In contrast, modern structural designs such as prisms, propellers, 2-solenoid, super-roll, clam, trefoil and box are not widely distributed and were probably adopted to perform specialized functions. Our timelines therefore uncover a universal tendency towards protein structural complexity that is remarkable.The spatial arrangements of secondary structures in proteins, irrespective of their connectivity, depict the overall shape and organization of protein domains. These features have been used in the CATH and SCOP classifications to hierarchically partition fold space and define the architectural make up of proteins. Here we use phylogenomic methods and a census of CATH structures in hundreds of genomes to study the origin and diversification of protein architectures (A) and their associated topologies (T) and superfamilies (H). Phylogenies that describe the evolution of domain structures and proteomes were reconstructed from the structural census and used to generate timelines of domain discovery. Phylogenies of CATH domains at T and H levels of structural abstraction and associated chronologies revealed patterns of reductive evolution, the early rise of Archaea, three epochs in the evolution of the protein world, and patterns of structural sharing between superkingdoms. Phylogenies of proteomes confirmed the early appearance of Archaea. While these findings are in agreement with previous phylogenomic studies based on the SCOP classification, phylogenies unveiled sharing patterns between Archaea and Eukarya that are recent and can explain the canonical bacterial rooting typically recovered from sequence analysis. Phylogenies of CATH domains at A level uncovered general patterns of architectural origin and diversification. The tree of A structures showed that ancient structural designs such as the  sandwiches, bundles, barrels, prisms, solenoids, and propellers. The appearance and diversification of general structural designs and their confirmation from published literature defines a unique chronology of structural innovation. The study also uncovers a recent trend of architectural sharing between Archaea and Eukarya and benchmarks the phylogenomic analysis of CATH domains with SCOP domains.Proteins are vital and central macromolecular players necessary for the functioning of the cell. The redundant and highly conserved structural makeup of proteins reflects their ability to act as genomic repositories of evolutionary history. These structures are fundamental subjects for the study of molecular evolution. Structural biologists have demonstrated the existence of a wide array of compact 3-dimensional fold structures, the protein domains. Their classification resulted in hierarchical taxonomies that describe protein fold space, most notable SCOP, CATH and FSSP. Studies have shown that certain types of protein shapes are more abundant than others and this uneven distribution implicates processes by which new shapes are discovered. Our evolutionary genomic research explores the evolution of protein domains at the deeper levels of classification. However, we have not embarked in a systematic study of the origin and evolution of general structural designs. These designs include topologies such as  The polypeptide chains of proteins generally fold into highly ordered and well-packed three-dimensional (3D) atomic structures Protein domains are compact, recurrent, and independent folding units of protein structure that sometime combine with other domains to form multi-domain proteins. They are considered evolutionary units and are the basis for several protein structure classification schemes. Two of them, CATH and SCOP, are accepted as gold standards and share a number of common features Protein structures are evolutionarily conserved and capable of preserving an accurate record of genomic history sandwiches, bundles, barrels, solenoids, propellers) and published literature define in this study a unique chronology of structural innovation. Structural phylogenies of domains at T and H levels of structural abstraction uncover global evolutionary patterns of structural distribution in the world of organisms. The study benchmarks previous phylogenetic analysis of SCOP-defined domains and again reveals the early origin of the archaeal superkingdom. Congruent patterns of diversification derived from protein structure provide remarkable support to the ancient history of the cellular world, and trees of life confirm the primordial evolutionary patterns.While we have studied how F, FSF and FF domains appeared and distributed in the world of organisms, we have not embarked in a systematic study of the origin and evolution of general structural designs. Here we study how these designs evolve in trees of domain structures, this time focusing on the CATH classification. The appearance and diversification of general protein structural designs at A-level  than lower taxonomical levels, confirming the contention that they are evolutionarily more conserved. Second, the highest level of structural abstraction (CATH A) does not contain a single superkingdom-specific taxonomic group, suggesting that these groups represent sets of structures that are late evolutionary additions to the protein repertoire. Finally, ABE and BE domain structures are consistently the dominant taxonomic groups at all hierarchical levels, from FF to A. This final observation suggests they represent the most ancestral and common taxonomical groups. The most parsimonious corollary of these evolutionary patterns of domain distribution is that the ancient BE taxonomical group must arise by loss of archaeal-specific domain structures, suggesting Archaea is the most ancient superkingdom. As we will now show, this suggestion can be confirmed by phylogenomic reconstruction.nd) was measured on the trees as a relative distance in nodes from the hypothetical ancestor of domains at the base of the trees, and used to build the timelines. Since our method produces rooted trees that are highly unbalanced and reject the Yule and random speciation models nd was considered a good and most-parsimonious proxy for time. To study how domain structures distribute in proteomes, we calculated a distribution index (f), the number of species that use each structure given on a relative 0–1 scale. The f index was plotted along the timelines of domain structures, i.e. against nd (And = 0–0.068), fifteen Ts (Tnd = 0–0.061) and fifteen Hs (Hnd = 0–0.049) were present in all proteomes examined (f = 1) and were the most ancient in the timeline. A list of the fifteen Hs is given in f of As decreased with increasing age. The f of Ts and Hs decreased with their increasing age until f approached zero at Tnd = 0.55 and at Hnd = 0.55, respectively. We term these ages “crystallization points” of the T and H structural chronologies, borrowing the idea of a phase transition from physics. At these time points, a steady decrease in f results in a large number of structures being specific to a small number of organisms. After crystallization, an opposite trend takes place, in which Ts and Hs increase their representation in genomes. In contrast, the architectural chronology that describes the appearance of As remained unaffected by the crystallization event since the losing trend of As started at And = 0.56–0.60 but rarely reached zero  with the membrane-bound lytic murein transglycosylase D (chain A) H domain (3.10.350.10). Its appearance is congruent with the loss of the first SCOP FSF in Archaea (FSFnd = 0.174), the LysM domain (d.7.1), observed in previous studies f-value) also occurred in Bacteria and Eukarya, but involved fewer and younger structures. Analysis of domain distribution in Bacteria shows that AB and B structures (dark yellow) started to increase representation after the crystallization point, leading towards their diversification and specification. Similarly, the eukaryotic chronology showed that comparatively younger architectures [e.g. BE (blue) and E (green)] increased their popularity among the eukaryal lineages. The appearance and distribution of the seven taxonomical groups of T and H structures was unfolded in the timelines using boxplots describing the range of Tnd and Hnd values and measures of central tendency for each group (nd = 0) to the present (nd = 1). These structures represent instantiations of the domain content of the urancestor but their late appearance may also indicate events of horizontal transfer between lineages. Boxplots for BE, AE and AB explain relationships among superkingdoms over time. The BE boxplot is the most ancient of the three, suggesting Archaea diversified early by reductive evolution. The A, B and E boxplots reflect the history of ‘signature’ structures that are unique to individual superkingdoms. These signatures appear first in Bacteria and then concurrently in Archaea and Eukarya, an observation that is congruent with timelines derived from SCOP domains f values, suggesting these molecular designs were acquired as adaptations to new environments and lifestyles. The appearance of structures shared by only two superkingdoms was also revealing. For example, the AE boxplot's upper whisker approached Hnd = 1, implying a recent relationship between Archaea and Eukarya. Comparatively, the nd values for SCOP FSFs for the AE taxonomical group was FSFnd = 0.85, supporting the late appearance of the interaction To uncover hidden patterns of organism diversification in our dataset, we divided structures according to their distribution in superkingdoms and constructed three separate structural chronologies for the genomes of each superkingdom at A, T and H levels of structural abstraction . Taxonomch group . Only doHnd<0.176), H structures of intermediate ancestry corresponding to the superkingdom specification epoch (0.176<Hnd<0.55) and H structures that are derived and reflect the organismal diversification epoch (0.55<Hnd). The proteome tree reconstructed from the most ancient H structures was rooted paraphyletically in Archaea, reflecting their early segregation through the minimalist strategy. Reconstructions from H structures of intermediate ancestry produced trees with three clades corresponding to the three superkingdoms that were rooted in Archaea. Finally, reconstructions from H structures that were derived yielded the canonical tree of life rooted in Bacteria. It is noteworthy that the rooting of these trees reflects the early appearance of Bacteria-specific domain structures  sharing of protein architectures between archaeal and eukaryal genomes. The timeline reveals that while AE domain structures appeared for the first time when Archaea and Eukarya acquired their superkingdom-specific signature structures, the vast majority of them appeared quite late in evolution e.g., . This waf values indicate for example their presence in most of archaeal and eukaryotic proteomes  sandwich (3.40) is the most abundant and ancient of all proteins. The orthogonal bundle (1.10) and the α/β-complex (3.90) are equally abundant and are the second and third most ancient architectures. Remarkably, the phylogenomic tree of As shows that comparatively simpler shape structural designs are more favored than complex designs and in general are more ancient, appearing at the base of the tree. Architectural complexity was here evaluated on empirical grounds by focusing on the topology and regularity of spatial arrangements of secondary structures in a structural design. For example, the most ancient 3.40 and 1.10 architectures involve simple arrangements of secondary structure that can be very diverse in different structural variants while more recent shape designs are spatially more convoluted and regular (And = 0.76) with the very complex Clam architecture, and then in Archaea and Bacteria. We note that Archaea loses four As quite late and in a row, showing that the pervasive reductive trends of Archaea described above extend almost to the present. This also reflects the conservative nature of extremophilic Archaea, which are not in need of modern structural designs. Bacteria loses the most recent A structural design, Box (2.80), at And = 1, which is shared by both archaeal and eukaryal genomes. Box is involved in nucleotide excision repair, a molecular function that has a unique place in cellular defense because of its wide substrate range and its ability to virtually remove all base lesions from a genome. Ögrünç et al. f index for Box in Archaea (f = 1) and Eukarya (f = 0.96) again indicates a recent sharing of structural designs between archaeal and eukaryal organisms. Architectures constitute the second highest level of structural abstraction in CATH, and because of their high conservation it is difficult to clearly delimit the three epochs of the protein world. In contrast, our results indicate CATH H and SCOP FSF are the most suitable levels to uncover the evolution of domain structures in genomes. These levels of abstraction are structurally and evolutionarily conserved. They preserve deep phylogenetic signatures and are variable enough to dissect evolutionary history of proteomes and molecular functions.The architectural chronology of As is evolutionarily more conserved than chronologies of Hs and Ts . The tim regular . As time regular . The fewsandwiches, bundles, barrels, prisms, horseshoes, rolls, solenoids, propellers, complexes and other   and rather simple structural designs , prisms, trefoil and box, have low f values (f = 0.85–0.10) and are very complex. Under the assumption that widespread and abundant designs are old, complex folds appear to have evolved later than simpler folds. We also mapped the appearance of T and H structures harboring individual A designs, plotting Hnd and Tnd values for Hs and Ts belonging to each of the 38 known As  . We foun designs . In turnknown As .3-layer (αβα) sandwich (3.40) architecture  The structural makeup of the most ancient itecture  represenorthogonal bundle (1.10) and α-β-complex (3.90) appear immediately after the 3-layer (αβα) sandwich (3.40) design. The orthogonal bundle consists of a 3–4 α-helix bundle and is found in a number of different proteins, most of which associate with membranes. Due to physical constraints imposed by the lipid bilayer of membranes the list of possible membrane protein structures is limited to either bundles interleukin-2, interleukin-4, human growth hormones, and the granulocyte-macrophage colony-stimulating factor (GM-CSF)α/β-complex architecture groups together all those designs that include significant α and β secondary structural elements in a mixed fashion. Examples of α/β-complex proteins include bacterial and mammalian pancreatic ribonucleasesZn metallo-proteases and DNA topoisomerasesbarrel structures are the most ancient and abundant in the protein world, the α/β-barrel (3.20) and the β-barrel (2.40)And = 0.13). The α/β-barrel is composed of eight α-helices and parallel β-strands that alternate along the peptide backbone. The α/β-TIM barrel is the most prominent example of α/β-barrel and is widely present in enzymes of central metabolism β-barrel is a large β-sheet that twists and coils to form a closed structure in which the first strand is hydrogen bonded to the last. β-strands in β-barrels are typically arranged in an antiparallel fashion. Barrel structures are commonly found in porins and other proteins that span cell membranes and in proteins that bind hydrophobic ligands in the barrel center, such as lipocalinsroll is a complex nonlocal structure in which 3–4 pairs of antiparallel β-sheets, only one of which is adjacent in sequence, are ‘wrapped’ in 3D space to form a barrel shape And = 0.3.The solenoids, horseshoes, prisms, propellers and trefoils. Solenoid proteins, with their arrays of repeating motifs, tend to have elongated structures that contrast with the majority of globular proteins whose polypeptide chains follow more complex trajectories armadillo repeats And = 0.46. The α-horseshoe protein appears at And = 0.4, is a super helical structure made up of a number of three α-helical orthogonal bundle repeats. The α-β horseshoe appeared at And = 0.56, consists of several α/β-repeating units ribonuclease inhibitor, a cytosolic protein that binds strongly to any ribonuclease that may leak into the cytosol, takes the concept of the repeating α/β unit to the extreme Prisms are similar to solenoids in geometry but completely different in connectivity. A more self-contained β-sheet forms each face of a triangular prism. They appear late at And = 0.86. The trefoils consist of an unusual β-sheet formed by six β hairpins arranged with three fold symmetry into ‘Y’ like structures And = 1).A number of distinct and more complex architectures appear later on in the chronology, including 3-layer (αβα) sandwich (3.40), harbors the most ancient and abundant topology, the Rossmann fold (3.40.50) and the most ancient and abundant superfamily, the P-loop containing nucleotide triphosphate hydrolases (3.40.50.300). Despite differences of topology and ranking within databases P-loop containing nucleotide triphosphate hydrolase” FSF (c.37.1) of SCOP The most ancient and popular architecture, the We note that levels of structural abstraction above H and FSF unify domains that may not be necessarily homologous. In other words, T and A in CATH and F in SCOP may show episodes of structural convergence. This could complicate evolutionary interpretations. The fact that the same evolutionary patterns observed using H domain structures in this study  sandwich cytosolic architecture and the orthogonal bundle that often associates with membranes are the most ancient and are preferentially involved in metabolic activities. The origin of proteins thus lies at the interface of primordial membranes and cytoplasm. Bundles and barrels that populate membranes soon follow. Metabolic and membrane proteins thus appear crucial for the early biochemistry of primordial cells Structural designs that are architecturally simpler are ancient and highly abundant in the extant world of proteins and organisms. We find the prisms, propellers, 2-solenoid, super-roll, clam, trefoil and box are derived and less favored in the world of organisms. These designs are generally specific to groups of organisms and have been probably adopted for specialized functions.Structural designs that are architecturally complex, such as Although CATH and SCOP differ significantly in their protein domain definitions and in the hierarchical partitioning of fold space, we find that both protein structural classifications classify a protein on very similar theoretical grounds by taking into account their structural, functional and evolutionary roles. Remarkably, CATH and SCOP structures harbor similar phylogenetic signatures and reveal patterns of origin and diversification that are congruent.Structural chronologies provide evidence that Archaea established the first organismal divide by losing a substantial number of domain structures early in evolution. We speculate this reductive evolutionary process reflects the environmental pressure of an ancient extremophilic lifestyle that forced maintenance of a minimal domain repertoire. Our analysis provides additional support to the archaeal rooting of the tree of life obtained from evolutionary analysis of SCOP domains e.g. , RNA strBox (2.80) design that is involved in nucleic acid repair.Structural chronologies uncover a recent trend of sharing of domain structures between Archaea and Eukarya that continues to the present and involves complex architectures such as the Finally, we also speculate that this modern archaeo-eukaryotic architectural sharing pattern is the most probable reason for the bacterial rooting of the canonical tree of life reconstructed from changes in sequence. In contrast to structure, sequence evolution is more dynamic and prone to phylogenetic signal loss We note that these conclusions entrust CATH with the ability to properly apportion domain structures in fold space and are only valid if assumptions of character argumentation are valid.Our trees of domain structures define timelines that trace back the history of innovation, diversification and distribution of protein structural designs. Our finding that protein architectures tend to become more complex in evolution is very significant. In a previous study, analysis of β-barrel structures revealed that the curl and stagger and complexity of the connectivity of supersecondary structures increases in evolution Gene3DGene3D is a repository of manually curated HMM predictions with a false positive prediction rate of only 0.2–0.6%. As with SUPERFAMILYGene3D were searched against HMM libraries using the iterative Sequence Alignment and Modeling System (SAM) method. Data matrices of genomic abundance (G) of domains at A, T and H levels were assembled for phylogenetic analysis. Empirically, G values represent numbers of multiple occurrences of an A, T and H domain in a genome, ranging from 0 to thousands and resembling morphometric data with large variances. Because existing phylogenetic programs can process only tens of phylogenetic character states depending on user's CPU performance, the space of G values in the matrix was reduced using a standard gap-coding technique with the following formula:G values on a 0–20 scale  test with a tree length distribution obtained from 1,000 random trees.Phylogenomic trees were reconstructed using the maximum parsimony (MP) optimality criterion in PAUP* with 1,000 replicates of random taxon addition, tree bisection reconnection (TBR) branch swapping, and maxtrees unrestricted. Phylogenetic confidence was evaluated by the nonparametric bootstrap method with 1,000 replicates . The degree of phylogenetic signal for taxa was measured using the skewness (nd) from the hypothetical ancestral architecture at the base of the trees in a relative 0–1 scale. nd was calculated by counting the number of internal nodes along a lineage from the root to a terminal node (a leaf) of the tree on a relative 0–1 scale with the following formula:a represents a target leaf node , r is a hypothetical root node, and m is a leaf node that has the largest possible number of internal nodes from node r. Consequently, the nd value of the most ancestral taxon is 0, whereas that of the most recent one is 1. Node distance can be a good measure of age given a rooted tree because the emergence of protein domains  is displayed by their ability to diverge (cladogenesis or molecular speciation) rather than by the amount of character state change that exists in branches of the tree (branch lengths).Since trees of domain structures are rooted and are highly unbalanced, we unfolded the relative age of protein domains directly for each phylogeny as a distance in nodes Click here for additional data file.Figure S1A methodological flowchart illustrating the reconstruction of phylogenies of proteomes and protein architectures using protein domain census data.(PDF)Click here for additional data file.Protocol S1By hand example of protein domain data normalization and encoding. The objective is to build a phylogenomic matrix for the reconstruction of a tree of domain structures.(PDF)Click here for additional data file.Table S1List of 15 most ancient and popular Hs.(PDF)Click here for additional data file.Table S2List of 492 organisms with their genome id and genome names.(PDF)Click here for additional data file.Table S3List of 295 FL proteomes used to reconstruct proteome trees.(PDF)Click here for additional data file.


The GFOGER motif in collagens (O denotes hydroxyproline) represents a high-affinity binding site for all collagen-binding integrins. Other GxOGER motifs require integrin activation for maximal binding. The E318W mutant of the integrin α2β1 I domain displays a relaxed collagen specificity, typical of an active state. E318W binds more strongly than the wild-type α2 I domain to GMOGER, and forms a 2:1 complex with a homotrimeric, collagen-like, GFOGER peptide. Crystal structure analysis of this complex reveals two E318W I domains, A and B, bound to a single triple helix. The E318W I domains are virtually identical to the collagen-bound wild-type I domain, suggesting that the E318W mutation activates the I domain by destabilising the unligated conformation. E318W I domain A interacts with two collagen chains similarly to wild-type I domain (high-affinity mode). E318W I domain B makes favourable interactions with only one collagen chain (low-affinity mode). This observation suggests that single GxOGER motifs in the heterotrimeric collagens V and IX may support binding of activated integrins. Additional contacts with the α2 I domain are made by phenylalanine and arginine residues of the GFOGER peptide. GFOGER constitutes a high-affinity motif for all the collagen-binding integrins reported to date Conformational change in the α2 I domain was revealed by its co-crystallisation with a collagen-like, triple-helical, peptide of sequence [GPO]2+ ligand T221 to alanine, disrupting the MIDAS Various strategies have been adopted to produce constitutively active or inactive forms of the α2 I domain. Inactivation has been achieved by mutating the MgHere we report a functional and structural characterisation of the α2 I domain E318W mutant. Unlike the wild-type I domain, the E318W mutant binds the GFOGER peptide with 2:1 stoichiometry. Because the three chains of a collagen triple helix are not topologically equivalent, two distinct binding modes are observed crystallographically, one resembling that of the wild-type I domain and the other sacrificing one of the two favourable contacts with the phenylalanine residues of the GFOGER motifs. The structure suggests how activated integrin α2β1 might bind to heterotrimeric collagens harbouring a high-affinity motif in only one of the three chains of the triple helix.We previously used a solid-phase binding assay to investigate binding of α2 I E318W to the Collagen Toolkit III, which represents the entire triple helix of collagen III as a set of overlapping peptides We also investigated the binding of α2 I E318W to a larger panel of triple-helical Gxx'GEx'' peptides than previously used Binding of α2 I E318W to triple-helical peptides in solution.2GxOGER[GPO]3,×  = F, M, A) in solution using analytical size exclusion chromatography (SEC). Because the collagen-like peptides do not absorb at 280 nm, they can be added in molar excess without obscuring the elution of the protein peak. The wild-type α2 I domain formed a complex with the GFOGER peptide, as evidenced by a 0.6 ml reduction in elution volume , as well as to the ligated conformation of the wild-type α2 I domain Consistent with the SEC data, the asymmetric unit of the crystals contains one GFOGER peptide interacting with two molecules of α2 I E318W, termed A and B . The col2+ ion of the MIDAS; the same chain also provides the phenylalanine that makes van der Waals contacts with N154 and Q215, and the arginine that interacts electrostatically with D219  compared with molecule A (530 Å2).Molecule B also interacts with two of the three collagen chains, but because the three chains in a triple helix are not topologically equivalent, some of the interactions made by molecule A are not possible. The leading chain interacts with molecule B in the same way as the trailing chain with molecule A . In contWe considered whether it is theoretically possible to achieve identical binding modes in a 2:1 complex. We modelled a complex in which I domain A is bound to the leading and middle chains, and I domain B is bound to the middle and trailing chains . In contrast to the crystal structure, this modelled arrangement leads to clashes between the two I domains in a region that is key for allosteric regulation (residues 285–291) 2+ ion of the MIDAS. Binding of α2 I E318W to shorter high-affinity peptides was similar to wild-type, probably because the assay was saturated. With lower affinity peptides, notably GMOGER, a substantial increase in binding of α2 I E318W was observed , as a heterotrimer with GLOGER in the α2(I) chain, and in collagens α1(II), α1(III), α2(V) and α3(IX). In collagens V and IX, GMOGER is present without a corresponding motif in the other constituent α-chains. Recent advances in producing register-specific collagen heterotrimers The GST-tagged recombinant human α2 I domain-encoding plasmids (pGEX-2T-α2 I and pGEX-2T-E318W-α2 I) for binding studies were a generous gift from Dr D. Tuckwell . For crystallisation and SEC, the His-tagged E318W I domain ORF was cloned into the bacterial expression vector pDEST-N110 (a kind gift of Dr. M. Dyson) as described E. coli cells (Novagen) were transformed with the respective expression plasmid and a 50 ml overnight culture used to inoculate 1 litre of Luria Broth medium containing 50 μg/ml ampicillin (Melford). After growing the culture for 2 h at 37°C, expression was induced with 0.5 mM isopropyl β-D-thiogalactopyranoside (Melford), and the culture grown overnight at 25°C. The cells were harvested by centrifugation for 20 min at 4500 g and the pellet was gently resuspended in 25 ml of ice-cold TES buffer  containing 1 tablet of EDTA-free protease inhibitor cocktail (Roche Applied Science), 50 mg of lysozyme (Sigma-Aldrich) and 25 mg of protamine sulphate (Sigma-Aldrich). The suspension was left on ice for 10 minutes and centrifuged for 10 min at 6000 g (4°C). The supernatant was collected and the pellet gently resuspended in 25 ml of ice-cold 5 mM MgSO4 containing 1 tablet of EDTA-free protease inhibitor cocktail, 50 mg of lysozyme and 25 mg of protamine sulphate. After 10 min on ice the suspension was centrifuged for 10 min at 6000g (4°C) and the pellet discarded. The pooled supernatants were centrifuged for 20 min at 16000g (4°C) and filtered through a 0.22 μm filter. Imidazole (Merck) was added to a final concentration of 50 mM and the solution loaded onto a 5 ml HisTrap FF column  equilibrated in PBS  using an ÄKTA Purifier system . The proteins were eluted with PBS containing 150 mM imidazole, concentrated using Vivaspin centrifugal devices (Sartorius) and further purified by SEC using a Superdex 75 10/300 GL column  and a running buffer consisting of 100 mM Tris-HCl pH 7.5, 150 mM NaCl and 2 mM MgCl2 (SEC buffer). The final protein yields were ∼10 mg/litre of culture.The wild-type and E318W α2 I domains were produced in the same way for binding studies; recombinant proteins were expressed as described 5−[insert]-(GPP)5GPC. For Toolkit peptides, the insert contains 27 amino acids of primary collagen sequence, each subsequent peptide advancing 18 residues along the collagen triple-helical domain creating a 9−residue overlap between adjacent peptides in the set 2−[insert]-(GPO)3, the GPO flanking sequence providing greater thermal stability. Peptides, as C-terminal amides, were synthesised on TentaGel R-Ram resin using an Applied Biosystems Pioneer peptide synthesiser as described previously The general format of the peptides for solid-phase binding assay is GPC(GPP)2 or EDTA for 1 h at room temperature. Wells were washed five times with 200 μl of adhesion buffer containing MgCl2, before adding 100 μl of adhesion buffer containing Amersham anti-GST-HRP conjugate  at 1:10,000 dilution for 1 h at room temperature. After washing, colour was developed using an ImmunoPure TMB Substrate Kit (Pierce) according to the manufacturer’s instructions. Replacing Mg2+ with EDTA abolished binding (not shown). Data for short peptide binding were compared using 2-way ANOVA to establish the overall effect of the E318W mutation, and using repeated measures 1-way ANOVA for specific comparisons .I domain adhesion was determined colorimetrically using a solid-phase binding assay, modified slightly from our previous studies The lyophilised triple-helical peptides  were dissolved in 500 μl of 1 mg/ml stock solutions of either wild-type or E318W α2 I domains. The amount of peptide was chosen so as to yield a greater than 2-fold molar excess of peptide. After incubation for 30 min at 4°C, the solutions were injected onto a Superdex 200 10/300 GL column and run at 4°C at a flow rate of 0.5 ml/min in SEC buffer.2, 0.1 M bis-Tris pH 5.5, 25% PEG 3350 as precipitant. Crystals were flash-frozen in liquid nitrogen directly from the crystallisation drop.The α2 I E318W-GFOGER complex was formed by dissolving 3.1 mg of lyophilised GFOGER peptide in 2 ml of a 3.5 mg/ml solution of α2 I E318W in SEC buffer (∼2:1 peptide:protein ratio). After incubation for 30 min at 4°C, the solution was subjected to SEC as in the analytical experiments. The fractions corresponding to the α2 I E318W-GFOGER complex were pooled, concentrated to 9 mg/ml and stored at 4°C overnight. A substantial precipitate was observed on the following day and pelleted by centrifugation. The clear supernatant was stable at 5.3 mg/ml and screened for crystallisation using a Mosquito nanolitre robot (TTP LabTech). Crystals grew after 2 days with 0.2 M MgClfree and the smallest difference between Rwork and Rfree. Mg2+-ligand distances were restrained to 2.07 Å. Difference density indicated the presence of a Mg2+-bound water molecule in each I domain; due to the low resolution of the diffraction data, water molecules were not included in the model, however. Crystallographic statistics are summarised in www.pymol.org). Surface areas were calculated with AREAIMOL Diffraction data were collected from a single α2 I E318W-GFOGER crystal at 100K on beamline I04 of the Diamond Light Source  using a wavelength of 0.9795 Å and a CCD detector (ADSC). The diffraction data were integrated with XDS


Collagen is modified by hydroxylation and glycosylation of hydroxylysine residues. This glycosylation is initiated by the β1,O galactosyltransferases GLT25D1 and GLT25D2. The structurally similar protein cerebral endothelial cell adhesion molecule CEECAM1 was previously reported to be inactive when assayed for collagen glycosyltransferase activity. To address the cause of the absent galactosyltransferase activity, we have generated several chimeric constructs between the active human GLT25D1 and inactive human CEECAM1 proteins. The assay of these chimeric constructs pointed to a short central region and a large C-terminal region of CEECAM1 leading to the loss of collagen galactosyltransferase activity. Examination of the three DXD motifs of the active GLT25D1 by site-directed mutagenesis confirmed the importance of the first (amino acids 166–168) and second motif (amino acids 461–463) for enzymatic activity, whereas the third one was dispensable. Since the second DXD motif is incomplete in CEECAM1, we have restored the motif by introducing the substitution S461D. This change did not restore the activity of the C-terminal region, thereby showing that additional amino acids were required in this C-terminal region to confer enzymatic activity. Finally, we have introduced the substitution Q471R-V472M-N473Q-P474V in the CEECAM1-C-terminal construct, which is found in most animal GLT25D1 and GLT25D2 isoforms but not in CEECAM1. This substitution was shown to partially restore collagen galactosyltransferase activity, underlining its importance for catalytic activity in the C-terminal domain. Because multiple mutations in different regions of CEECAM1 contribute to the lack of galactosyltransferase activity, we deduced that CEECAM1 is functionally different from the related GLT25D1 protein. Collagen undergoes several post-translational modifications before formation of a right-handed triple helix in the endoplasmic reticulum. These modifications include the hydroxylation of selected proline GLT25D1 and GLT25D2 as encoding collagen galactosyltransferase  enzymes Caenorhabditis elegans have shown that the inactivation of the lysyl hydroxylase gene let-268 yields a lethal phenotype related to abnormal collagen type-IV secretion D2045.9 gene, the putative ortholog to human GLT25D1 and GLT25D2, is associated to growth defects and multiple morphologic abnormalities Whereas the roles of proline and lysine hydroxyllation in collagen biology are well established, the functional relevance of collagen glycosylation is presently unclear. Recently, we have identified the two genes The sequences of the GLT25D1 and GLT25D2 proteins were found to be strongly similar to the cerebral endothelial cell adhesion molecuzle (CEECAM1) protein In the present work, we investigate the rationale for the lack of ColGalT activity in the CEECAM1 protein by domain swapping and single point mutation. By permutation with portions of the active GLT25D1 galactosyltransferase, the production of GLT25D1-CEECAM1 chimeric proteins enables the identification of the regions responsible for the loss of galactosyltransferase activity. In parallel, this approach allows narrowing down the regions of GLT25D1 that are essential for the catalytic activity.http://toolkit.tuebingen.mpg.de/hhpred) Escherichia coli (PDB ID: 2z86). The region ranging from amino acids 24 to 466 of human GLT25D1 produced a hit with an E-value of 6.2E-09 and P-value of 2.7E-13. This bacterial chondroitin polymerase belongs to the CAZY GT2 family of glycosyltransferases and contains two functional domains adopting a glycosyltransferase GT-A fold The CEECAM1 protein is structurally similar to the ColGalT enzymes GLT25D1 and GLT25D2. The comparison of the three polypeptides shows 55% sequence identity between human CEECAM1 and human GLT25D1 and 50% between human CEECAM1 and human GLT25D2 To identify the molecular basis for the lack of collagen glycosyltransferase activity in CEECAM1, we constructed chimeric proteins between GLT25D1 and CEECAM1 based on the three domains mentioned here above. The corresponding chimeras were named CEECAM1-Nter, CEECAM1-Mid and CEECAM1-Cter . The chiTo delineate more precisely the regions of CEECAM1 and GLT25D1 that are essential for enzymatic activity, we have produced four additional chimeric constructs for the central domain and five additional constructs for the C-terminal domain. From the four chimeric constructs containing portions of the CEECAM1 central domain, only the construct, in which the stretch between amino acids 232 and 282 of GLT25D1 was replaced by the corresponding stretch of CEECAM1 (named CEECAM1-MidC), was found to be enzymatically active, yet with decreased ColGalT activity to 32% of wildtype GLT25D1 levels . The anaTo study the impact of the C-terminal domain on ColGalT activity, five chimeric constructs encompassing parts of the CEECAM1 C-terminal domain in GLT25D1  were exp2+ chelation The presence of DXD motifs is typical for glycosyltransferases utilizing nucleotide-activated sugars as donor substrates. Such DXD motifs are usually involved in the binding of the nucleotide diphosphate moiety through MnAs noted in our domain swapping experiments, a portion of the CEECAM1 central domain abolished the enzymatic activity when replacing the GLT25D1 stretch encompassing amino acids 280 to 342 . In an aThe comparison between the C-terminal domain of GLT25D1, GLT25D2 and CEECAM1 isoforms from various animal genomes pointed to 26 amino acids diverging between GLT25D1 and GLT25D2 on the one hand and CEECAM1 on the other hand . As showm and Vmax parameters of the partially active chimeric constructs CEECAM1-MidC and CEECAM1-CterC to assess whether the decreased activity can be related to altered interaction with the substrates. This kinetic analysis showed that the low ColGalT activity of CEECAM1-MidC was due to decreased Vmax  and the α1,4 N-acetylhexosaminyltransferase EXTL2 (CAZY family GT64) 2+ and with the diphosphate moiety of the donor substrate.The N-terminal domain of GLT25D1 and CEECAM1 were interchangeable without losing the galactosyltransferase activity, which did not mean that this domain was dispensable for the enzymatic activity. In fact, the DXD motif in this domain was shown to be essential to the activity of GLT25D1. Since the N-terminal domain of CEECAM1 could yield an active chimeric protein when combined with GLT25D1, the experiment showed that both proteins are structurally closely related and that such chimeric constructs are neither instable nor inactive Most of the central domain of CEECAM1 failed to yield any enzymatic activity when combined to GLT25D1. The kinetic analysis of the CEECAM1-MidC construct showed that this region was important for the rate of the galactosyltransferase reaction. Considering the predicted GT-A fold of the GLT25 family proteins, this finding suggests that the central domain is important to associate the two lobes of the active enzymes to encompass the donor and acceptor substrates.The C-terminal domain of the active GLT25D1 enzyme includes two DXD motifs, which are missing in the corresponding domain of the inactive CEECAM1 protein. Our mutagenesis experiments showed that the first of these two DXD motifs is required for the ColGalT activity of GLT25D1, whereas the second one is dispensable. This fact suggests that the C-terminal domain is also involved in the recognition of acceptor substrates and that critical residues for this function are missing in the CEECAM1 sequence. The importance of the C-terminal domain in the recognition of the donor and acceptor substrates was confirmed by the kinetic analysis performed on the partially active chimeric construct CEECAM1-CterC.Bacterial glycosyltransferases of the CAZY family GT25, such as Lex2B β1,4-glucosyltransferase and LpsB β1,2-galactosyltransferase CEECAM1 orthologous genes in all mammalian genomes would argue against CEECAM1 as representing a pseudo-gene or being the consequence of a late gene duplication event specifically found in the human genome.The CEECAM1 protein was first described as a cerebral adhesion protein Taken together, our results provided insights on the functional organization of the GLT25D1 ColGalT by showing that two regions of the protein are essential for the enzymatic activity. Our work also showed that the inactivity of CEECAM1 as a ColGalT relies on multiple amino acid changes, thus suggesting different substrate specificity or different biological activity. The determination of the exact structure of mammalian GLT25 proteins or of homologous bacterial proteins by crystallization will clarify the structural basis of the CEECAM1 functions.14C]Gal was from GE Healthcare . Restriction enzymes and DNA modifying enzymes were from New England Biolabs .Collagen type I from bovine Achilles tendon, UDP-Gal and the anti-Flag M2 monoclonal antibody were from Sigma-Aldrich . UDP-Gal, 10 mM MnCl2, 20 mM NaCl, 50 mM morpholinepropanesulfonic acid pH 7.4 and 1 mM DTT. Reactions were incubated for 3 h at 37°C then stopped by addition of 500 µl of 5%TCA/5% phosphotungstic acid. The precipitated assay products were applied on glass fiber filters , washed with 10 ml of 50% ethanol, dried for 30 min and counted in a scintillation β-counter (Packard).Recombinant baculoviruses were produced in After determination of protein concentration, samples corresponding to 10 µg proteins were denatured at 95°C, loaded on 8% SDS-PAGE, and transferred to PVDF membranes . Flag proteins were detected using an anti-Flag M2 monoclonal antibody  (Sigma-Aldrich), then by a secondary antibody, anti-mouse peroxidase conjugate  (Sigma-Aldrich), followed by detection using SuperSignal West Pico chemiluminescent substrate .http://prodata.swmed.edu/ promals3d/promals3d.php) http://toolkit.tuebingen.mpg.de/hhpred) http://www.ebi.ac.uk/Tools /msa/clustalw2/) Protein sequences corresponding to human GLT25D1 (Swiss-Prot:Q8NBJ5), human GLT25D2 (Swiss-Prot:Q81IYK4) and human CEECAM1 (Swiss-Prot:Q5T4B2) were used as input for the PROMALS3D multiple sequence and structure alignment tool  including restriction sites used for cloning.(TIF)Click here for additional data file.Figure S2Schematic representation of the PCR fragments used to generate chimeric CEECAM1-CterA/B/C/D/E constructs. The top panel shows the full length GLT25D1 cDNA with the restriction sites used for cloning. Below the C-terminal domain of CEECAM1-Cter-short and the CterA construct are represented as in supplemental (TIF)Click here for additional data file.


Synaptotagmin I (Syt I) is a vesicle-localized protein implicated in sensing the calcium influx that triggers fast synchronous release of neurotransmitter. How Syt I utilizes its two C2 domains to integrate signals and mediate neurotransmission has continued to be a controversial area of research, though prevalent hypotheses favor independent function. Using differential scanning calorimetry and fluorescence lifetime spectroscopy in a thermodynamic denaturation approach, we tested an alternative hypothesis in which both domains interact to cooperatively disseminate binding information. The free energy of stability was determined for C2A, C2B, and C2AB constructs by globally fitting both methods to a two-state model of unfolding. By comparing the additive free energies of C2A and C2B with C2AB, we identified a negative coupling interaction between the C2 domains of Syt I. This interaction not only provides a mechanistic means for propagating signals, but also a possible means for coordinating the molecular events of neurotransmission. This complex process is mediated by several key proteins including synaptobrevin, syntaxin-1, SNAP-25, complexins, and synaptotagmin I 2+ and lipid ligands as well as from proteins within its immediate vicinity to facilitate vesicle and plasma membrane fusion is not well understood, with conflicting evidence both for and against domain cooperation How Syt I utilizes two C2 domains to rapidly transmute binding information from Caint) that collectively facilitate coupling. The sign of Δgint describes the nature of the inter-domain coupling and how the domains communicate with one another. In positive coupling, both domains will experience a similar effect upon introducing a perturbation. With negative coupling, the two domains experience opposite effects. If, for instance, the perturbation is ligand binding and Δgint is positive, both domains will experience a stabilizing effect. Regardless of Δgint sign, introducing a perturbation that changes the conformer distribution  of one domain will directly impact the ensemble of the adjacent domain.Recent theoretical work In light of the theoretical work above, we propose that rather than having an additive coincidence detector function, the C2 domains of Syt I cooperatively propagate binding information through inter-domain coupling. This type of interaction would be of significant functional relevance as each domain would become an allosteric regulator of the other. Ligand binding to C2B, for instance, would not only redistribute C2B’s ensemble, but would also increase or decrease the probability of binding-competent conformers of C2A being occupied.int’s necessarily small magnitude. This problem is best appreciated in the context of the C2 domain’s restricted volume. The nine residue linker that tethers C2A and C2B together , change in heat capacity (ΔCp), and melting temperature (Tm) were then used to determine free energies of stability at physiological temperature (ΔG°37°C) using the Gibbs-Helmholtz equation  and calculated (ΔHTm) enthalpies. By comparing the additive free energies of the individual C2 domains with the C2AB construct, we were able to identify a negative coupling interaction between the C2 domains of Syt I. The quantitative description of this interaction provides a mechanistic means by which Syt I cooperatively propagates binding information throughout both domains and may represent a capacity for coordinating the molecular events of neurotransmitter release.Since our hypothesis is based upon subtle differences in protein energetics, two highly sensitive methods were employed to test it, namely differential scanning calorimetry (DSC) equation ). The va2+ and lipid ligands, there are clear differences in domain stability, with C2B being generally less stable  The Syt I C2AB construct was thermally denatured in the presence and absence of Cacan rate . In all nfolding . This confolding  [30], .(4)Tm, Tm, and ΔCp are the fit parameters in this model, however, to further constrain this fit, ΔCp was fixed using an empirical approximation method that showed good agreement with experimentally-derived values p values for C2A, C2B, and C2AB were 1.92, 2.19, and 4.53 kcal/mole, respectively. The parameters resulting from the global fit were subsequently substituted into n = 8, when achievable) were used in the global fit for each set of conditions. Each data point within each technique is the average of the 4 replicates with the associated error plotted as 95% confidence interval. The error associated with fit parameters and the model line, which represents the global fit for all 8 data sets, were determined using the SolverAid function of Excel in an approach by De Levie The terms ΔH37°C, ΔHTm, and the physiological temperature of 310 K of each protein construct under each set of ligand conditions using the Gibbs free energy equation:Lastly, the entropy change associated with the unfolding transition (ΔS) was calculated using the ΔG°Figure S12+ (red). Note that the enthalpy distributions of an N/D pair shift in response to ligand, consistent with C2AB malleability and a ligand-induced change in conformer distribution.Application of the fluctuation dissipation theorem to C2AB. Qualitative representation of native (N) and denatured (D) enthalpy distributions for C2AB in the absence of any ligand (blue), in the presence of POPC:POPS (60∶40) liposomes (green), and in the presence of Ca(TIF)Click here for additional data file.Figure S2Effectiveness of the C2B purification protocol used for this study. Prominent band in lanes 2, 3, and 5 are MBP-C2B after cell lysis (2), during elution of unwanted protein (3), and during 250 mM imidazole elution (5). Lane 6 shows separation of MBP from C2B. Lane 7 shows pure C2B after passing cut MBP-C2B over column. Ladder  has molecular weights (kDa) of 10, 15, 20, 25, 37, 50, 75, 100, 150, and 250.(TIF)Click here for additional data file.Table S1Complete list of calorimetric enthalpies used to assess concentration dependence of the C2B domain.(DOC)Click here for additional data file.Table S2Complete list of calorimetric enthalpies used to assess concentration dependence of the C2AB cytosolic fragment.(DOC)Click here for additional data file.Table S3em of 345 nm. Analogous controls were performed for C2A. When FLT integrated fluorescence intensity was normalized and globally fit, there was no substantial variation in calculated fit parameters. This indicated that water fluorescence (water raman at 328 nm) at 340 nm was negligible.Thermodynamic parameters and associated errors for C2B and C2AB using FLT λ(DOC)Click here for additional data file.Text S1Two-state derivation, heat capacity fluctuation dissipation theorem, C2B purification, and non-linear least squares regression analysis.(DOC)Click here for additional data file.


E.coli HtpG. These chaperones are studied in complex with the natural ligands ATP, ADP and in the Apo state. Common key aspects of their functional dynamics are elucidated with a novel multi-scale comparison of their internal dynamics. Starting from the atomic resolution investigation of internal fluctuations and geometric strain patterns, a novel analysis of domain dynamics is developed. The results reveal that the ligand-dependent structural modulations mostly consist of relative rigid-like movements of a limited number of quasi-rigid domains, shared by the three proteins. Two common primary hinges for such movements are identified. The first hinge, whose functional role has been demonstrated by several experimental approaches, is located at the boundary between the N-terminal and Middle-domains. The second hinge is located at the end of a three-helix bundle in the Middle-domain and unfolds/unpacks going from the ATP- to the ADP-state. This latter site could represent a promising novel druggable allosteric site common to all chaperones.Understanding how local protein modifications, such as binding small-molecule ligands, can trigger and regulate large-scale motions of large protein domains is a major open issue in molecular biology. We address various aspects of this problem by analyzing and comparing atomistic simulations of Hsp90 family representatives for which crystal structures of the full length protein are available: mammalian Grp94, yeast Hsp90 and  all chaperones. For the three molecules, two corresponding regions are singled out as ligand-susceptible hinges for the large-scale internal motion. On the basis of this and other evidence it is suggested that these regions represent functionally relevant druggable substructures in the discovery of novel allosteric modulators.Understanding the connections between structure, binding, dynamics and function in proteins is one of the most fascinating problems in biology and is actively investigated experimentally and computationally. In the latter context, significant advancements are possible by exposing the causal link between the fine atomic-scale protein-ligand interactions and the large-scale protein motions. One ideal avenue to explore this relationship is given by proteins of the Hsp90 chaperones family. Their dynamics is regulated by ATP binding and hydrolysis, which activates the onset of large-scale, functional conformational changes. Herein, we concentrated on three homologs with markedly different structural organization—mammalian Grp94, yeast Hsp90 and prokaryotic HtpG—and developed a novel computational multiscale approach to detect and characterize the salient traits of the functionally-oriented internal dynamics of the three chaperones. The comparative analysis, which exploits a novel highly simplified, yet viable, description of the protein internal dynamics, highlights fundamental mechanical aspects that preside the ligand-dependent conformational arrangements in  In recent years, experimental and theoretical studies have increasingly focused on promiscuous proteins One notable instance is represented by the 90 kDa heat shock (Hsp90) family of proteins HtpG, Hsp90 and Grp94 have a mutual sequence identity of about 45% which reflects in the good structural alignability of their individual domains. However, the preferential relative orientation of the domains varies significantly depending on the reference organism and cellular compartment Figure 1In general, Hsp90 functions depend on ATP binding and hydrolysis, and play a pivotal role for controlling multiple signaling networks associated with cell viability In a recent atomistic molecular dynamics (MD) study of Hsp90 The above-mentioned studies This approach could have an even greater potential if it could exploit or target the functional dynamical aspects shared by the homologue chaperones despite their different overall structure. Such mechanistic properties, being at the intersection of the functional dynamics of different proteins, would expectedly be crucial for the molecules' activity and their characterization could help the discovery of potent inhibitors.To make progress in this appealing avenue, which complements previous approaches where chaperones' differences rather than similarities were analyzed Here we take a first step towards formulating a dynamics-based, multiscale comparative scheme that is more general and hence applicable to structurally-different proteins whose internal dynamics has a very large amplitude. Specifically, the internal dynamics of different proteins is compared from the fine level of inter-residue fluctuation to the much larger one of domain motion. The latter is captured with a minimalistic quasi-rigid domain description which allows for a transparent and intuitive dynamics-based comparison.The strategy is applied to the atomistic simulations of the three Hsp90 chaperones in the ATP-, ADP- and Apo states, covering a total time span of 900 ns. It is found that two regions are arguably involved in modulating the well-known ligand-dependent large-scale motion of the three chaperones Proteins of the Hsp90 family are homodimers. Each protomer consists of three structural domains: an N-terminal regulatory Domain (NTD), responsible for ATP binding, a Middle Domain (M-domain), which completes the ATPase site necessary for ATP hydrolysis and binds client proteins, and a C-terminal Domain (CTD) that is involved in dimerization Figure S1), and the rest of the dimeric interface involves a few contacts, such as α-helix2 and α-helix3 from the NTD, one of the edges of the M-large subdomain and the tips of the ATP lids.It is seen that the full-length construct of Grp94, in complex with either ATP or ADP, is a semi-open, V-shaped dimer held together by a twisted C-terminal interface The available crystal structures of full-length Grp94, Hsp90 and HtpG were used as starting points for extensive atomistic MD simulations, see Figure S2. Such difference is not expected a priori given the rather closed character of the dimeric conformation of nucleotide-bound Hsp90.Specifically, the MD trajectories of yeast Hsp90 were obtained by extending to 100 ns those carried out by some of us in a previous study S6.The behavior of the Grp94 and HtpG trajectories is much richer and it indicates that the relative positioning of the N-terminal and Middle domains is strongly affected by the type of bound ligand, as detailed below. The final structures of all simulations are provided as supplementary information files , S3).In the ATP-state of Grp94, the initial proximity of the N-terminal and Middle domains is maintained mostly by the electrostatic attraction of the nucleotide and the oppositely-charged Lys residues  and by the interaction of hydrophobic residues such as Val416, Phe417 and Ile418 (M-domain) with Leu117 (N-term domain). In the course of the MD evolution the relative orientation of the N-terminal and Middle domains is also preserved. The ATP-lid, which completes the nucleotide binding site in the NTD, populates mainly the starting “open” state and, towards the end of the trajectory, the “extended open” state. The latter lid conformation is induced as the NTD of one protomer approaches the N- and M-domain of the opposite protomer Figure 2For the ATP-bound HtpG the number of contacts between the NTDs, which initially involve only the tips of the ATP-lids (residues 106–115), increases during the MD evolution. The tightening of the NTDs interface is favored by the relative rotation of the two protomers which, in addition, causes an increase of the inter-protomer distance between the Middle and C-terminal domains and exposes residues 11,12,15 in α-helix1 to the interaction with the ATP lid of the partner protomer Figure 3Figures S4). Moreover, the N-terminal domain is canted with respect to the Middle one. This rearrangement is promoted by the fact that the electrostatic attraction of Lys364, Lys404 and Lys405 (in the Middle domain) with the nucleotide bound in the NTD is weaker for ADP compared to ATP. Because of the increased separation of the N-terminal and M-domains (S5), the catalytic loop is less constrained and Arg448, Glu103 lose the preferential orientation towards the nucleotide-binding site. The effect is illustrated by the ADP induced variation of the Lys-nucleotide minimum distances reported in Figure S6). Although the time-span covered by the present simulations is, as expected, not sufficient to reach a state with fully-detached NTDs, our analysis highlights specific interactions responsible for the onset of the motions inducing N-terminal dissociation.Significant conformational changes occur in the MD trajectory started from the ADP-bound structures. In Grp94 the ATP-lid, which was initially modeled in the “open” state , undergoes a conformational change that populates mainly the “extended open” state S2 and Figure S5).Consistently with previous observations for Hsp90, the C-terminal solvent accessible surface in the ATP-state is smaller than in the ADP-bound one because the mobility of the two C-terminal loops is restricted by the nearby M-small subdomain .Finally, the effects of the ligands on HtpG C-terminal interfaces are consistent with that observed for the eukaryotic cases, despite the different structural organization of the terminal domains in the prokaryotic protein. Namely, ADP induces an increase of the C-terminal solvent accessible surface with respect to ATP . In Hsp90 this behavior is not observed due to the extremely long simulation time it would take to unwind the tightly packed N-terminal interface. Analogous considerations also apply to the Apo structure of HtpG, where the initially-contacting NTDs presumably restrict the conformational space visited in the simulation timescale. In fact, as noted before, because of the necessarily limited duration of atomistic MD simulations, the starting conformations of the models may have a significant impact on conformational sampling of these complex molecular assemblies. Notwithstanding these expected difficulties, it is remarkable that sizeable structural rearrangements are observed across these trajectories and therefore provide valuable indications into the innate inter-domain motions that the chaperones can sustain in thermal equilibrium.Interestingly, in the Apo-state of Grp94, the ATP-lid is very flexible and locally unfolded and visits both the “open” and the “extended open” conformations, indicating that no specific conformational preference exists in the absence of ligand. The N-terminal domain can move significantly with respect to the Middle one in the absence of the nucleotide  and of segment 448–510, the principal helix of the Middle domain, with respect to the M-small and C-terminal domains, as indicated by higher values of distance fluctuation (the average distance fluctuation between the stretch 448–510 and residues over 510 is 1.7 Å2 with ATP and 3.2 Å2 with ADP). This segment includes part of a cluster of aromatic residues at the end of the H4–H6 three-helix bundle , located at the boundary between the M-large and M-small structural subdomains Figure S7). Interestingly, this local unfolding shortly precedes the opening transition of the dimeric clamp .More in detail, the change of ligand increases the mobility of the catalytically crucial residue Glu103 (on average 0.1 Å2 on average) to ATP (1 Å2 on average).Local mobility of the flexible linker connecting the M-small and C-terminal domain, around residue 603, increases in the presence of ATP with respect to ADP: the average distance fluctuations within segment 590–610 nearly double going from ADP . In particular, changing the nucleotide from ATP to ADP results in the unpacking/unfolding of the end of the H4–H6 three-helix bundle in the M-domain, corresponding to residues 427–432 in Hsp90 and residues 360–390 in HtpG. In addition, the increased fluctuation amplitude of the linker connecting the M-small domain and the C-terminal domain (around residue 525 in Hsp90 and 500 in HtpG) is also observed.Additional insight into mechanistic functional aspects of Grp94, Hsp90 and HtpG is obtained by considering their ligand-dependent geometric strain, that is the ligand-dependent local deformations of the residues' contact networks see  [41].Figure S9.It is seen that a recurrent buildup/release of geometric strain occurs in regions that either are directly involved in chaperone's functional activity or correspond to exposed loops. Restricting considerations to the first type of regions, one has that the strain hotspots in the ADP state correspond to (i) the linker connecting the N- with the M- interface (residues 340–343), (ii) the catalytic loop around Arg448, (iii) the interface region separating the M- and C-terminal domains (residues 573–575) and finally (iv) the C-terminal loops (residues 652–682) in the vicinity of a previously identified C-terminal binding pocket for allosteric inhibitors These results have a parallel in the Hsp90 strain pattern, also shown in Analogous results are found for HtpG too. As seen in all systems, the catalytic Arginine  shows an increase of conformational strain in the presence of ADP with respect to ATP. The increased strain reflects a reduced correlation of the motion of the catalytic loop and the bound nucleotide. The inspection of the MD trajectories indicates that ADP-dependent increased fluctuation of the catalytic loop reverberates on the motion of the proximal Middle domain helix, which triggers the local rearrangement at the Middle domain three-helix bundle H4–H6 . This suggests that the catalytic Arginine acts as a sensor for the nucleotide state. Indeed, in the presence of ADP, the increased local mobility of the catalytic Arginine releases initial structural/electrostatic constraints on the principal helix (H3) of the Middle domain, triggering the motion of this helix as a rigid body. Finally, no ligand-specific strain signal is evident for the highly-charged linker, connecting N-terminal and M-domains.In The insight gained so far into specific common aspects of the chaperones' internal dynamics is here used to introduce and apply a novel comparative analysis scheme that can be aptly used to make manifest the relatedness of the functionally-oriented movements in Hsp90, Grp94 and HtpG.To the best of our knowledge, the only presently available quantitative method for detecting and exposing correspondences in the internal dynamics of structurally different proteins is the dynamics-based alignment scheme An alternative and physically appealing approach is suggested by the marked block character of the distance fluctuation matrices in In order to ascertain the possibility to use a quasi-rigid domain description to capture the protomer motion across all simulations, we proceeded by first identifying a set of residues that can be put in one-to-one correspondence among the proteins. The set, from which a handful of highly mobile exposed residues were removed, consisted of 525 amino acids per protomer . Next, all the simulated MD trajectories were filtered by retaining only the Cα atoms of the 525 corresponding amino acids and combined into a “meta-trajectory” spanning a total of 1800 ns (900 ns per protomer). Because the “meta-trajectory” combines (reduced) protomeric conformations of the three-chaperones in Apo, ADP-bound and ATP-bound states, its conformational breadth is very large. In fact, individual conformations are at ∼10 Å RMSD from the reference “meta-trajectory” conformation (the one closest to the time-averaged structure) shown in Figure S10). These values are remarkably small compared to the typical RMSD differences reported above.The fluctuations of pairwise Cα distances in the “meta-trajectory” were used to optimally divide the corresponding amino acids into quasi-rigid domains The main corresponding large-scale movements across the three chaperones can be gleaned by restraining the motion of the two terminal quasi-rigid domains to pure rotations around an axis that is fixed with respect to the central domain see . Figure,and Video S3. This is a notable result because it is comparable to the resolution typically achieved in cryo-em or SAXS studies of flexible proteins of several hundred amino acids.The restricted domain motion still allows for capturing ∼55% of the covariance of the original “meta-trajectory”. Any configuration in the Grp94, Hsp90 or HtpG simulations can still be matched to an average RMSD of 7.4 Å, see The results highlight that the fundamental aspects common to the three chaperones are the rotary motions of the combined M-small+C-terminal macrodomain and the N-terminal domain relative to the M-large one. Notice that the axis of the macrodomain passes through the end of the H4–H6 bundle, which was discussed in a previous section in connection with other ligand-dependent properties. This is noteworthy, because the axis location and orientation was not constrained a priori to lie exactly at the boundary of the quasi-rigid domains.This fact vividly illustrates that, in spite of the diversities in the initial structural arrangements of the molecules, a common mechanism presides their ligand-dependent large-scale motion. In fact, the hinge located at the interface between the M-large and the M-small domains is highlighted in all proteins as a mechanical hotspot that is capable of modulating the motion of the C-terminal domain in a ligand-dependent manner. It is worth stressing that such key mechanical role is not evident from the standard structural partitions of the protomeric domains.The function of a large number of proteins and enzymes is determined by the synergic interplay of structure and dynamics, often modulated by substrate binding or covalent modifications. Although many mechanisms have been elucidated Here, we focus on a specific aspect of this general problem by considering how the large-scale internal dynamics of members of the Hsp90 family is affected by the bound nucleotide. In fact, the conformational turnover of these chaperones depends primarily on ATP binding and hydrolysis  Importantly, across all three chaperones, four common regions were identified: the nucleotide binding site in the N-terminal domain , the aromatic cluster in the H4–H6 helix bundle located in the Middle domain, the core of the C-terminal domain and finally the C-terminal loops defining the allosteric binding pocket for C-terminal inhibitors of Hsp90 , and , S3).By analyzing the various terms of the proteins' internal energy it is established that the ligand-dependent changes in the protomeric structures correlate with the strength of the electrostatic interaction between the bound nucleotide and the positively charged Arginine in the Middle domain  and, more notably, the cluster of aromatic residues at the end of the H4–H6 three-helix bundle in the Middle domain. In fact, going from the ATP to the ADP state, the increased separation of the nucleotide-binding domain and the catalytic loop in the Middle domain causes the end of the H4–H6 bundle to partially unfold/unpack. This, in turn, leads to the opening of the dimeric clamp. By contrast, the presence of ATP favors the stabilization of the aromatic cluster in an optimally-packed organization., , , , , , The fluctuation analysis of the 1800-ns-long “meta-trajectory”, obtained by combining the atomistic trajectories of the single protomers of the three chaperones in all considered ligand-bound states, was next used to subdivide the protomers of the Hsp90, Grp94 and HtpG chaperones into corresponding quasi-rigid domains. The subdivision into quasi-rigid domains lends naturally to describe the large-scale motion of the chaperones with a minimal number of degrees of freedom. In fact, as much as 61% of the “meta-trajectory” mean square fluctuations can be accounted for by the relative rigid-like movements (translations and rotations) of merely 3 quasi-rigid domain see , , , 7A minimalistic, though viable, description of the large-scale dynamics that is shared by the three chaperones is obtained by restricting the motion of the two side quasi-rigid domains to consist exclusively of rotations around axes that are fixed (same position and orientation) to the corresponding core of Hsp90, Grp94 and HtpG. By doing so, only two degrees of freedom (the instantaneous rotation angles) are needed to parameterize the “meta-trajectory” and yet it is still possible to account for 55% of its covariance.The location of the two optimal axes of rotation for the quasi-rigid domains was next used to single out regions that have a crucial mechanical role in the large-scale motion of all three chaperones and hence ought to represent good target sites for inhibiting drugs. Consistently with this observation, one of them is the interface between the N-terminal and Middle domains which corresponds to a previously identified functional site Based on the above results it is possible to formulate a consistent, unified framework for the conformational mechanisms of Hsp90, Grp94 and HtpG.The primary direct effect ascribable to the specific nucleotides in all three proteins is the modulation of the Coulombic interactions with the positively charged residues in the catalytic loop and in Middle domain see Table 1.Switching from ATP to ADP, the catalytic loop containing Arg448 in Grp94, Arg380 in Hsp90 and Arg336 in HtpG experiences particularly high geometrical strain. The deformation of the contact network of this loop correlates to that of the ordered cluster of aromatic residues at the basis of the H4–H6 three-helix bundle in the Middle domain. The electrostatic attraction of the N-terminal and Middle domains decreases and the protomers relax from the closed state (reference structure Hsp90-ADP), through a rotation that causes a partial unfolding of the basis of the three-helix bundle. This sequence results in an opening of the structures with increased mobility of the Middle domains  and in the collapse of the N-terminal domains over the M-domains, as well as in the population of the “open-compact” conformations described by Agard and coworkers (Grp94-ADP). In this framework, the catalytic loop provides a direct mechanical response to the type of bound nucleotide.Finally, the release of the nucleotide leads to states with no specific dynamic signatures, which can be correlated to the absence of conformational preferences in the absence of ligand. The release of the ligand induces a strain that favors a reduced twist at the C-terminal interface in all Apo systems, converging to the values typical of HtpG, which can be related to the tendency of the proteins to evolve to fully open structures.As discussed hereafter, the results of the present multiscale analysis are in accord with several experimental findings and contribute to establishing a comprehensive view of the functional mechanics of these chaperones.The Neckers group identified a conserved hydrophobic motif in β-strands at the C-terminal boundary of the N-domain, whose mutation abrogated Hsp90 function. Hsp90 mutants lacked chaperone activity in vitro and failed to support yeast viability. Chaperone activity was restored by truncation of the charged linker connecting the N-terminal and Middle domains, consistently with the key “mechanical” role of hydrophobic contacts in the N-M hinge region found in our study In a different study, Vasko and co-workers identified Sansalvamide A (SanA) as a structurally novel Hsp90 inhibitor. Through the use of pull-down experiments in the presence of the three isolated domains of Hsp90, a construct containing only the N-terminal/Middle domains, and the WT protein, the authors were able to show that SanA binds exclusively to the N-terminal/Middle construct of the protein Finally, Matts and coworkers The three-helix bundle in the Middle domain and the N-M interface hinge have also been shown to be involved in the interaction with the ATPase stimulating co-chaperone Aha1 In this context, results from the Agard group support the importance of the HtpG region around residue 450 as important in regulating conformational changes. Using small-angle X-ray scattering and EM studies, the authors showed that the conformational equilibrium of HtpG can be shifted with pH, and identified His446, in the immediate proximity of the identified dynamic hotspot, as a key residue that controls this pH-dependent equilibrium. Mutagenesis of His446 was shown to successfully modulate the conformational equilibrium at neutral pH.With regards to the dynamics of the C-terminal domains, the calculated ATP-induced increase in motion amplitude in the core of the C-terminal domain correlates well with the observed increase of hydrogen-deuterium exchange in the C-terminal core of HtpG upon ATP binding Finally, our model of the ATP-induced transition towards a compact state is consistent with available biochemical and structural data for Grp94. In this context, Dollins and coworkers E.coli HtpG, was studied by advanced numerical schemes. For each chaperone, the dynamics in the ATP-bound, ADP-bound and Apo states was studied using extensive atomistic molecular dynamics simulations, covering a total time span of 900 ns.The ligand-dependent internal dynamics of three Hsp90 chaperones with markedly different structural organization, mammalian Grp94, yeast Hsp90 and The collected data were analyzed within a novel comparative scheme that is aptly used to single out the salient, common traits of the chaperones' internal dynamics. The proposed analysis scheme has a multiscale character in that it allows to bridge between the ligand-dependent local modifications of the chaperones and the resulting large-scale conformational rearrangements of the chaperones' protomers. Compared to other powerful techniques - such as essential dynamics or principal component - the proposed approach overcomes the structural distortions  caused by reconstructing the very large amplitude internal dynamics by the linear superposition of very few modes. In fact, it is shown that most of the covariance observed across the 900 ns-long combined trajectories can be captured - with no structural distortion - by using merely two degrees of freedom, which are the angles of rotation of the two side quasi-rigid domains relative to the middle, core one. Based on this result we envisage that the same strategy could be profitably used in cryo-em and SAXS contexts as an effective computational aid for structural modeling.From this analysis, which is general and hence transferable to other contexts, it emerges that the ligand-dependent modulation of the structural rearrangements in both chaperones is governed by a common underlying mechanism. Specifically, the bound ATP favors the closed protomer conformation by the electrostatic attraction of the N-terminal and Middle domains. The interaction strength decreases significantly going from the ATP-state to the ADP-state and finally to the Apo one. As a result, the relative positioning of the N-terminal and Middle domains is progressively less constrained and favors the open and open-extended conformations, respectively.all three chaperones despite their different conformational organization and with a distance of about 40 Å from the nucleotide-binding site, is a possible novel allosteric site. Targeting this site by mutations or small molecules might affect the mechanisms of conformational transition that are crucial for chaperone activity. The strategy to discover putative new inhibitors has been described in The closing/opening rearrangements that are crucial for the chaperones' activity cycle are made possible by the presence of two primary hinge regions at the interface of the quasi-rigid middle domain with the two side ones. These hinges appear to be key players in modulating the protomeric conformations of all Grp94, Hsp90 and HtpG chaperones in response to the type of bound ligand. The first hinge site is located at the interface of the N-terminal and Middle structural domains, while the second is located at the end of the H4–H6 helix-bundle at the boundary between the M-small and the M-large structural subdomains. The first hinge site has already been validated as a potential target for inhibition of the Hsp90 activity The overall approach we propose may represent a novel and effective means of modulating the functions of different members of the Hsp90 family with drugs that intervene by specifically addressing regions crucial for the functional dynamics of the molecule other than the classically targeted active site.The same starting structure was used for the simulations of the ATP-, ADP-, and Apo states of each of the three proteins.3 and contains about 71000 solvent molecules and overall more than 228000 atoms. All simulations and the analysis of the trajectories were performed using the 4.0.3 version of the GROMACS software package The Grp94 structure used for MD simulations is the full length dimer of the canine protein crystallized by Dollins et al. The HtpG structure used for MD simulations is the extended full length dimer modeled starting from the crystal of the bacterial protein by the Agard group ɛ = 1, and a time step of 2 fs were used. All atoms were given an initial velocity obtained from a Maxwellian distribution at the desired initial temperature of 300K. The density of the system was adjusted performing the first equilibration runs at NPT condition by weak coupling to a bath of constant pressure  Following the same protocol used for the Hsp90 simulations, for which we refer to For Hsp90, the simulation protocol is analogous and is fully described in A:ijd is the (time-dependent) distance of the Cα atoms of amino acids i and j and the brackets indicate the time-average over the trajectory. Notice that A is invariant under translations and rotations of the molecules and, unlike the covariance matrix, does not depend on the choice of a particular protein reference structure. The A matrix, and various quantities derived from it, can be used to characterize the salient elasticity and plasticity properties of a protein undergoing structural fluctuations.For each MD trajectory we computed on the time interval 20–100 ns (the first 20 ns trajectory are removed in order to avoid equilibration artifacts), the matrix of distance fluctuations A. In fact, pairs of amino acids belonging to the same quasi-rigid domain should be associated with much smaller distance fluctuations than amino acid pairs in different domains The presence of quasi-rigid domains in the protein of interest should reflect in specific properties of i, is defined asj runs over all protein amino acids, and f is a sigmoidal function that restricts the contribution to the sum to amino acids that are within about 5 Å from amino acid i: x is expressed in Ångstroms. Hinge regions will be characterized by high values of p because in the course of the dynamical evolution their local network of contacts appreciably changes due to the relative motion of neighboring domains.Consistently with the above observation, the hinge regions for the quasi-rigid domain motion can be identified by inspecting the geometric strain profile introduced in ref. In order to compare the salient features of the internal dynamics of Hsp90, Grp94 and HtpG we first identified the set of their equivalent amino acids. Because of the different length and structural organization of the two chaperones we used to this purpose the FATCAT Specifically, a FATCAT alignment was obtained separately for Hsp90 and HtpG with Grp94; subsequently, we considered the intersection of these two subsets of residues. For each chaperone, we chose as representative protomer the conformation that is closest to the average protomeric structure of the combined Apo, ADP and ATP MD simulations.The flexible alignment returned 525 corresponding residues (corresponding to ∼82% of each protomer). Next, the individual configurations sampled during each MD trajectory of the proteins were “filtered” by removing the non-corresponding residues. These “reduced” 525-amino acid long configurations in various ligand states were combined into a single “meta-trajectory”. By suitable analysis of this trajectory, it is possible to obtain valuable insight into those aspects of internal dynamics that are common to all chaperones.A suggests that the internal dynamics of all three chaperones can be described in terms of relative movements of few quasi-rigid domains connected by hinge regions. The automated identification of a given number of domains was performed, according to the PiSQRD method discussed in refs. q, of quasi-rigid units. In a nutshell, this partitioning is identified by minimizing the intra-unit mean-square fluctuations of pairwise distances of amino acids compared to inter-unit pairwise distances. A figure of merit for the viability of the rigid partition is found for each considered number of quasi-rigid domains, q = 2,3,4 etc. and based on that the subdivision with the least viable number of domains is retained.The inspection of matrix To provide the background for discussing the rotofit procedure described later, we give hereafter a concise account of the PiSQRD algorithm.q putative quasi-rigid domains. Because of the putative quasi-rigid character of the domains, it is expected that (i) the intra-domain structural fluctuations are negligible and (ii) the internal dynamics should mostly consist of relative rotations and translations of the domains.Let us suppose to start with a tentative (e.g. random) partitioning of amino acids into i belonging to the quasi-rigid unit l with respect to the pure roto-translational motion that best describes the collective movement of all amino acids in the same domain. In the above expression, i-th amino acid in a reference structure, To ascertain the extent to which these conditions are realized one calculates the quasi-rigid fit “error”The best quasi-rigid domain subdivision is found by minimizing the above error over the tentative assignments of amino acids to the quasi-rigid domains, the latter being explored in a stochastic search in the partition space.Because of the large-amplitude character of Grp94 and Hsp90 motion the essential dynamical spaces were used only as a first step in our analysis. The returned subdivision was then used as a starting point for minimizing Cα-trace of a protein should be well described by a purely-rotational motion around a suitable axis passing through one or more Cα's. To identify the dominant rotation axis of a given domain we accordingly enforced that the motion of each amino acid could be well approximated by a pure rigid-body rotationtime-independent vector providing the orientation of the rotation axis which is further constrained to pass through the point, 2 as in (3). To make the optimization computationally feasible, the Cα atoms and 500 orientations of Because the internal dynamics of a protein preserves the chain connectivity, it is intuitively expected that the motion of the Notice that: (i) the rotation axis does not necessarily pass through the domain center of mass and that (ii) the rotation matrix depends on time only through the instantaneous angle, because the location and directionality of the rotation axes are fixed.Figure S1A, the structural superposition of the C-terminal domains from each protomer of Grp94 (blue), Hsp90 (red) and HtpG (green) shows the different twisting of the interfaces. Subpanel B shows the dihedral angle used for the calculation of the twisting of one CTD with respect to the other. Subpanel C reports on the time evolution in the different ligand states . The color coding for the time evolution of the trajectories is the same as for A.(TIF)Click here for additional data file.Figure S2A, Grp94. B, Hsp90. C, HtpG. The 20 ns long equilibration stage is indicated with a grey overlay. The interface is defined by residues: 660–675 and 723–735 in Grp94; 591–605 and 651–661 in Hsp90; 552–562 and 602–613 in HtpG.Time evolution of the Solvent Accessible Surface Area calculated at the C-terminal interface in the presence of ATP (black line) and of ADP (red line). (TIF)Click here for additional data file.Figure S3A, Time evolution of the electrostatic interaction energy between the nucleotides and charged residues in the Middle domain of Grp94. B, Starting structure (left) and 100 ns snapshot (right) showing the interactions between positively charged residues of the Middle domain and bound ATP at the N-domain of Grp94. Lysines and the putatively catalytic Arginine (Arg448) are shown. C, van der Waals interactions between Leu117 and hydrophobic residues of the Middle domain .(TIF)Click here for additional data file.Figure S4Representative structures of the most populated clusters of the N-terminal domain in Grp94, highlighting the relative populations and the conformations of the ATP-lid (red tubes).(TIF)Click here for additional data file.Figure S5A, Representative structures of the most populated clusters of the N and M-large domains, highlighting the relative orientations of the NTD with respect to the M-domain in the different Grp94 ligand states. B, Representative structures of the M-small and C-terminal domains, highlighting the relative orientations of the M-small with respect to the C-terminal domain in the different Grp94 ligand states. In this case, one single cluster accounts for most of the motion. The arrow highlights the contact between M-small and C-terminal domain.(TIF)Click here for additional data file.Figure S6Representative snapshots of the structural evolution in the Hsp90-ADP simulation.(TIF)Click here for additional data file.Figure S7A Left, Grp94 starting conformation. Right, end conformation detail of the three helix bundle, with Phe484 and Phe432 shown as VdW spheres, in the ATP and in the ADP simulation respectively. B, same as above, but for Hsp90. Phe364 and Phe421 are shown as VdW spheres. C, HtpG. Phe320 and Phe378 are shown as VdW spheres.Local unpacking of aromatic residues observed at the Middle domain in the presence of ADP. (TIF)Click here for additional data file.Figure S8Upper plot: Time evolution of the distance between the centers of mass of the two ADP molecules, bound to the N-terminal domains of Grp94, showing the opening of the dimer clamp during the MD trajectory. Lower plot: Time evolution of the distance between the sidechains of Phe484 and Phe432 of each protomer during the same MD trajectory, showing the increased sidechain separation due to the unpacking of the Middle domain aromatic core.(TIF)Click here for additional data file.Figure S9A), Hsp90 (B) and HtpG (C) monomer in the presence of ADP. The units are Å2.Time evolution of geometrical strain of a Grp94 ((TIF)Click here for additional data file.Figure S10RMSD between real meta-trajectory protomer configuration and corresponding rigid domain fit as a function of time. RMSD between each protomeric configuration (“frame”) of the meta-trajectory and its best-fit approximation obtained by relative rigid displacement of the domains in the template protomeric structure (see (TIF)Click here for additional data file.Text S1Coordinate file for the final structure of the Grp94-ADP simulation.(TXT)Click here for additional data file.Text S2Coordinate file for the final structure of the Grp94-ATP simulation.(TXT)Click here for additional data file.Text S3Coordinate file for the final structure of the Hsp90-ADP simulation.(TXT)Click here for additional data file.Text S4Coordinate file for the final structure of the Hsp90-ATP simulation.(TXT)Click here for additional data file.Text S5Coordinate file for the final structure of the HtpG-ADP simulation.(TXT)Click here for additional data file.Text S6Coordinate file for the final structure of the HtpG-ATP simulation.(TXT)Click here for additional data file.Video S1Solid backbone: Grp94 meta-trajectory. The meta-trajectory consists of all protomeric snapshots  of Grp94 in the Apo, ADP- and ATP-bound states. The faint backbone represents the best fit to the Grp94 protomers obtained by suitably rotating the two terminal quasi-rigid domains of protomeric template see Figure 6(MPG)Click here for additional data file.Video S2Solid backbone: Hsp90 meta-trajectory. The meta-trajectory consists of all protomeric snapshots  of Hsp90 in the Apo, ADP- and ATP-bound states. The faint backbone represents the best fit to the Grp94 protomers obtained by suitably rotating the two terminal quasi-rigid domains of protomeric template see Figure 6(MPG)Click here for additional data file.Video S3Solid backbone: HtpG meta-trajectory. The meta-trajectory consists of all protomeric snapshots  of HtpG in the Apo, ADP- and ATP-bound states. The faint backbone represents the best fit to the Grp94 protomers obtained by suitably rotating the two terminal quasi-rigid domains of protomeric template see Figure 6(MPG)Click here for additional data file.