGAT1 have the capacity to bind the above ligands, we show the entire region, amino acid residues 1–113, is needed for optimal binding and the secondary structural changes associated with this event.In this study, we investigated the interaction of CoA and oleoyl-CoA with the cytoplasmic N-terminal region  or the polypeptide beyond amino acid residue 113 which contains the multiple TM domains. DisoPred31  is intrinsically disordered23. Although there are 14 predicted phosphorylation sites in DGAT1 from Arabidopsis thaliana  N-terminal domain (residues 81–113)1-113, and the autoinhibitory domain, BnaDGAT11-80, with N-terminal His-tags , were recombinantly expressed in BL21(DE3) strain of Escherichia coli and purified using Ni-NTA affinity chromatography. The mobility of these recombinant proteins on SDS-PAGE was atypical as they migrated slower than expected, based on their expected molecular masses   and attributed to the lack of hydrophobic amino acid residues and anomalous interaction with SDS38. It was observed that the purified proteins were prone to oligomerization and aggregation in a concentration and time dependent manner. Additionally, these purified constructs were susceptible to proteolysis despite the addition of protease inhibitors. In preliminary experiments, TEV protease was added to cleave His tag from BnaDGAT11-113, but this required long incubation times (2 hours to overnight), after which degradation and nonspecific oligomers were observed when proteins were applied on a size exclusion column. Furthermore, concentrating the protein (>200 μM) after Ni-NTA affinity chromatography also led to oligomerization. To mitigate these issues, several steps were taken. Because milligram amounts of protein were required for the biophysical studies presented in this manuscript, the TEV protease cleavage step was omitted, and we worked with His-tagged DGAT1 proteins. Notably, the CD spectrum indicate the disordered nature of our untagged23 and His-tagged proteins in solution, however, as expected, differences in the spectral properties can be seen  to avoid aggregation upon storage at 4 °C.The full-length cytoplasmic domain, BnaDGAT11-113 and BnaDGAT11-80 yielded a monodisperse peak on SEC  for DGAT1 constructs were calculated from the standard curve using folded globular protein standards. The values were 2.2 nm and 1.7 nm for BnaDGAT11-113 and BnaDGAT11-80 respectively. The apparent molecular mass calculated from above was 21 kDa and 10.5 kDa respectively. Given the predicted non-globular (extended) nature of these proteins, the exact molecular mass and oligomeric state was calculated using in-line classical light scattering in tandem with SEC (SEC-MALLS)  and 8.6 kDa (BnaDGAT11-80). The apparent molecular mass calculated from SEC-MALLS were lower than that calculated using SEC standards demonstrating the nonglobular and monomeric nature of the purified constructs. In addition, BnaDGAT11-113 or BnaDGAT11-80 preincubated with CoA or oleoyl-CoA were assessed using the above biophysical method to validate whether any change in conformation occurs in the presence of ligands. Peaks eluted at similar positions compared to their unbound forms suggested that the interaction with ligand did not cause a major change in the overall monomeric state or its hydrodynamic radius.Freshly purified samples of BnaDGAT1SEC Fig. . The hyd81-113 are involved in the interaction with CoA23. However, the thermodynamics of interaction of CoA ligands with the cytosolic domain of BnaDGAT1, or any other DGAT1, has not been reported. In preparation for isothermal titration calorimetry (ITC), standard buffer optimization was assessed using fluorescence-based thermal shift assay39, which demonstrated that BnaDGAT11-113 was stable in sodium phosphate buffer (pH 7.5), with a Tm of ~38 °C   Fig. . The the1-113 and BnaDGAT11-80 as a consequence of binding oleoyl-CoA or CoA. The CD spectra of His-tagged BnaDGAT11-113 is non-zero between 250-270 nm  observed in the NMR studies23. Contrary to the above, BnaDGAT11-80 showed a decreased helicity upon ligand binding, along with a decrease in random coil nature as demonstrated by the decrease in negative ellipticity at ~200 nm  experiments were performed to obtain detailed 3D structural information on this disordered region of DGAT1. SAXS data was collected after the protein had been passed over an inline SEC in order to separate the components of the mixture  derived from the Guinier analyses for apo- BnaDGAT11-113 was 32.14 +/− 0.82 Å for the protein. Subtle differences for BnaDGAT11-113 with different ligands were observed; indicating absence of overall large scale structural changes with this domain when incubated with acyl-CoA or CoA in solution. Given the experimental radius of gyration collected from Guinier analysis, we calculated the theoretical Rg using simple power-law relationship for intrinsically disordered proteins40 as stated below:The scattering profiles, for the various forms of BnaDGAT1DRs Fig. . The conDRs Fig. . The rad2] as a function of vector [q] denotes the Kratky plot, which is a qualitative analysis of the scattering profile42. The Kratky plots do not have maxima, but instead have a short plateau followed by a monotonic increase at higher value of [q]. This indicates the presence of a highly extended particle for BnaDGAT11-113, thus confirming the intrinsically disordered nature of the N-terminal region of BnaDGAT11-113 .q113 Fig. .23 was employed to generate a rigid model of residues 81–113, and then the program AllosMod-FoXS, which can fill missing fragments, was used to generate a full-length initial model for MultiFoXS. It is unlikely that the system does exist in one conformation as suggested by the Kratky plot  is intrinsically disordered. Our NMR structure of BnaDGAT181-113 demonstrated that this is the minimal structural unit in the cytosolic domain consisting of a defined loop (Ala94-Pro102) followed by a short helix (Leu103-Ser112)23. Furthermore, bioinformatic analysis of the N-terminal domain indicates the presence of 14 potential phosphorylation sites , with hexa-His tag at the carboxyl end, leads to a protein segment that self-associates28, the N-terminally tagged recombinant protein segments used in this study favored the monomer in solution. During SEC used to purify these recombinant proteins, it was noted that there was, however, a tendency for these to aggregate if the sample concentration applied to the SEC column was above 200 μM. Our analyses show that the N-terminal domain of BnaDGAT1 has higher binding affinity for oleoyl-CoA than CoA. This is in line with our previously conducted kinetic analysis on the above region23. Interestingly, BnaDGAT1-80 is also capable of interacting with CoA and oleoyl-CoA, but there is a ten-fold decrease in affinity compared to the full cytosolic domain BnaDGAT11-113. Our previous in vivo analysis of truncated forms of BnaDGAT1 suggested that BnaDGAT11-80 represents an autoinhibitory region of the cytosolic domain of BnaDGAT123. The fact that this region also interacts with CoA or acyl-CoA supports the above proposed regulatory role of this region.In our previous study, we demonstrated with NMR titration analysis, docking studies and mutagenesis that BnaDGAT128. More recently, we further demonstrated full-length BnaDGAT1 forms a dimer in yeast membranes23. Upon truncation of this disordered region, the first 80 residues, of the cytoplasmic N-terminus of BnaDGAT1, crosslinking was no longer observed, suggesting dimerization occurs via this N-terminal region23. Furthermore, the N-terminal domain of murine DGAT1 was also shown to be important for dimerization and tetramerization28. It should be noted, however, that there are slight differences in sequence between the N-terminal domains of these two species (25% sequence identity). This agrees with previous findings that long IDRs have high sequence variability44. Nonetheless, we previously found that the nature of the amino acid residues within the N-terminal domain identified to bind CoA is conserved between animal and plant sequences23. In the current study, however, where we study the first eighty residues, we primarily observe a monomeric species for the N-terminal region of BnaDGAT1. Taken together, we suggest that under the current conditions the BnaDGAT11-80 region alone is not sufficient for dimerization. It is possible that tethering to the membrane allows for close proximity of the cytoplasmic N-terminal residues, which may facilitate dimerization. It is therefore likely that the N-terminal domain structure is influenced by the presence of the DGAT1 membrane domain or the lipid bilayer interface. Indeed, we have recently demonstrated that the negatively charged lipid phosphatidate in mixed micelles enhanced BnaDGAT1 activity, and this activation was attributed to the interaction of the N-terminal domain of BnaDGAT1 with this lipid45.Early studies on the N-terminal domain of BnaDGAT1 suggested that they primarily formed monomers, with dimer and tetramer species observed with crosslinking. SEC analysis of the N-terminal domain also supporting self-association, however at this time the nature of the intrinsic disorder in this region was not known1-11628. In addition, although the amino acid sequences of BnaC.DGAT1.a and BnaA.DGAT1.b exhibit a very high level of identity, most differences in sequence occur in the protein segment encoded by the first exon46. Thus, the nature of truncation tagging  and/or differences in sequence may have influenced possible oligomerization versus preservation of the monomeric species. We conclude, given our current understanding of this region from this work and our more recent papers, that the N-terminus is the preferred location to insert an affinity tag to obtain a stable homogeneous species for biophysical characterization and given the structural features at the C-terminus of the N-terminal domain. Interestingly, adding an N-terminal tag to amino acid sequences representing native BnaDGAT1 isoforms has previously been shown to mask the deleterious influence of the native N-terminal sequences, resulting in increased production of recombinant BnaDGAT1 polypeptides in vivo in a yeast system42. The resulting yeast microsomes also exhibited an increase in in vitro DGAT activity of to about 150-fold.The BnaDGAT1 truncations used in the current study involved N-terminal tagging of the first 80 amino acid residues of isoform BnaC.DGAT1.a, whereas an earlier study was based on a C-terminally tagged BnaA.DGAT1.1b1-113 upon titration with trifluoroethanol had been previously reported23. Trifluoroethanol is known to stabilize protein secondary structure by lowering the dielectric constant of the solution thus mimicking a ligand bound state47. Our CD results indicate that indeed there is a gain in helicity upon ligand binding in BnaDGAT11-113 and a decrease in random coil structure in case of BnaDGAT11-80. It is interesting to note that the CD profile of full length BnaDGAT123 exhibited predominantly helical nature and the truncated N-terminal fragments (BnaDGAT11-113 and BnaDGAT11-80) used in the previous23 as well as current study indicate predominantly disordered structure. This could again indicate that the presence of membrane domain and/or lipid bilayer could promote the structure of this cytosolic domain.Owing to their inherent flexibility, very few biophysical techniques can be implemented to explore the structural details of IDRs. The increase in helix propensity of BnaDGAT11-113. Transient local secondary structure could be induced upon interaction with effectors while preserving the extended conformation of the protein in solution. However, these minor changes are not typically visualized using SAXS with IDRs containing extended conformation and high structural heterogeneity. This scenario has been previously observed with the adaptor protein, Juxtanodin38.For 3D-structural analysis, SAXS data collection was carefully performed in order to minimize radiation damage and concentration-dependent inter-particle interactions, followed by further assessment of background-subtracted scattering profiles. Because of the intrinsically disordered nature of the hydrophilic N-terminal domain of BnaDGAT1, conformational heterogeneity made the data interpretation challenging. Our multi-state ensemble modeling using the program MultiFoXS fitted well to the experimental profile. This is consistent with the SEC profile that the samples are all monomeric in solution. The resulting over-fitting was avoided by computing ensembles of multi-state models that fitted the SAXS data, thus highlighting the conserved feature, which was the C-terminal region of BnaDGAT148. The latter have been reported as molecular recognition elements (MoRFs), known to play a role in binding events50. MoRFs could fold as helices observed in short amphipathic stretches within long disordered sequences, β-strand51, or form irregular structures. A correlation between the SEC-MALLS and SEC-SAXS data is observed as the hydrodynamic radius and the radius of gyration did not change substantially upon incubation with the ligands. Our CD experiments indicate, however, that the binding event is subtly coupled with folding. Although the overall tertiary structure is in the extended conformation, there is a slight gain in the local secondary structure upon incubation with ligands. The interaction with the ligands could lead to formation of transient secondary structure. Such intricate secondary structural changes, however, are not large enough to be captured at present with low resolution SAXS data.It has been previously reported that IDRs can stochastically fluctuate among multiple states such as coil-like or one with localized secondary structures1-80 is unstructured according to CD experiments and is predicted to have a cluster of phosphorylation sites, reflecting a possible role in the regulation of the enzyme by the N-terminal domain. As an effect of the structural heterogeneity, the IDR in BnaDGAT1 could form modular assemblies of independently foldable regions, partially folded regions, or regions that never fold. Such a scenario has been observed in nuclear pore complexes where the IDRs containing multiple Phe-Gly motifs adopt various conformations for specific functions52.The IDR BnaDGAT153. Additionally, IDRs are known to interact with their targets with high specificity and low affinity, which is also observed in our case as the binding occurs with effectors in micromolar range. This rapid association, initiation of signaling and easy dissociation allows the IDRs to coordinate regulatory events in space and time. As mentioned previously, the level of BnaDGAT1 activity during seed development can have a substantial effect on the flow of carbon into TAG. Considering the presence of multiple phosphorylation sites necessary for signaling45, and the ability to interact with acyl-CoA and/or CoA, this work adds to the growing body of literature that the cytoplasmic N-terminal domain of BnaDGAT1 not only regulates the enzyme activity by interacting with multiple effector proteins and ligands but also could aid in metabolic “pathway crosstalk” with nonrandom conformational preferences upon association with putative partners. Thus, this work sets the foundation for identifying other interactors of the IDR of BnaDGAT1 and other forms of the enzyme from various organisms that produce TAG.The structural adaptability of IDRs facilitates their interaction with diverse partners with overlapping binding sites, a consequence of its linear conformation. This could provide an increased interaction surface area and avoid steric clashes. Consequentially, the speed and efficiency of the above interactions are also increased because of less stringent spatial orientation requirements. It has also been observed that IDRs could adopt different local secondary structures upon binding to different binding partners. The disordered C-terminal domain of p53 forms helix, sheet, or two different types of coils upon interaction with four different targets31, Globplot232 and FoldIndex33. Disorder enhanced phosphorylation predictor35 was used to predict the potential phosphorylation sites in this domain.The analysis for the folding properties of cytoplasmic N-terminal domain of BnaDGAT1 was performed using DisoPredE. coli (Invitrogen) was used as an expression system for recombinant pET16b encoding 10 x His-BnaDGAT11-113 or 6 x His-BnaDGAT11-80. Genetic constructs were designed such that each recombinant polypeptide segment contained an N-terminal tag as shown in the supplemental data. Preinnoculum was prepared by inoculation of single colony to 100 ml of Luria Broth media with 100 mg/mL ampicillin grown for overnight in a rotary shaker. Overnight culture (20 mL) was used to inoculate 1 litre of LB with ampicillin and 6 litre of inoculated media was grown at 37 °C until the optical density (OD600) reached 0.7 to 0.8. Expression of recombinant protein was induced by addition of 1 mM of isopropyl-b-thiogalactopyranoside (IPTG). After 5 hrs of incubation at 30 °C, the cells were harvested by centrifugation at 4000 × g , resuspended in 100 ml of buffer A  supplemented with protease inhibitor tablet (Roche scientific). The lysate was emulsified using Avestin Emulsiflex and further clarified by centrifugation . Ni-NTA affinity chromatography was performed using Ni-NTA beads (Qiagen) incubated with the clarified lysate. The protein was eluted using a gradient mix of buffer A and buffer B  glycerol and 2 M imidazole). The presence of purified protein was observed on Coomassie stained SDS-PAGE. To further obtain monodispersed preparation, SEC was performed in a Superdex 200 (16/60)  with Superdex buffer containing 25 mM sodium phosphate pH 8.0, 300 mM NaCl, 5% (v/v) glycerol.The BL21(DE3) strain of ® rEX Refractive Index Detector, QELS (Quasi Elastic Light Scattering), UV detector (Wyatt Technology).The central peak fractions after the size exclusion column were used for molecular mass determination by inline size-exclusion chromatography-multi-angle laser light scattering (SEC-MALLS). Samples, 50 μl, were loaded at 5 mg/ml onto the SEC-MALLS and the above superdex buffer was used for elution and for differential refractometry. Data was analyzed with ASTRA V software (Wyatt Technology). BSA was used to normalize the MALLS detector. Experiments were performed at 25 °C on an FPLC-managed Superdex 200 10/300 size exclusion chromatography column , coupled to a Wyatt DAWN EOS light scattering detector, Optilab54. Screens were designed with a varied range of buffers pH (3.0 - 10.0) combined with different concentrations of sodium chloride , with and without 5% (v/v) glycerol. Aliquots, 2 µl, of protein (5 mg/ml) were mixed with equal volume of 1X dye solution. Aliquots, 2 µl, of the above mixture was added to 18 µl of each screen solution placed in 96 well plates. Each reaction was performed in triplicates. Fluorescence was monitored on an Applied Biosystems 7500 FAST RealTime PCR System with an excitation range of 510–530 nm while the temperature was held for 1 min per degree from 24–95 °C. The emission signal at 567–596 nm was used for analysis. Similar analysis was performed with protein incubated with oleoyl-CoA at different molar ratios . The shift in melting temperature (Tm) indicated binding.In order to characterize the stability of the BnaDGAT1 N-terminal domain in different aqueous conditions, thermofluor assay using SYPRO Orange dye was conductedKd was obtained using χ2 minimization on a model assuming a single set of binding sites. Three floating variables: stoichiometry (N), binding constant (Kd) and the change in enthalpy of interaction (∆H) were finally obtained55. For the system containing BnaDGAT11-113 and oleoyl-CoA, MicroCal ITC200  was used  and the data obtained was analyzed using SEDPHAT56. The experiments were performed in duplicate. Data shown is mean with standard error.Binding studies for both constructs were performed using VP-ITC Microcal Instruments . Oleoyl-CoA or CoA was dissolved in ITC buffer  at concentration of 130 µM and the resulting solution was placed in the analysis cell. Protein at 1.13 mM was titrated against the cell. Titrations were carried out at 20 °C using 10 µL of syringe solution injected at an interval of 200 s. Control experiments were performed by injecting protein into the cell containing the buffer to rule out the possibility of self association of protein at high concentration 57 and the CDSSTR algorithm58 and SP175 reference data set.Jasco-810 spectropolarimeter was used to measure the far-UV CD spectra corresponding to peptide bond absorption at 10 °C. Spectra were collected for 10 µM of protein in 10 mM of sodium phosphate buffer pH 7.3 in a Quartz SUPRASIL cuvette (Hellma) with a path length of 1 mm. Measurements were made with an increment step of 0.5 nm, an integration time of 4 s per step and a bandwidth of 2 nm. Each spectrum was an average of 5 accumulations. The protein was incubated with ligand at 1:1 molar ratio. The signal due to buffer alone and buffer with ligand was subtracted from that of the protein and protein with ligand respectively. The proportions of secondary structures of the protein was estimated from the [Θ] values between 190 and 240 nm using the DichroWeb server  and for averaging profiles every 5 frames. The final averaged curves were selected, following further assessment for inter-particle interactions and/or aggregations using the program PRIMUS62.SEC-SAXS studies were performed at SSRL Beamline 4–2 to mitigate aggregation, as described previously1-113, the multi-state modeling was performed using the program MultiFoSX, which has been specially designed for samples showing conformational heterogeneity63. To generate an initial model for MultiFoXS, the previously reported NMR structure23 was employed for a rigid model of residues 81–113 and then the program AllosMod-FoXS  was used to fill other missing fragments. 10,000 conformations were generated for conformational sampling. The SAXS profile of each conformation is calculate for SAXS scoring and then multi-state model enumeration is performed iteratively. It simulated up to 5-state models and displayed top 100 and 25 best scoring models for 1–4-state and 5-state, respectively.Since initial analysis indicated high flexibility of the BnaDGAT1Supplementary information