The genetic code shapes the genetic repository. Its origin has puzzled molecular scientists for over half a century and remains a long-standing mystery. Here we show that the origin of the genetic code is tightly coupled to the history of aminoacyl-tRNA synthetase enzymes and their interactions with tRNA. A timeline of evolutionary appearance of protein domain families derived from a structural census in hundreds of genomes reveals the early emergence of the ‘operational’ RNA code and the late implementation of the standard genetic code. The emergence of codon specificities and amino acid charging involved tight coevolution of aminoacyl-tRNA synthetases and tRNA structures as well as episodes of structural recruitment. Remarkably, amino acid and dipeptide compositions of single-domain proteins appearing before the standard code suggest archaic synthetases with structures homologous to catalytic domains of tyrosyl-tRNA and seryl-tRNA synthetases were capable of peptide bond formation and aminoacylation. Results reveal that genetics arose through coevolutionary interactions between polypeptides and nucleic acid cofactors as an exacting mechanism that favored flexibility and folding of the emergent proteins. These enhancements of phenotypic robustness were likely internalized into the emerging genetic system with the early rise of modern protein structure. The catalytic aminoacylation domain of the enzyme contains an amino acid binding site capable of activating a specific amino acid by condensation with ATP to form aminoacyl-adenylate. This activated molecule then esterifies the 2′ or 3′-hydroxyl group of the ribose in the 3′ end of the acceptor arm of tRNA. The activation process is highly specific and involves proofreading Aminoacyl-tRNA synthetases (aaRSs) are multidomain protein enzymes that attach L-amino acids to their cognate tRNAs with high specificity structuralclassificationofproteinsThe evolutionary accretion of structure is the process by which molecules add parts such as substructures or structural modules (domains) to their molecular makeup. The process can take millions of years and is usually driven by improvements in biological function and stability [5.6]. Timelines that describe the gradual appearance of domain structures in evolution have shown that the editing and anticodon-binding domains of aaRSs are late additions to the central catalytic role of their aminoacylation domains The phylogenomic strategy can also link evolution of proteins and RNA . The ageHere we focus on the accretion of proofreading and specificity domains of aaRSs and their interaction with evolving tRNA. We find that the most ancient aaRS domains harbor archaic dipeptidase and ligase activities, suggesting that the primordial peptides and polypeptides they originally produced conditioned the amino acid makeup of modern proteins. To test this hypothesis, we study the sequential overlap of dimers in the amino acid sequence of proteins and consider that these ‘dipeptide’ constituents are relics of ancient amino acid composition. Each dipeptide defines one of ∼400 pairs of two sequential residues or ‘peptide bond types’ that make up protein sequence. We show that dipeptides of protein domains appearing in evolution before the first anticodon-binding domain are significantly enriched in amino acids that are subject to aaRS editing. Remarkably, results drawn directly from phylogenomic and structural information uncover a hidden link between the emergence and expansion of the genetic code and protein flexibility. This link provides important clues to understand one of the most challenging problems that exist in biology.We use timelines that describe the gradual appearance of protein domains in the protein world and substructures in RNA molecules to: (i) unfold the history of aaRS domains and their associated functions, (ii) determine if aaRS domains and tRNA molecules coevolve, (iii) retrodict the origin and evolution of the genetic code, and (iv) find connections between the rise of genetics and patterns embedded in the structure of proteins. Our approach is mostly ideographic  and is strongly grounded in cladistics and reciprocal Hennigian illumination ndFF) was calculated from a published tree of domain structures ndFF was used to construct a timeline of domains, with time flowing from the origin of proteins (ndFF = 0) to the present (ndFF = 1). These ndFF values are good proxy for geological time when trees of domains are used as molecular clocks ndFF = 0–0.024), all of which harbor Rossmann-like α/β/α-layered topologies ndFF = 0.020. The FF of class I enzymes (d.104.1.1) appeared immediately after at ndFF = 0.024. Our timelines indicate that the process of domain accretion started with the CP1 ‘ValRS/IleRS/LeuRS editing domain’ FF  ∼3.3 Gy ago and that class II enzymes were the first to add anticodon-binding domains ∼ 3 Gy ago  . Accretion spanned all three major epochs of the protein world The relative age of individual domains (∼ 0.200) . DomainsSaac) against the age of aaRS domains (ndFF) endowed with editing activities. We assume the age of the FF domain is the age of the associated molecular function Sacc = (2.40±0.79) ndFF; 2R = 0.741; F = 9.10; P<0.0117; total set; Sacc = (1.17±0.17) ndFF; 2R = 0.814; F = 47.87; P<0.0001). The regression line unfolds an evolutionary timeline of archaic editing functions. TyrRS, SerRS, and LeuRS interact with the oldest cognate tRNAs, type II tRNA molecules harboring a long variable loop necessary for tRNA recognition. Their catalytic domains appear very early at ndFF = 0.020–0.024. The most ancient editing functions are pre-transfer and post-transfer mechanisms mediated by TyrRS, SerRS, LeuRS, ProRS, LysRS and MetRS  as a function of the age of anticodon-binding domain FFs (ndFF) and reveals coevolution during the ndFF = 0.196–0.249 interval and late tRNA structural recruitments occurring initially in parallel with recruitment of ribosomal protein domains in the ribosome. An ‘idealized’ timeline partitions the code in three anticodon tRNA-aaRS binding interaction expansions: (A) Pro–Ala, Thr, Gly and His, from ancient to derived (involving class IIa anticodon-binding domains); (B) Val, Met–Ile, Cys, Gln, Arg and Leu (involving class Ia and Ib anticodon-binding domains); and (C) Asp, Asn–Lys (class II), Ser, Glu, Tyr, Lys (class I) and Phe–Trp, resulting from late recruitments.Only 10 aaRSs harbor editing functions, and most amino acid sieving functions of editing domains appeared before anticodon-binding domain recognition of identity elements in the anticodon arm of tRNA . We explnd MetRS . Saac vGroup 1, the most ancient) and type I tRNA , and more modern domains interacting with the bottom half of tRNA  (vis-á-vis each other and describes major and minor groove modes of tRNA recognition by aaRSs (vis-á-vis representation exhibit a coordinated complementarity of C2-G72 and G2-C72 base pairs in the acceptor stem (with an exception of only 3 of 32 comparisons) (vis-á-vis code representation), complementary base pairings are overrepresented in G for major-major groove RGN-NCY and major-minor YGN-NCR comparisons, while being drastically underrepresented in the major-minor RUN-NAY and slightly underrepresented in the minor-minor YUN-NAR complementarity relationships. Representation patterns in the GC alphabet of the operational code are mostly due to aaRSs with amino acid editing properties that are ancient, suggesting an archaic imprint of tRNA identity elements in emerging translation  . We mappby aaRSs . Each starisons) . Within nslation . Since anslation , our patvis-á-vis code representation To unfold the origin and evolution of the ‘standard’ genetic code at the base of the decision tree, we mapped the ancestries of anticodon tRNA-aaRS binding expansion groups A, B and C onto the condensed Group 1 domains are substantially less affected by mutational manipulation than type I tRNA elements interacting with Group 2 and 3 domains  has retained ancestral recognition features of limited specificity, which in biochemistry are considered ancestral An analysis of identity elements in cognate tRNA and their respective role in aminoacylation  domains . MoreoveWe mapped code expansion in sense-antisense codon exchange graphs that retain secondary structure information Group 1 catalytic domains have atypical functions. Amino acid-[Acyl-Carrier-Protein]-ligases (aaACPLs) homologous to SerRSs aminoacylate 4′-phosphopantheteinyl and other prostetic groups of carrier proteins Groups 1 and 2 domains  makeup of protein structures . DipeptiGroup 1, 2 and 3 structures, constant along the FF timeline , with Group 1 amino acids showing the largest and Groups 3 the smallest frequencies , with patterns that were remarkably consistent throughout the timeline. Thus, dipeptides display ancestral specificities of aaRS domain history.Since amino acid and dipeptide signatures manifest in individual folds along the timeline, any composition bias must critically influence emerging protein structure. The analysis revealed a statistically significant progression of frequencies of amino acids specified by quencies . Since lde bonds . Again, ndFF ≤0.2) to those in the entire timeline and identified dipeptides that were specifically overrepresented using the hypergeometric distribution (P<0.01). With only one exception, half of enriched dipeptides involved one and the other half involved two amino acids specified by the ancient editing domains , together with Ala, Val, Ile, Leu, were highly represented in the Urey-Miller experiments To confirm this important finding, we compared dipeptides appearing before anticodon-binding domains , involved the oldest type II cognate tRNAs, which harbor a long variable loop necessary for tRNA recognition. An analysis of identity elements in the acceptor stem of tRNA suggests that these archaic charging specificities are founders of mirror modes of tRNA acceptor stem recognition and the ancient operational code (Ser(CGA) in selected fungal species can be aminoacylated with Ser and Leu by SerRS and LeuRS Group 2 amino acid charging specificities, which involve Val, Ile Met, Lys, and Pro.We find that tRNA isoacceptors coevolve with aaRS domains with pre-transfer and post-transfer editing and trans-editing activities , which wnal code . Class Inal code . Class Inal code  and pattnal code . Primordnal code . We notendFF ∼ 0.2–0.25) confirms that the full bottom half of tRNA and its anticodon loop identity elements, especially complementary triplets (anticodons), unfolded completely ∼3 Gy ago  responsible for modern protein synthesis and the establishment of a fully functional ribosomal machine ∼2.8–3.1 Gy ago Interactions of tRNA with the CP1 domain 3 Gy ago . Protein3 Gy ago . This laGroup 1 TyrRS, LeuRS and SerRS structures, some of which embed atypical molecular functions. Most notably, SerRS-homologous aaACPLs carry ligase functions resembling those of NRPS aminoacylating modules Groups 1 and 2 aaRSs to make a range of cyclodipeptide molecules Group 1 and 2 amino acids were enriched in proteins originating before the appearance of anticodon-binding functions and modern genetics. The enriched dipeptides in the ancient proteins were statistically disfavored in loops and their boundaries with regular structures, but favored in boundaries with turns  capable of forming loops and van der Waals locks, and then proteins smaller than the size of an average compact domain (∼100 residues in length) If indeed, the rates of generation of domains from prior forms were high, what mechanisms could have enhanced domain innovation in the emerging protein world? In our model, we propose that the initial drivers for the production of early peptides and proteins were membrane and vesicle stability, a concept that was previously elaborated A late development of genetics occurring after the inception of protein-catalyzed peptide bond synthesis requires that information in primordial peptides and proteins that preceded mature forms of dipeptidases and ligases be coopted by the emerging genetic system E of our conjecture h, which is derived from phylogenies and timelines, explains evidence e given background knowledge b according to Popper’s formula:p denotes the probability (and likelihood) of e given both h and b, and p the probability of e given b.Our ‘bold’ conjecture for the origin of genetics departs from standard ‘world’ paradigms dominated by ancient RNA-based replicators, which we consider unrealistic and incapable of withstanding severity of test h is a conjunction ensemble that includes phylogenomic trees, timelines of aaRS domains and tRNA structures, minimum number of additional steps required to force hypotheses of monophyly, and statistical links between amino acid and dipeptide compositions associated with protein secondary structure and historical statements, all of which explain the actual patterns and processes of origin.Here, hypothesis e include matrices of FF structural homologies and tRNA substructural features from information in genomes and molecules, biases in amino acid and dipeptide compositions of FFs, genetic code complementarity and degeneracy and mappings to amino acid physicochemical properties, and functional and structural evidence from biochemistry and structural biology that support amino acid charging specificities. A number of assumptions relate to e that we want to make explicit: (i) Structural homologies at FF level and tRNA substructure level rely on the ability of hidden Markov models (HMMs) to encapsulate the structural design of molecules even in the presence of continuities in the space of protein and RNA sequence E cutoff values capable of maximizing consistency and information content of thousands of phylogenetic characters, optimizing methods of topological correspondence, and increasing the machine learning power of HMMs of structural recognition. (ii) The sampling of the world of proteins is sufficiently trusted to draw reliable evolutionary conclusions, even in the presence of biases in the choice of organisms with genomes that have been sequenced and the overrepresentation of microbes with parasitic lifestyles Evidence b includes support for character transformation series defined by the evolutionary model of growth of molecular structures and hypotheses of character polarization (rooting)  the popularity of domain structures (abundance) increases in time at global levels in the protein world, through gene duplication, de novo creation, cooption and rearrangements, as a general consequence of mutational diffusion of macromolecules in sequence space Background knowledge ing) see, the exiing) see and otheh, some of which we have already discussed, including the effect of compositional biases in mutational robustness. In particular, history of dipeptides and other elementary units of molecular structure are expected to manifest in fast-evolving genomic sequences responsible for de novo creation of genes Important predictions can be made from structuralclassificationofproteins (scop) 1.73; scop 1.75) reconstructed from a census of 989 genomes (A989) hmmscan of the profile HMMER3 package scans genomic sequences (with probability cutoffs E of 10−4) against a library of advanced linear HMMs of structural recognition in superfamilyIn this study we mapped the evolution of aaRS domains in a published evolutionary timeline of domain appearance at fold family (FF) level of structural abstraction 1g) of the length distribution of 1,000 random trees concise classification strings (ccs). For example, the catalytic domain of tyrosyl-tRNA synthetase [EC 6.1.1.1] corresponds to the c.26.1.1 FF, in which c represents the protein class (α/β proteins), 26 the F , 1 the FSF (nucleotidylyl transferase superfamily), and 1 the FF .Despite robust evolutionary trends across phylogenies nd) was calculated directly from the rooted trees using a script that counts the number of nodes from the root (base) of the tree to each leaf and provides it in a relative zero-to-one scale. These nd values take advantage of the highly imbalanced nature of the trees of domain structures, as recently discussed nd values are also good proxy for geological time. The molecular clocks for protein domains at fold (F)(t = –3.802 Fnd +3.814) and fold-superfamily (FSF)(t = –3.831 FSFnd +3.628)(11) levels were used to calculate the geological ages of selected families , provided that FFs were the most ancient in each group. We note that extending the clock to FFs showed that domain age continued to be proportional to time but with larger dispersion at high ndFF values. Clocks were calibrated with geological ages derived from the study of fossils and geochemical, biochemical, and biomarker data, which are affected by the validity of the assumptions used in each and every one of the supporting studies The relative age of protein structures (trees of molecules and trees of substructures that portray the history of the system (molecules) or its component parts (substructures), respectively (The ages of tRNA molecules here used were derived from published timelines of amino acid charging and encoding generated from trees of tRNA structure ectively . The phyS) required to force groups of tRNA taxa in trees  defined measures of ancestrality of individual groups and were used to build evolutionary timelines of amino acid charging and amino acid encoding. Hypotheses with smaller S were considered less affected by recruitment and represented processes that were more ancient. Using this approach, chronologies of amino acid charging and codon discovery were directly derived for isoacceptor (Saac) and anticodon-specific (Scod) tRNAs, respectively. The validity of character argumentation and the assumption that groups that require lower number of steps are deemed more ancient was derived from the rooted trees and the model of character polarization Phylogenetic constraint analysis restricts the search for optimal trees of tRNAs to pre-specified topologies FFnd) were plotted against the ages of isoacceptor tRNAs (aacS) and anticodon-specific tRNAs (codS). We found that correlations were significant in all instances (P<0.0067). Regression lines unfolded evolutionary timelines of archaic editing functions and anticodon-binding specificities, once editing and other accessory domains were identified. Specifically, catalytic and editing domains appearing before anticodon-binding domains (ndFF <0.196) and editing domains for AlaRS, ThrRS and PheRS appearing after that age  were included in correlation studies. Regression timelines were also compared to a conservative idealized timeline that spans domain age and underweights tRNA evolution . Two groups of aaRSs appearing close together in the idealized timeline are part of well-established superclusters defined by sequence and structural analysis Saac values of tRNAs interacting with structurally related ProRS, LysRS and MetRS makes them evolutionarily derived when compared to SerRS, a fact that is made explicit by the coevolutionary patterns. A recent sequence-based analysis of SerRS, ProRS and ThrRS shows indeed the late appearance of ThrRS The ages of protein domain structures at FF level (Km)wt/(k/Km)mutant, with rate constants being either kcat or VmaxPutative imprints in the primordial complementarity proposed to exist in the genetic code were borrowed directly from Rodin and Rodin n >100), it is appropriate to use a parametric method (ANOVA) to test whether there is significant difference among the frequencies of the three amino acid groups  in every secondary structure.We analyzed amino acid frequencies in secondary structures for a non-culled and a culled set of protein entries from the Protein Data Bank (PDB) . SecondasuperfamilyIn order to calculate amino acid frequencies in different FFs, we assigned structures to PDB sequences with the HMMs from FF <nd0.2) when compared to the set of 2,384 sequences and 1,475 FFs appearing throughout the timeline (nd = 0–1). For each of the two sequence sets, we counted the numbers of multiple occurrences of amino acids or dipeptides and then calculated the probability of enrichment of every amino acid or dipeptide that was present in the ancient 305-sequence set using the hypergeometric distribution and the following equation We also examined whether or not each of the 20 amino acids or the 408 possible dipeptides that included 2 ambiguous amino acids, Z and X, was respectively enriched in FFs that were more ancient than the oldest anticodon-binding domain of the timeline  implies the chance that a random variable X has k multiple occurrences of amino acids or dipeptides for a given amino acid or dipeptide. Referring to the equation and previous literature P values for every amino acid or dipeptide that has k/n larger than M/N, and evaluated statistical significance of enrichment of each amino acid or dipeptide in the ancient FFs at 99% confidence levels (P<0.01).Observed values superfamilyscop IDs scop, Interpro, Pfam, SwissProt, and literature sources (http://supfam.cs.bris.ac.uk/SUPERFAMILY/function.html). Domain architectures were queried in the PDB database http://www.rcsb.org/pdb/home/) and annotated using Gene Ontology (GO) http://www.geneontology.org). GO terms define a vocabulary of molecular functions, biological processes, and cellular components. We examined GO terms linked to molecular functions of translation. Manual annotations also involved queries in the UniProtKB (proteinknowledgebase) database (http://www.uniprot.org/) and HMM-based structure assignments. Annotations were mapped onto the domain timeline.Molecular functions linked to FFs were annotated using the hierarchical classification of Structural alignments of individual protein structural entries against structural sets were performed using DALI conservation mapping Figure S1Evolutionary accretion of domains in aaRS enzymes. A. One of nine most parsimonious phylogenomic tree reconstructions describing the history of the aaRS protein domains analyzed in this study. Terminal leaves are colored according to aaRS class  and indexed with aaRS domains labeled with concise classification strings (ccs). The tree matches the corresponding subtree in the global tree of FFs described in the next panel. B. Optimal most parsimonious phylogenomic tree of FFs  reconstructed from an analysis of the proteomes of 420 free-living organisms. Terminal leaves are not labeled in the tree since they would not be legible. The Venn diagram shows occurrence of FFs in the three superkingdoms. C. Evolutionary timeline of domain innovation. Domain ages (arrowheads) are mapped along a timeline of FF domain appearance derived from the global phylogenetic tree of FFs. For reference, the timeline is indexed with landmarks derived from domain history 1 of a periodic table of structures Tyr deacetylase (DTD). The enzyme hydrolyzes D-Tyr-tRNATyr  in TyrRSs, which lack internal editing functions (TIF)Click here for additional data file.Figure S2The origin and evolution of the standard genetic code. The ancestries of the three anticodon tRNA-aaRS binding expansion groups  were mapped onto a degenerate genetic code table.(TIF)Click here for additional data file.Figure S3Distribution of age groups of domains with editing  and anticodon-binding  functions, groups in exchange graphs, and active site participation in Venn diagrams of amino acids describing their physicochemical properties. Venn diagrams show that the origin of amino acid charging in Group 1 specificities was associated with a polar, turn-inducing and active-site promoting amino acid (Ser) and hydrophobic aromatic (Tyr) and aliphatic (Leu) counterparts. In turn, the start of genetic encoding was associated with small turn-inducing amino acids. We note however that ancient Groups 1 and 2 domains charge amino acids with low active site-participation frequencies, the only exception being Ser, while Group 3 exhibits the opposite trend. The origin of the standard genetic code derived from expansion groups A, B and C (Group H (helix) is uniquely enriched in large hydrophobic and polar amino acids (3 of each) that can be buried and made non-polar by H-bond formation in α−helices and β−strands, respectively Group T (turn)  and Group E (strand)  are rather balanced in the overall distribution of properties Click here for additional data file.Figure S4Analysis of the number of identity elements in cognate tRNA interacting with Groups 1, 2 and 3 aaRS domains and their aminoacylation role estimated by loss of aminoacylation efficiency upon mutation. Note that identity elements associated with Group 1 domains retain ancestral features of poor specificity. These elements include N73 discriminator base and the N4:N69 base pair of the acceptor stem, which appear to be a diagnostic identity element of aaRSs with ancient editing functions. SerRS, LeuRS, IleRS ValRS and MetRS uniquely recognize the N4:N69 base pair element. The 3′ terminal A76 of tRNALeu plays an important dual role in aminoacylation and editing of LeuRSs, as well as in other class I aaRS systems. In SerRS, as in other Class II aaRS systems, the interactions with the acceptor stem of tRNA are limited and those with the anticodon loop absent. A recent study reveals that the phosphate backbone of these few identity elements interact with amino acid side chain residues of SerRS in a solvent-related manner through a conserved network of water molecules (TIF)Click here for additional data file.Figure S5Evolutionary pathways of code expansion and their possible impact on protein structure. The standard genetic code maps a set of 64 base triplets (codons) to 20 standard amino acids (plus Sec and Pyl for subsets of organisms), and 3 translation stop signals. Zull and Smith 10-helices (G), π-helices (I), turns (T) and bends (S). A. Analysis of frequencies of total amino acids belonging to the three exchange graphs (top diagrams), Group H , Group T , and Group E , in the secondary structures of 6,828 protein domain sequences. Bars headed by the same letters are not significantly different (P = 0.05) following an ANOVA and the Tukey post-hoc test. The analysis revealed that each group is indeed enriched in amino acids that participate in helix, turn and strand secondary structures, respectively. The non-culled set of 204,531 domain sequences  displayed the same global trends (data not shown). Venn diagrams of chemical properties show that Group H is enriched in large hydrophobic and polar amino acids that can be buried in α-helices and β-strands, Group T in small amino acids necessary for turns and bends, and Group E in hydrophobic amino acids necessary to bury strand elements in sandwiched or barrel conformations , Group 2  and Group 3  domains. Dissection of exchange graphs into the three domain age groups indeed confirms that Group T is consistently overrepresented in turns and bends throughout code history. In turn, Group H is overrepresented in domain age Groups 1 and 3, while Groups E is only overrepresented in Group 2. These patterns show the initial importance of helical and turn structure in early protein evolution (seeGroup T) uniquely implements bends and turns throughout genetic code history is highly significant. Sequence motifs that exist in these loop regions of proteins are the most ancient in the protein world Groups 1 and 2 domains charge amino acids with low active site-participation frequencies Click here for additional data file.Figure S6Structural alignments of amino acid-[acyl-carrier-protein]-ligases (aaACPLs) and cyclodipeptide synthases (CDPSs) to homologous aaRSs using DALI conservation mappingand structural entries of the Astral compendium.A. RMSD-Z score plots of 601 structural neighbors of aaACPLs  with Z scores above 2. The closest structural neighbor of aaCPLs  is an unsusual SerRS enzyme from a metanogenic archaeon, Methanosarcina barkeri , align to the d.104.1.1 catalytic core of the class II SerRS enzyme. B. RMSD-Z score plots of 785 structural neighbors of CDPSs  with Z scores above 2. The closest neighbors of CDPSs are TyrRSs  from archaeal microbes. We note that lower Z scores recover matches to aaRSs from bacteria and eukaryotes, a tendency that supports the ancient origins of the structures. In all cases, CDPSs align to the c.26.1.1 catalytic core of class Ic enzymes but lack ATP binding sites. C. Structural comparisons of AlbC CDPS to the protein folds in ASTRAL using an advanced algorithmic implementation, GANGSTA+ Methanocaldococcus jannaschii (1J1U), is displayed . Overall results show that the structural cores and crucial sequence sites that contact ligands are highly conserved . Thus, functions of aaACPL and CDPS structures appear to be relics and founders of archaic Group 1 aaRS biosynthetic activities.(TIF)Click here for additional data file.Figure S7The frequencies of amino acids specified by Groups 1, 2 and 3 aaRS structures in 2,384 protein sequences  were mapped along the evolutionary timeline of FFs (scatter chart) and studied for central tendencies .(TIF)Click here for additional data file.Figure S8Models of origin of mirror modes of tRNA acceptor stem recognition by aaRSs. The class I FF domain appears in the timeline concurrently with the GP-binding domain of elongation and initiation factors, the G protein domain (c.37.1.8), at ndFF = 0.020. The class II FF domain appears immediately after at ndFF = 0.024. The almost concurrent emergence of domains necessary for tRNA aminoacylation and for the formation of ternary complexes with tRNA and other proteins is striking. The finding fulfills coevolutionary assumptions of three non-mutually exclusive models: A. Complementarity of ancient genes reflects complementary aaRS interactions: The existence of mirror modes of tRNA acceptor stem recognition has been suggested to reflect complementarity (head-to-tail) of ancient aaRS genes Complementary aaRSs protected primordial tRNA from degradation: Mirror recognition of tRNA by complementary aaRSs could have protected primordial tRNA from destruction by nucleases and phosphate bond-cleaving metal ions Recognition resulted from aaRS-driven polypeptide biosynthesis: aaRSs could have acted in concert with other aaRSs and translation factors . For the Rv2275 enzyme that produces cYY Tyr, which is followed by nucleophilic attack by a Ser88 hydroxyl group of the enzyme on the ester carbonyl, which produces a covalently tyrosinoylated enzyme and free Tyr-tRNA. A second Tyr-tRNATyr binds after a possible rotation of the side chain that holds the tyrosinoylated catalytic residue and a second nucleophilic attack likely produces a YY dipeptide intermediate, which is then subjected to an unknown mechanism of cyclization chemistry. A similar overall mechanism has been dissected for the AlbC enzyme that produces cFL sensu factors  to perfo(TIF)Click here for additional data file.Table S1The viz-a-viz representation of the genetic code that puts complementary codes head-to-head with each other. Light gray and black cells mark the two modes of tRNA recognition from the minor and major sides of the acceptor stem of tRNA, respectively. Numbers 1, 2 and 3 denote codon positions and N2 the consensus of the second nucleotide from the 5′ end of the acceptor arm of tRNA. Shades denote the relative age of the ‘operational code’ (taken from (PDF)Click here for additional data file.Table S2Risks of confusion of complementary anticodons see  under fo and their relative ages .(PDF)Click here for additional data file.Table S3Dipeptide sequences enriched in ancient domains. Dipeptides are identified by participating amino acids using one-letter codes and listed together with statistical significance values of enrichment (p) and subsets specified by intervening amino acids corresponding to Group 1, 2 and 3 domain structures (one of 9 subsets possible).(PDF)Click here for additional data file.


Centrioles organise centrosomes and template cilia and flagella. Several centriole and centrosome proteins have been linked to microcephaly (MCPH), a neuro-developmental disease associated with small brain size. CPAP (MCPH6) and STIL (MCPH7) are required for centriole assembly, but it is unclear how mutations in them lead to microcephaly. We show that the TCP domain of CPAP constitutes a novel proline recognition domain that forms a 1:1 complex with a short, highly conserved target motif in STIL. Crystal structures of this complex reveal an unusual, all-β structure adopted by the TCP domain and explain how a microcephaly mutation in CPAP compromises complex formation. Through point mutations, we demonstrate that complex formation is essential for centriole duplication in vivo. Our studies provide the first structural insight into how the malfunction of centriole proteins results in human disease and also reveal that the CPAP–STIL interaction constitutes a conserved key step in centriole biogenesis.DOI:http://dx.doi.org/10.7554/eLife.01071.001 Organisms—and individual tissues—grow and develop by dividing their cells. However, the process of cell division does not have to be symmetric, and the fates of the cells can be very different if cellular contents, including RNAs or proteins, are exclusively retained in the ‘mother’ or passed to her ‘daughter’. Organelles known as centrioles can play an important part in influencing whether cell division is symmetric or asymmetric.Centrioles contain ordered assemblies of various proteins, and mutations in some of these proteins can cause developmental defects in humans. For example, mutations in the centriolar proteins CPAP and STIL cause a syndrome known as microcephaly, in which the brain is smaller than normal. Although CPAP and STIL are known to bind each other, how they interact on a molecular level to form centrioles—and how this interaction is disrupted in microcephaly—is not well understood.Cottee et al. have now used structural and biochemical assays to explore how these two proteins bind to each other, and have identified specific amino acid residues that enable this interaction. These residues are highly conserved across many organisms, and a mutation in one of them has previously been associated with microcephaly in humans. Now, Cottee et al. demonstrate that this mutation weakens the interaction between CPAP and STIL in vitro.To explore these processes in vivo, Cottee et al. studied mutant fruit flies in which the interactions between CPAP and STIL were weaker than normal, and found that these mutations prevented the normal formation of centrioles. Furthermore, there was a striking correlation between the ability to form centrioles in fruit flies and the ability of CPAP and STIL to bind each other, based on the structural model and in vitro binding studies.Cumulatively, these findings reinforce the importance of CPAP and STIL in centriole formation, and suggest that one reason for the development of microcephaly may be defects in the proper formation of centrioles.DOI:http://dx.doi.org/10.7554/eLife.01071.002 Centrioles are small cylindrical organelles whose outer walls contain a ninefold symmetric array of microtubule triplets. These structures form the basal bodies that template the assembly of cilia and flagella, and they also organise a proteinaceous matrix termed the pericentriolar material (PCM) to form centrosomes, the main microtubule organising centres in animal cells. These organelles play an important part in many aspects of cell organisation, and centriolar dysfunction is linked to a plethora of human diseases, including cancer, obesity, macular degeneration and polycystic kidney disease .Drosophila, here called dCPAP) and STIL  in flies lead to defects in the asymmetric division of larval neural stem/progenitor cells  is a rare condition where patients are born with small brains . All eigor cells . Mutatioor cells . Moreoveor cells . Thus, tCentrioles are complex structures, but work in several model systems revealed only a small number of conserved proteins to be important for centriole assembly. These include PLK4/SAK, SAS-6, STIL/Ana2, CPAP/CenpJ/SAS-4, Cep152/Asl, and CEP135 . SeveralOf particular interest in this regard is the putative centriolar CPAP–STIL complex, as mutations in both components result in MCPH . CPAP anDanio rerio CPAP and used isothermal titration calorimetry (ITC) to test its ability to bind to a fragment of D. rerio STIL that spanned CR2 (residues 404–448)  and C-terminal region (residues 429–448). The N-terminal region exhibited an only slightly weaker binding (KD ∼4 μM) to the TCP domain, whereas the C-terminal region showed a very weak binding (KD > 500 μM)  can interact with a ∼400 amino acid (aa) region (residues 231–619) of human STIL . To try 404–448) , Table 1 500 μM) ; Table 1D. rerio CPAP937–1124, both on its own and in a complex with D. rerio STIL408–428  used to study β-rich self-assemblies . In contThe structure of the CPAP–STIL complex revealed that the STIL peptide binds in a polyproline II helical conformation along one edge of the TCP domain β-sheet. The STIL peptide binds to CPAP by four main mechanisms . First, D. rerio) that would be well positioned to bind to these aromatic residues in an analogous way (D. rerio) may be bound all along the edge of the TCP domain. Although our ITC experiments suggest that these putative additional contacts are insufficient to establish strong binding between STIL and CPAP  is mutated to valine in some MCPH patients. To test whether this mutation disrupts the organisation of the TCP domain, we obtained the crystal structure of mutation ; Table 2 binding . Circula binding . ITC exp 40-fold , while mD. rerio STIL404–448 peptide and tested their ability to interact with the WT D. rerio TCP domain in ITC experiments  (residues 700–901) in complex with the region of Ana2 (dSTIL) equivalent to CR2 (residues 1–47) . Indeed, all copies of the complex obtained in the structures from both species superimposed well and exhibited the same four major groups of binding interactions as described for the D. rerio structure. This conservation includes the contact made by the E792 residue in dCPAP . Together, these data allow us to determine a consensus CPAP binding motif in metazoan STIL proteins  . The dSTPRxxPxP,  and suggDrosophila, we turned to D. melanogaster as a model system to address the functional relevance of this interaction in vivo. In flies, the lack of dCPAP or dSTIL leads to centriole loss and a consequent severe uncoordinated (unc) phenotype due to the lack of basal bodies and so cilia in Type I sensory neurons. These flies lack all mechano- and chemo-sensation and, although viable, they usually die shortly after eclosion, as they cannot feed or move in a coordinated fashion (Since the binding mechanism of CPAP and STIL is conserved between zebrafish and  fashion . We exam fashion .10.7554/dSTIL mutant (dSTIL mutation while the double mutant failed to do so (data not shown). The single mutants P11A and R12A were also able to partially rescue the centriole loss phenotype, whereas the double mutant P11A:R12A showed only a poor rescue (We first expressed a version of dSTIL that lacks the first 45 aa (including the PRxxPxP motif required for the interaction with dCPAP). GFP-tagged wild-type dSTIL (dSTIL_WT-GFP) served as a control. Both proteins were expressed at similar levels and localised strongly to centrosomes in the presence of endogenous dSTIL . Only thL mutant ; Table 7L mutant . All thrL mutant . Both sir rescue ; Table 7We next deleted the entire TCP-domain of dCPAP (dCPAP_ΔC), or expressed GFP fusion proteins carrying mutation clusters (MCs) altering 3–4 residues in different regions of the TCP domain . MutatioC. elegans homolog of the STIL proteins in flies and vertebrates, but there is little sequence homology between these proteins (C. elegans CPAP homologue) is functionally important. We used the Mos single-copy insertion system  or a form in which the C-terminal TCP domain (aa 557–808) had been deleted (SAS-4ΔTCP::GFP); both transgenes contained a 497 bp resequenced region in their N-terminal coding region (preserving codon usage) that rendered them resistant to RNAi-mediated depletion , but not by the ΔTCP transgene (aa 1–556)  . While Smaterial . Thus, tΔTCP::GFP to become incorporated in the centriole outer wall could be due to an inability to interact with SAS-5, we performed a pull-down assay to determine whether 35S-labelled in vitro translated SAS-4 fragments could interact with the N-terminal or C-terminal regions of SAS-5 bound to beads , our structural analysis revealed that the interaction interface is conserved, confirming the previous proposal that fly Ana2 is the functional homologue of vertebrate STIL  recognition-domain that specifically binds to a short target motif in STIL. Although the overall sequence identity of the CPAP and STIL proteins between ate STIL . Our chaDrosophila Ana2 and C. elegans SAS-5 suggests that STIL homologs are under particularly strong lineage-specific selection. Despite the many sequence changes between Drosophila Ana2 and vertebrate STIL, our work suggests that the interaction interface between Ana2/STIL and dSAS-4/CPAP TCP domain has been retained, highlighting its importance. Even in C. elegans, which is the most divergent of the functionally characterised STIL homologs, our work indicates that the SAS-4 TCP domain is essential for centriole assembly, and that a TCP-domain dependent interaction between SAS-4 and SAS-5 has been conserved. Nevertheless, as the SAS-4 TCP domain is not sufficient for interaction with SAS-5 and we were unable to identify a PRxxPxP interaction motif in worm SAS-5, more work will be needed to understand the SAS-4—SAS-5 interaction in C. elegans and its relationship to the CPAP–STIL interaction in other metazoans.The high degree of sequence divergence between vertebrate STIL, C. elegans SAS-4 (A surprising aspect of our findings is that the E792V (MCPH) mutant and all three of the mutation clusters (MCs) that we analysed in dCPAP localise poorly to centrosomes. For the E792, MC1, and MC2 mutations this could be expected, as these are all predicted to perturb the interaction between dCPAP and dSTIL (as is the case with similar mutations in zebrafish CPAP in our in vitro binding assays), and this would be predicted to perturb the recruitment of dCPAP to centrioles. The MC3 cluster, however, is not predicted to lie in a strong interaction interface and, unlike the MC1 and MC2 mutation clusters, it can rescue the centriole duplication defect in dCPAP mutant cells nearly as efficiently as the WT protein. Possibly, an interaction with another protein that plays some part in recruiting dCPAP to centrioles might be perturbed by these mutations. Alternatively, similar to the situation with ns SAS-4 , dCPAP mOur studies provide the first structural insight into the nature of the link between centrioles and human microcephaly. It is unclear why mutations in genes encoding key centriole or centrosome proteins can lead to such a specific neuro-developmental disorder in humans. It is widely assumed that some aspect of centriole/centrosome function must be particularly important in human neural progenitor cells, and that the failure of these cells to proliferate in an appropriate manner underlies the small brain size in affected individuals . One posA previous analysis of the behaviour of various CPAP mutant proteins (modelled on MCPH mutations) in human cells revealed some surprising findings . The delD. rerio CPAP937–1124 was cloned from D. rerio cDNA. Proteins were expressed in Escherichia coli BL21 (DE3) Rosetta as N-terminally His-tagged constructs, and purified via immobilised metal ion affinity chromatography , proteolytic tag cleavage, followed  by size-exclusion chromatography and ion-exchange chromatography using standard methods. The selenomethionine derivative protein was expressed in selenomethionine supplemented M9 medium and purified in the same way. Purified constructs contained the sequence GPHM at the N-termini that stem from the cloning and protease cleavage sites.D. rerio STIL404–448 was cloned from IMAGE clone 7147918 and expressed in E. coli C41 BL21 (DE3), fused to two His-tagged lipoyl domains from Bacillus stearothermophilus dihydrolipoamide acetyltransferase at both the N- and C terminus. The peptide was purified via NiNTA chromatography, proteolytic cleavage of the His-lipoyl domains, and ion-exchange chromatography. The purified constructs contained a G (GG for D. rerio STIL404–448 and its point-mutants) at their N-terminus and the sequence EFGENLYFQ (ENLYFQ for D. rerio STIL408–428 and D. rerio STIL411–428) at their C-terminus. These extra sequences stem from the cloning and protease cleavage sites.D. rerio constructs were introduced into the expression vectors by site-directed mutagenesis.Mutations of the Drosophila dSTIL1–47 was genetically fused to the N-terminus of Drosophila dCPAP700–901 via 3-way ligation. The fusion protein was expressed in E. coli B834 (DE3) as an N-terminally His-tagged fusion, and purified via NiNTA chromatography, proteolytic tag cleavage and size exclusion chromatography. The selenomethionine derivative protein was expressed using SelenoMethionine Medium  and purified in the same way.Codon-optimised  D. rerio CPAP937–1124 was crystallised in sitting drops in 80 mM Tris pH 8.5, 160 mM MgCl2, 20% PEG-4000, 18% Glycerol, 1 mM DTT at 19.5°C. The drops were set up using 1 μl of the protein solution and 0.5 μl of the reservoir solution. Crystals were mounted after 3 days and flash-frozen in liquid nitrogen.Native D. rerio CPAP937–1124 E1021V was crystallised in sitting drops in 80 mM Tris pH 8.5, 160 mM MgCl2, 24% PEG-4000, 20% glycerol at 19.5°C. Crystals were mounted after 3 days and flash-frozen in liquid nitrogen.D. rerio CPAP937–1124 crystals were obtained using the sitting drop method with a reservoir solution of 80 mM Tris pH 8.5, 160 mM MgCl2, 26% PEG-4000, 18% glycerol, 1 mM DTT at 19.5°C. Drops were set up using 1 μl protein solution and 1 μl of reservoir solution. Native CPAP937–1124 crystals were used for streak-seeding into these drops and crystals allowed to grow for 7 days before mounting and flash-freezing them in liquid nitrogen.SeMet D. rerio CPAP937–1124 and D. rerio STIL408–428 were initially obtained using the LMB screening set-up with the Clear Strategy 2 pH 8.5 screen . Crystals were used to streak seed into sitting drops consisting of 1 μl of a reservoir solution of 100 mM Tris pH 8.5, 200 mM CaAcetate, 17% PEG-2000 MME and 1 μl of protein/peptide mixture (0.25 μl protein + 0.75 μl peptide) at 19.5°C. Crystals were grown for 2 days before mounting them in 100 mM Tris pH 8.5, 200 mM CaAcetate, 17% PEG-2000 MME, 25% glycerol and flash-freezing them in liquid nitrogen.Crystals of the complex of D. rerio CPAP937–1124 used for crystallisations were measured by the Bradford assay with BSA as a standard and were 39.9 mg/ml (apo-CPAP937–1124), 46.2 mg/ml (CPAP937–1124 E1021V), 30.6 mg/ml (SeMet CPAP937–1124) and 81 mg/ml CPAP937–1124 . The concentration of STIL408–428 (CPAP/STIL complex) was determined by amino acid analysis and was 11.8 mg/ml (3.8 mM).The protein concentrations of D. melanogaster dSTIL1–47-dCPAP700–901 was crystallised using the sitting drop approach, using the Morpheus screen (Molecular Dimensions). Crystals grew after approximately 3 weeks . Crystals typically grew after 3–4 weeks. Some crystals were used for microseeding of further screens including an optimisation screen. Seed stock was generated using a Seed bead kit . Details of crystallisation conditions are shown in Native D. rerio datasets were integrated and scaled using MOSFLM  in 50 mM HEPES pH 7.5, 100 mM NaCl at 25°C. Samples were stored by the instrument in 96-well microtiter plates at 5°C prior to loading and performing the titrations. Standard experiments used 19 × 2 μl injections of STIL peptide into CPAP protein preceded by a single 0.5 μl pre-injection. Heat from the pre-injection was not used during fitting. Data were analysed manually in the Origin software package provided by the manufacturer and fit to a single set of binding sites model. All measurements were corrected using control ITC experiments in which the peptide studied was injected into buffer only. The small endothermic heats of injection in these experiments were fitted to a linear function that was subsequently subtracted from the equivalent integrated heats of the peptide–protein binding experiment before fitting. The concentration of CPAP in the cell was typically 40 μM but varied maximally between 20 and 100 μM. The concentration of STIL used in the syringe was typically 700 μM but varied maximally between 600 and 2600 μM depending on the affinity of the peptide interaction being studied.ana2169 , ana2719    , sas-4s2d dCPAP) , pUbq-dCll types . Flies w1–724 and dSTIL_ΔN46–420, respectively. Single point mutations and mutation clusters were introduced into full length dCPAP and dSTIL using site-directed mutagenesis . Constructs were injected either by Genetic Services Inc.  or Cambridge DNA Injection Service .GFP-tagged full length versions of dCPAP and dSTIL used in this study were made by cloning the full length dCPAP cDNA and the dSTIL cDNA into the pUbq-GFP(C-terminus) destination vector using the Gateway System . PCR with dCPAP_WT and dSTIL_WT as template was used to make dCPAP_ΔCdCPAP or dSTIL169/dSTIL2719 mutant background, and desired pupae were collected from vials and transferred to filter paper  for analysis.All constructs were tested for their ability to rescue the uncoordinated phenotype, which is a feature of flies lacking centrioles . For thawww.fiji.sc/Fiji, II for WT SAS-4; and unc-119(ed9)III; ltSi177II for SAS-4ΔTCP.g MosSCI . To rendsas-4 RNA was generated as described , a 63X 1.4 NA Plan Apochromat objective, and a QuantEM:512SC EMCCD camera (Photometrics). Adult worms were dissected in M9 buffer, and embryos were mounted onto 2% agarose pads for imaging. 11 × 1 μm z-stacks were collected in the GFP channel , along with one central DIC section.35S-Met labelling.SAS-4 constructs were cloned into a pET21a vector for in vitro transcription/translation. Proteins were expressed using the T7 TNT Quick Coupled Transcription/Translation System  with E. coli Rosetta2(DE3) cells and purified on Ni-NTA agarose (Qiagen) using standard protocols. For pull-down experiments, proteins were dialysed into 25 mM HEPES, 100 mM NaCl, 20 mM imidazole, 1 mM DTT, 10% sucrose, 0.02% Tween-20, pH 7.4.SAS-5 fragments were cloned into a pRSET-A vector with a C-terminal 6xHis tag. Proteins were expressed in SAS-5 fragments were pre-incubated with 20 μl Ni-NTA beads for 45 min at 4°C. 10 μl of the SAS-4 IVTT product was added to the beads with 190 μl buffer and incubated at 4°C for 30 min. The beads were washed with 3 × 200 μl buffer and resuspended in 100 μl SDS-PAGE sample buffer. Samples were run on 10% SDS-PAGE gels and either stained with Coomassie or dried and exposed to a phosphor screen overnight. Phosphor screens were analysed on a Personal Molecular Imager System . review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  forms a single beta sheet that is reminiscent of the structure of amyloid fibrils. The distinction is that amyloid fibrils are formed by strands from multiple protein molecules, whereas here a single protein forms the sheet. Also, in amyloid fibers the sheets pack against each other whereas the CPAP TCP domain forms a single sheet. This structure may well be unprecedented, and the finding is important because it extends our understanding of protein architecture.In this paper the authors provide crystal structures for a central part of the CPAP/STIL protein complex from The TCP domain is found to form a novel proline-recognition domain. The authors identified a ∼40 amino acid region in STIL that is proline rich and conserved. They dissected this region to find that a peptide segment corresponding to the entire region binds with 2µM affinity to CPAP, but that a shorter segment (411–428) binds with only slightly diminished affinity. This shorter segment was crystallised with the TCP domain, using the zebrafish sequences, and also on its own. Remarkably, the single beta sheet formed by the TCP domain lacks a hydrophobic core, and is stabilised principally by hydrogen bonding. Sequence repeats have been identified previously in the domain and these are shown to form the beta-hairpin repeats in the structure. There are no extensive crystal packing interfaces, and so the structure suggests that the functional unit of TCP is a monomer. This is verified by biophysical data.The STIL peptide adopts a PP-II helix conformation and interacts with the TCP domain via conventional interactions, including the packing of proline side-chains from the peptide into hydrophobic surfaces on the TCP domain. Additional conservation in the TCP domain, beyond that required for the interaction with the short crystallised peptide, suggests that the complete interface is more extensive.Drosophila, revealing strong structural conservation despite weak sequence similarity. There is extensive analysis of the implications of the structure for centriole formation, using cell-based assays in the Drosophila system.The importance of the crystallographic interface between the TCP domain and the STIL peptide has been verified by mutagenesis and by determining the structure of the equivalent complex from eLife. The paper represents a significant advance, and it is well validated and well described.The novelty of the structural fold of the TCP domain and the new mechanism revealed for recognition of proline-rich peptides revealed by the structures of the complexes makes this paper suitable for Minor comments:1) In one place the authors call the interaction with the peptide 'unique' and in another 'highly unusual.' It would strengthen the paper to give quantitative evidence for the lack of similarity of this structural element to previously determined structures, and for a brief discussion of any similar structures. At the very least, the authors may wish to give somewhat more prominence to their very unusual structural finding. It is quite rare these days to determine a structure of a soluble protein and really find something new.2) The paper is very clearly written and can be accepted with very minor changes. One suggestion regarding clarity is that the paper will be of interest to structural biologists who are not familiar with the cell-based assay systems, and so these should be explained better. To start with, the “unc” phenotype is mentioned in the Results with no explanation. Presumably, this is the “uncoordinated phenotype” mentioned in the earlier paragraph, but this should be explained. The use of this phenotype in the cell-based assays needs more explanation. Presumably, a general behavioral phenotype (“uncoordinated”) is being mapped into specific features of the embryonic cells? 1) In one place the authors call the interaction with the peptide 'unique' and in another 'highly unusual.' It would strengthen the paper to give quantitative evidence for the lack of similarity of this structural element to previously determined structures, and for a brief discussion of any similar structures. At the very least, the authors may wish to give somewhat more prominence to their very unusual structural finding. It is quite rare these days to determine a structure of a soluble protein and really find something new.We expanded the structural description of the TCP domain and included an additional figure supplement with information on its sequence repeats. We also commented on the similarity to other protein structures and added a figure supplement showing a side-by-side comparison of the TCP domain and the closest structural match in the PDB. It is noteworthy to mention that this structure is artificially engineered and is capped on both ends by globular domains that stabilise it.The TCP domain folds into an extended β-sheet meander. In general, β-meanders are frequently found in the PDB, e.g., in β-barrels , β-propellers or some α+β proteins where often α-helices pack against the meander sheet. What makes the TCP domain structure unique is that it consists solely of a freestanding meander β-sheet that is on both sides exposed to the solvent and entirely lacks a defined hydrophobic core. We also demonstrate that this domain is stable on its own and it is folded and predominantly monomeric in solution. Currently, there are only three natural proteins, structurally characterised and deposited in the PDB, that contain regions forming an elongated single meander β-sheet. These are: outer surface protein A (1osp), histone methyltransferase set7/9 (1h3i) and bacteriophage gp138 (PDB code 3pqh). In none of these, however, the meander β-sheet exists on its own. The 'nonglobular' β-sheet in each of these structures is flanked by other globular domains and in two of them the region of the β-sheet that is exposed to the solvent is relatively small (three strands in OspA and set7/9). In the bacteriophage gp138, one of the β-sheet faces is hydrophobic and buried in the trimer interface.As to quantitative evidence for the lack of similarity to other structures in the PDB, we decided not to include it as it would not be very meaningful in our opinion. We could have included the RMSDs of comparisons to other β-meander containing proteins. However, what would these numbers really mean, if the overall protein architecture and the context in which the meander is found are so different? We could have used the Z-scores of the DALI server . However, these scores suffer from similar shortcomings. Furthermore, these servers are well known to miss structural similarities and it is not clear to us therefore what the absence of close hits would signify. We would like to point out that one of the co-authors of this paper (AA) is a developer of the SCOP  database and therefore has a good overview of the available protein structural repertoire.2) The paper is very clearly written and can be accepted with very minor changes. One suggestion regarding clarity is that the paper will be of interest to structural biologists who are not familiar with the cell-based assay systems, and so these should be explained better. To start with, the “unc” phenotype is mentioned in the Results with no explanation. Presumably, this is the “uncoordinated phenotype” mentioned in the earlier paragraph, but this should be explained. The use of this phenotype in the cell-based assays needs more explanation. Presumably, a general behavioral phenotype (“uncoordinated”) is being mapped into specific features of the embryonic cells?Drosophila experts. Please also note that we include in our manuscript  that we completed analyzing while our manuscript was under review. The corresponding phenotypes of these mutants further strengthen our argument and strongly confirm our structural model.We clarified the corresponding sections in detail to make the manuscript clearer and easier to follow for non-nuscript  three mo


Here, the crystal structure of the non-catalytic domain of PDIR was determined to 1.5 Å resolution. The structure adopts a thioredoxin-like fold stabilized by a structural disulfide bridge with a positively charged binding surface for interactions with the ER chaperones, calreticulin and ERp72. Crystal contacts between molecules potentially mimic the interactions of PDIR with misfolded substrate proteins. The results suggest that the non-catalytic domain of PDIR plays a key role in the recognition of protein partners and substrates.Protein disulfide isomerases comprise a large family of enzymes responsible for catalyzing the proper oxidation and folding of newly synthesized proteins in the endoplasmic reticulum (ER). Protein disulfide isomerase-related (PDIR) protein  is a specialized member that participates in the folding of α In order to acquire the proper arrangement of disulfide bonds, newly synthesized membrane and secreted proteins fold in the oxidative environment of the endoplasmic reticulum (ER) with the assistance of ER enzymes called protein disulfide isomerases (PDIs) N-linked glycosylation of nascent polypeptides during protein synthesis in the ER 1-antitrypsin binds to PDIR 1-antitrypsin Human protein disulfide isomerase-related (PDIR) protein, also known as PDIA5, was originally identified from a human placental cDNA library 1-antitrypsin b'domain of PDIA1 increases the chaperone activity of PDIR to PDIA1 levels, but decreases oxidative refolding of α1-antitrypsin PDIR is the only PDI composed of an N-terminal non-catalytic domain and three catalytic domains. The catalytic domains are unique in the sequences of their catalytic motifs: CSMC, CGHC and CPHC. Of these domains, the second catalytic domain (CGHC) exhibited the highest isomerase activity on model substrates, while the CSMC domain was most important for the folding of αHere, we present the crystal structure of the non-catalytic domain of human PDIR, determined at 1.5 Å resolution. The structure reveals a highly conserved positively charged surface that is involved in interactions with protein partners. NMR titrations show that the domain binds to the negatively charged P-domain of calreticulin, which suggests that PDIR can engage the calnexin cycle. Crystal contacts between PDIR molecules further identify a putative substrate-binding site on the PDIR non-catalytic domain. The observed interactions suggest that PDIR can bind substrates directly and also recruit them through interaction with calreticulin.E. coli BL21(DE3) in rich (LB) medium as a C-terminal His-tag fusion. For production of selenomethionine-labeled protein, the expression plasmid was transformed into the E. coli methionine auxotroph strain DL41 (DE3) and the protein was produced using LeMaster medium 2+-charged Sepharose resin followed by size-exclusion chromatography using HPLC buffer . Selenomethionine-labeled protein was purified in a similar manner. In addition to the PDIR sequence, the resulting protein contains an N-terminal methionine and a C-terminal extension Leu-Glu-His-His-His-His-His-His. The 15N-labeled P-domain of mouse calreticulin (residues 211–261) was expressed and purified as described earlier The non-catalytic domain (residue 29–150) of human PDIR was cloned into pET29a (Amersham-Pharmacia) and expressed in 2 cold stream (Oxford Cryosystem). The crystals belong to the p212121 space group and contain one molecule in the asymmetric unit (Z  =  4) corresponding to Vm  =  2.1 Å3 Da−1 and a solvent content of 42%.Initial crystallization conditions for the PDIR non-catalytic domain were identified utilizing hanging drop vapor diffusion using PACT screen (QIAGEN). The best crystals were obtained by equilibrating a 1.0 µL drop of a protein (6 mg/mL) in buffer (10 mM HEPES (pH 7.0), 0.1 M NaCl and 5 mM DTT), mixed with 1.0 µL of reservoir solution containing 0.15 M DL-malic acid (pH 7.0) and 20% (w/v) PEG 3350, and suspended over 1 mL of reservoir solution. Crystals grew in 3–14 days at 22°C. The identical conditions were used for growing selenomethionine-labeled protein. For cryoprotection, the reservoir composition with the addition of 20% glycerol was used. For data collection, crystals were picked up in a nylon loop and flash cooled in a Nhttp://pymol.sourceforge.net/). The coordinates and structure factors have been deposited in the RCSB Protein Data Bank (accession number 4i6x).Diffraction data from a SeMet-labeled crystal were collected using a single-wavelength (0.9791 Å) SAD regime on a MarMosaic CCD 300 detector at beamline 08ID-1 at the Canadian Light Source, SK, Canada. Diffraction data from a native crystal were collected at a wavelength of 0.9770 Å on an ADSC Quantum-210 CCD detector (Area Detector Systems Corp.) at beamline A1 at the Cornell High-Energy Synchrotron Source (CHESS) . Data pr15N-labeled 0.15 mM P-domain of calreticulin to the final molar ratio of 1∶4.4, or the unlabeled P-domain was added to 15N-labeled 0.15 mM non-catalytic domain of PDIR to the final molar ratio of 1∶8. For the high-salt titration, the proteins were exchanged into 10 mM HEPES, 0.5 M ammonium sulfate at pH 7.0 and the 15N-labeled P-domain was titrated with unlabeled PDIR domain to the final molar ratio of 1∶2. All NMR experiments were performed at 298 K using Varian 500 and 800 MHz spectrometers. NMR spectra were processed using NMRPipe NMR samples contained 0.15–0.5 mM protein in 10 mM HEPES, 100 mM NaCl at pH 7.0. For NMR titrations, unlabeled non-catalytic domain of PDIR was added to Hydra magnipapillata (XP_002159276) possesses eight. Within the family of PDI proteins, the PDIR non-catalytic domain is relatively unique with a sequence similarity of only 20% identity to its closest paralog, the a domain of ERp57.The domain organization of PDIR is unusual. Unlike the better known PDI family members, which contain four thioredoxin-like domains, the N-terminal domain of PDIR is non-catalytic. We used a BLAST sequence similarity search to identify homologous domains. As expected, most of the hits corresponded to PDIR orthologs from different species . Some ofWuchereria bancrofti and Brugia malayi. Mammalian RNA methyltransferases do not contain PDIR-like domains. Human PDIR has 43% sequence identity to the corresponding domains in the methyltransferases with a preponderance of conserved positively charged residues  with crystals appearing in 1–2 days. Unfortunately, the crystals had visible defects and yielded an anisotropic diffraction pattern. The use of a vector with a C-terminal His-tag improved the protein expression and the quality of the crystals.The low sequence similarity of PDIR to known structures prevented us from using molecular replacement for phasing of the diffraction data. We produced selenomethionine-labeled crystals and determined the structure using a single-wavelength anomalous dispersion (SAD). Consequently, this model was used to refine the native protein structure at 1.50 Å resolution . The asyThe structure shows a typical thioredoxin-like fold containing a five-stranded central β-sheet in the β1˜ββ3˜ββ2˜ββ4˜ββ5 arrangement; all the strands are parallel except for strand β4. The β-sheet is flanked on both sides by two α-helices that are parallel to each other . The doma of human ERp57 Structural similarity search DALI b'x structure of human PDIA1, where a tryptophan residue of the x linker between the b' and a' domains inserts into the substrate-binding site on the non-catalytic b' domain We analyzed intermolecular contacts in the crystal for physiologically relevant protein-protein binding surfaces. The C-terminal tail of the PDIR construct is instrumental in crystal formation wherein the side chains of Leu143 and Trp144 insert into a pocket between strand β1 and helix α1 of an adjacent PDIR domain . The walWe calculated the sequence conservation in the non-catalytic PDIR domain and mapped it onto the structure to identify the functionally important surfaces . The mos15N-labeled the P-domain fragment of calreticulin (residues 211–261) and monitored the changes in the NMR spectrum upon addition of the non-catalytic domain of PDIR. Addition of the PDIR domain caused a number of shifts in the 1H-15N correlation spectrum of the P-domain, indicating binding between the proteins  and unlabeled P-domain of calreticulin. The 1H-15N correlation spectrum of the PDIR domain showed excellent dispersion of signals characteristic of a well-folded domain (We also performed a reverse NMR titration using d domain . Additiod domain .Although PDIR was first discovered more than 15 years ago, to our knowledge, this is the first structural characterization for any part of PDIR. The non-catalytic domain is of interest because of its enigmatic function and low sequence similarity to proteins of known structure. The structural similarity of the PDIR non-catalytic domain to the N-terminal domains of ERp57 and yeast PDI suggests that the proteins evolved in parallel from a common precursor with four catalytic domains. Perhaps, the PDIR N-terminal domain lost its catalytic activity while gaining the ability to bind substrates and other chaperones. More intriguing is the occurrence and function of PDIR N-terminal domain in RNA methyltransferases in parasitic nematodes. While the exact function in roundworms is unknown, SpoU methylase domains from other proteins bind to stem-loops of target RNA. The positively charged residues of PDIR are conserved in the methylase proteins and likely increase the affinity for binding RNA substrates.Our structure shows that the PDIR non-catalytic domain adopts a typical thioredoxin-like fold but with several distinct features. One of these is the presence of an intramolecular disulfide bridge between Cys85 and Cys94. These cysteines are strictly conserved in PDIR which suggests a structural requirement for the disulfide. A similar disulfide bridge is implicated in the stability of the third catalytic domain of ERp46 d of 16 µM NMR titrations show that the PDIR non-catalytic domain interacts with the P-domain of the ER lectin chaperone calreticulin. This result is consistent with the recent study that identified calreticulin as a binding partner of PDIR In conclusion, our results demonstrate a role for the PDIR non-catalytic domain as the primary binding site for the major ER chaperone calreticulin and possibly other proteins and substrates. The occurrence of PDIR-like sequences in methyltransferases in some nematodes hints at a broader function for the PDIR domain or is at least an unusual example of gene transfer.


Lactococcus lactis, which is widely used for industrial milk fermentation processes . As a result, lactococcal phages have become one of the most thoroughly characterized class of phages from a genomic standpoint. They exhibit amazing and intriguing characteristics. First, each phage has a strict specificity toward a unique or a handful of L. lactis host strains. Second, most lactococcal phages possess a large organelle at their tail tip (termed the baseplate), bearing the receptor binding proteins (RBPs) and mediating host adsorption. The recent accumulation of structural and functional data revealed the modular structure of their building blocks, their different mechanisms of activation and the fine specificity of their RBPs. These results also illustrate similarities and differences between lactococcal Siphoviridae and Gram-negative infecting Myoviridae.The Siphoviridae family of bacteriophages is the largest viral family on earth and comprises members infecting both bacteria and archaea. Lactococcal siphophages infect the Gram-positive bacterium  Lactococcus lactis is a Gram-positive bacterium extensively used for the production of fermented milk products, such as cheese production buttermilk and sour cream. L. lactis-containing starter cultures are therefore used world-wide in large scale industrial processes, which create ideal ecological niches for bacteriophage infections and development. These infections have a major economic impact due to impairment of lactococcal fermentations and the resulting need to close and decontaminate the production plants. Addressing this problem has proven to be challenging as L. lactis virulent phages are ubiquitous in plant environments and pasteurized milk , and the structure of their components involved in adhesion was determined by X-ray crystallography. These structural data, together with functional studies, made it possible to reveal striking features of lactococcal phages concerning their baseplate activation, and the specificity of their receptor binding proteins (RBPs). Since the recent discovery of the  unravel .Figures 1A,B). Lactococcal phages TP901-1 and p2 EM structures could be determined by dissecting the phages in smaller parts: capsid, connector, tail segments, and tail tip. The EM single-particle reconstruction was performed individually and the structures of these parts were reassembled on a scaffold obtained from analysis of a few straight tailed phages  hexamer from phage HK97 . A striking difference between the two tails is the presence of decorations on the tail of phage p2. Sequence analysis of phage p2 MTP revealed that its N-terminus shares similarity with other MTPs from other phages, such as SPP1 and Lambda . A prominent feature of these modules is the long tape measure protein (TMP), which determines the length of the tail  is the last gene encoding for a structural protein and is located directly upstream the holin and lysin ORFs . The shoulder domain (residues 1–141) has a β-sandwich fold assembling two 4-stranded anti-parallel β-sheets. A long helix contributed by each domain allows the three shoulder moieties to associate tightly. Immediately following the shoulder domains, the neck domain forms a triple-stranded β-helix of three segments organized into four β-strands along the threefold symmetry axis (β-prism). This very rigid neck structure has already been observed in the gp12 short tail fiber of Escherichia coli phage T4 (Myoviridae family), in which it plays a similar role, linking the N-terminal domain and the receptor binding head . It exhibits low but significant similarity to RBPs of other viruses: the reovirus attachment protein σ1 trimer . This “stem” domain is much smaller than the corresponding shoulder domain in the phage p2 RBP . However, in the p2 RBP, the three parallel helices (residues 19–32) located close to the threefold axis, are in a similar location to those of the TP901-1 RBP .The phage TP901-1 RBP trimer structure was determined by X-ray diffraction . Its N-tFigures 3D,E). The β-prism neck domain interlaces three segments of each subunit, and each of its three faces is made of four β-strands from the three monomers, constituting a domain comparable to that of the RBP of phage p2  connects the α-helical domain (17–30) and the β-prism  is a β-barrel formed of anti-parallel β-strands. This domain is the only part that shares sequence similarity with the RBP of p2 (28% sequence identity), and they exhibit very similar structures . Noteworthy, the crystal structure of the RBP head domain from phage bIL170 (936 group) displays the same fold as in phages p2 and TP901-1  via three hydrogen bonds established between the His 232 and Asp 234 side-chains and the glycerol O1 atom, and between the Arg 256 (from the other monomer) guanidyl group and the glycerol O2 atom . Furthermore, the hydrophobic face of glycerol packs against Trp 244 side-chain, as often observed with sugar complexes (The high-resolution structure of the p2 RBP (1.7 Å resolution) revealed the presence of three glycerol molecules (originating from the cryoprotectant liquor) bound at the interface between the head domains . The glyomplexes . Consideomplexes .Figures 4A,B). A specific interaction is observed between Tyr 55 of the nanobody, penetrating deeply in the glycerol-binding site , at the exact position occupied by the latter in the isolated RBP structure, and establishing interactions with Trp 244a and Arg 256b on either side. The OH group from Tyr 55 superimposes with the OH1 of glycerol and establishes similar hydrogen bonds with His 232 and Asp 234.The structure of the complex between p2 RBP and VHH5 was also determined by X-ray crystallography and revealed that the nanobody covers a large area of the head domain  are stacked against Phe 145 and establish hydrogen bonds with His 133, Asp 135, and Arg 155. As inferred from sequence alignments, three of these residues in the TP901 RBP are identical to those in the RBP of phage p2, while the fourth one corresponds to a substitution of Phe 145 by Trp 244. Two hydroxyl groups from the glycerol molecule are therefore strongly bound to the RBP head. In contrast, the third hydroxyl group is free and points to the bulk solvent in both structures . This orientation strongly suggests that the saccharidic binding site harbors the terminal residue of the receptor polymer, the free hydroxyl group pointing in the direction of the rest of the receptor polymer attached to the host.Glycerol molecules were also observed bound at the interface between head domains in the crystal structure of the phage TP901-1 RBP at 1.6 Å resolution . GlyceroFigures 3C,F). Differences in amino acid residues are only observed in the second binding sphere, which modulates the binding crevice surface and volume and, hence, may influence the saccharidic specificity observed between different phages and bacterial strains. The absence of tryptophan residue in the vicinity of the glycerol-binding site in the phage TP901-1 RBP prevented us to directly perform binding studies using tryptophan fluorescence quenching. A Phe145Trp point mutant was therefore designed and characterized the affinity of the complexes formed with glycerol, phospho-glycerol, N-acetyl muramic acid, muramyl-dipeptide, and galactose. The obtained affinity constants were roughly comparable to those measured for the p2 RBP . In contrast, nanobody 17 binds the stem domain and does not interfere with the receptor binding site . Indeed, L. lactis mutants lacking this “pellicle” could not be infected by their specific phage and genomic studies demonstrated the presence of L. lactis strain-specific “pellicle” cassettes coding for glycosyl-transferases and other enzymes involved in polysaccharide phosphate synthesis (The affinity of glycerol and phospho-glycerol for the RBPs suggested that lipoteichoic acids (LTA) could act as receptors for lactococcal phages. Conversely, the observation that many sugars bind equally well to the RBPs and the fact that the structure of LTAs is too simple to explain the different specificities of hundreds of lactoccocal phages constituted arguments against this hypothesis. A recent report by ynthesis . FinallyL. lactis strain MG1363 (L. lactis strain was biotinylated and attached to a SPR chip (ligand) while either the RBP of phage p2 or of phage TP901-1 was injected  to monitor interactions. While a typical saturation curve was obtained for the p2 RBP, yielding a Kd value of 230 ± 40 nM, it was not possible to reach saturation with the TP901-1 RBP. More significantly, the dissociation time of the TP901-1 RBP with the L. lactis MG1363 pellicle is extremely short as compared to that of the p2 RBP. As a result, phage TP901-1 would remain in contact for a very short time with the specific host of phage p2, which would be insufficient for adhesion and further infection (Preliminary experiments using surface plasmon resonance (SPR) explained the specificity mechanism of phage p2 RBP for n MG1363 . The purnfection .orfs 15 (Dit), 16 , 17, and 18 (RBP) encoded baseplate-related proteins (Table 1). The contiguous cluster of four genes was cloned, expressed in E. coli and purified, yielding a macromolecular complex of ~1.0 MDa containing ORFs 15, 16, and 18 .Based on their genomic location, we hypothesized that , and 18 . ORF17 c, and 18 . The basFigure 7A). The N-terminal domains form a tight ring with two layers of β-strands. This ring delineates a 40 Å-wide channel to allow the transit of the dsDNA genome during infection. The C-terminal domains (residues 137–275) are located at the ring periphery, and do not contact each other . They display a galectin fold supplemented by a long extension  having a critical role in baseplate assembly via the formation of a three-digit hand that anchors the N-terminal domain of the RBP .ORF15 (Dit) is composed of two domains. The N-terminal domain (“ring domain” 1–132) shows a split barrel-like fold similar to that found in phage Lambda gpV  and Hcp,Figures 7B,C) and its fold is similar to gp27 of myophage T4 .ORF16  is a 398 residue-long protein harboring four domains . Furthermore, residues 2–7 of ORF18 protrude from each subunit, forming the first strand of the shoulder domain of the next subunit by domain-swapping .The structure of ORF18 (RBP) is similar to that of ORF18 crystallized alone, with one exception: the N-terminal residues 2–17 of ORF18 in the baseplate structure are ordered and visible in the electron density. This is due to a tight interaction with the three-digit hand from the ORF15 galectin domain , which harbor the receptor-binding sites, to point “downward,” i.e., in the direction of the host cell surface. Instead, the RBPs were observed in a “heads-up” conformation, a position not compatible with optimal adhesion. However, the baseplate crystal structure fitted without rearrangement in the baseplate region of the p2 virion reconstruction .2+, attempts were made to obtain crystals in the presence of Ca2+ or Sr2+ without VHH5. New crystal forms were obtained readily with both cations, and their structure determined. Both structures were found to be identical, although the Sr2+ complex diffracted to higher resolution . The most striking feature was that the RBPs have rotated by ~200°, to orient their receptor-binding sites toward the distal phage extremity, leading to a “heads down” conformation . The ring formed by the N-terminal domain of ORF15 superposed well between the Sr2+-free and Sr2+-bound structures, but the galectin, arm and hand domains had moved significantly (2+ ion (or Ca2+ ion) is located at the interface between the N-terminal and the galectin domains of ORF15, and is coordinated by side chains of residues Asn10, Asp12, Asn241, Asp246, and the main-chain carbonyl of Leu11 , large enough for dsDNA passage. This opening results from a outward rotation of ORF16 cores with respect to the channel axis, and the opening of a crevice between domains 1, 2, 4, and 3 in an iris-like movement . Domain 3 remains in close contact with the next ORF16 in the trimer . In the activated baseplate structure, extensive interactions are established between ORF16s and ORF18s whereas these protein components were not in contact in the BP-VHH structure. In fact, these contacts lock the ORF18s in their “heads-down” conformation, giving to ORF16 the role played by the VHH5 molecules in the BP-VHH complex. Remarkably, the head domains are also maintained by the arm extensions belonging to the proximal Dit domains in the virion before being released when activation occurs .Since it was noticed that in some cases lactococcal phages infection required Casolution . The comficantly . The Sr2of Leu11 . The armorfs located between the dit and the rbp genes (orfs 46–49). Although this strategy was unsuccessful, a complex comprising only ORFs 46, 48, and 49 , could be expressed and purified, and its crystal structure determined at 3.8 Å resolution . From the proximal to distal end, it is formed by a Dit hexamer (ORF46) surrounded by 18 copies of BppU (ORF48) holding 54 RBPs (ORF49) organized as 18 trimers . All together, it forms a complex of 78 proteins and 54 possible receptor binding sites, organized in six tripods each containing three BppU and three trimeric RBPs . Six domains are appended to this core without forming contact with each other . Each monomer is formed from a N-terminal domain (residues 1–145) with a β-sandwich, an α-helix, and a β-hairpin, followed by a C-terminal domain (residues 146–255) folded as a galectin-like β-sandwich . This Dit structure is similar to that of phages SPP1 . Each monomer is composed of a N-terminal globular domain , a linker (123–134), two helices joined by a kink (135/139–194) and a globular C-terminal domain . This structure binds to the three stem domains of three RBP trimers . Each BppU C-terminal domain deeply projects a loop (residues 217–234) in the crevice formed at the top of the RBP trimer to anchor it to the baseplate via electrostatic interactions . Moreover, three aliphatic/aromatic residues belonging to BppU  fill the center of the RBP crevice. The conservation of the residues involved in the BppU/RBP interactions suggests that common architectural themes are found among P335-phages . The six most peripheral RBPs do not establish any contact and appear to be highly mobile , which likely harbor different pellicles in terms of composition and structure.This baseplate structure is most probably shared, more or less closely, by several phages from the P355 group and beyond. EM studies have demonstrated the structural resemblance of the P335 phage Tuc2009 baseplate with that of TP901-1 . The majreceptor . Althoug2+ whereas TP901-1-like phages do not require Ca2+ for infection .Comparison of the structures of the p2 and TP901-1 baseplates revealed that the latter is already in a “ready to adsorb” conformation without requiring any conformational change. This observation could be correlated with functional data as p2-like phages are non-infectious in the absence of Canfection . These rges SPP1  and Lambges SPP1  affinityges SPP1 . This obL. lactis host strains can now be rationalized considering the diversity allowed by the chemical structure of the “pellicle” phospho-polysaccharide forming the most external layer surrounding these cells. Interactions between phage RBPs and the pellicle appear to be characterized by a moderate affinity but with long enough adhesion times (thanks to low koff) to allow initiating infection. Progresses are being made to decipher the RBP/pellicle recognition mechanisms in different phages, aiming to understand the molecular determinants of this specificity.The fine specificity of lactococcal phages for their L. lactis. This probably reflects the fact that many phages use sugars as receptors. The nominal weaker affinity for polysaccharides as compared to protein/protein interactions (e.g., phage T5 pb5 binds to FhuA receptor with sub-nanomolar affinity; According to the literature, a large number of phages exhibit a baseplate, beyond those infecting 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.