SUMO belongs to the ubiquitin-like family (UbL) of protein modifiers. SUMO is conserved among eukaryotes and is essential for the regulation of processes such as DNA damage repair, transcription, DNA replication and mitosis. UbL modification of proteins occurs via a specific enzymatic cascade formed by the crosstalk between the E1-activating enzyme, the E2-conjugating enzyme and the E3-ligase. An essential discrimination step in all UbL modifiers corresponds to the interaction between E1 and E2 enzymes, which is mediated by the recruitment of the E2 to the UFD domain (Ubiquitin-Fold Domain) of the E1 enzyme. To gain insights in the properties of this interface, we have compared the structures of the complexes between E1 UFD domain and E2 in human and yeast, revealing two alternative UFD platforms that interact with a conserved E2. Comparative sequence analysis of the E1 UFD domain indicates that the E2 binding region has been conserved across phylogenetic closely related species, in which higher sequence conservation can be found in the E2 binding region than in the entire UFD domain. These distinctive strategies for E1-E2 interactions through the UFD domain might be the consequence of a high selective pressure to ensure specificity of each modifier conjugation system. The post-translational modification pathway of proteins by UbLs (Ubiquitin-like modifiers) is characterized by the presence of specific enzymatic cascades , which results in the formation of an isopeptidic bond between the UbL and the protein target1316Kd = 1.2 μM)Kd = 87 μM)The first specificity step in the pathway corresponds to the interaction of the UbL modifier with its particular E1-activating enzyme8121314Protein sequence variations in the E1 UFD domain of different UbL modifiers are quite significant, especially in the binding region to the E2 enzyme. All reported structures of UFD domains display an analogous β-grasp structure, and the interaction of the UFD domain with the E2-enzyme occurs through the same side of the β-sheet structure131415181915Metazoa) or in the same order . Phylogenetic and homology analysis revealed that the region involved in Ubc9 binding displays a slightly higher conservation degree than the UFD domain between phylogenetically closely related organisms, although it also displays higher variability, highlighting the relevance of this interface in the protein-protein specificity for each type of UbL modification.Here we present a detailed structural comparison analysis of the two complexes between SUMO E1 UFD domain and Ubc9 from yeastThe interaction between the ubiquitin-fold domain (UFD) of the E1-activating enzyme and the E2-conjugating enzyme has been revealed as a crucial discrimination step in the conjugation pathway of UbL modifiers141823Human E1 ubiquitin-fold domain (UFD) was designed based on the structure of the full-length human SAE1-SAE2 E1-activating enzyme (PDB code 1Y8Q)2 and involves 26 and 19 residues of UFD and Ubc9, respectively, which is comparable to the analogous complex in yeast (1557 A2 interface)An overall structural comparison of free and bound Ubc9 structures displayed little variation (0.71 Å rmsd Cα deviation), although differences were observed in the binding region to UFD, displaying rmsd Cα deviations between 1.50 Å and 3.66 Å in the residues forming the β1-β2 loop (Lys30 to Met36). Similarly, free and bound E1 UFD structures are almost identical . According to the PISA serverIn contrast to the conservation proposed according to sequence homology between human and yeast UFD, 17% of identity, this homology is even lower, 11%, when a structural alignment is performed . Interes22The binding surface of Ubc9 in yeast and human is highly conserved, and is basically formed by similar backbone and side-chain interactions in the α1 helix and the β1-β2 loop of Ubc9 (yeast and human Ubc9 share 56% sequence identity). Specific side-chain interactions in Ubc9 include a hydrophobic patch formed by Leu6, Met36, and Leu38, and a basic patch formed by Arg13, Lys14, Arg17 and Lys18 . Interes25Low Homology region involved in E2 Binding 2 (LHEB2). The LHEB2 can be divided in two regions, each establishing interactions with the basic and hydrophobic patches of Ubc9 13As mentioned before, human Ile489 and yeast Leu485 adopt a similar conformation and engage identical backbone hydrogen bonds with the β1-β2 loop of Ubc9. Interestingly, despite the lack of sequence conservation, the equivalent residues in the ubiquitin UFD domain, namely Ser950-Leu951, also engage analogous backbone hydrogen bonds contacts with the β1-β2 loop of the E2 enzyme (Ubc4)18Saccharomycetales order, which include Saccharomyces cerevisae. The phylogenetic analyses of both domains, E1 UFD and LHEB2, show that clustering of the E1-UFD sequences in the phylogenetic tree reflects the taxonomical relationships of the species represented  or yeast sequences (conservation within the group). Similarly, metazoan UFD or LHEB2 sequences were compared with the corresponding yeast (conservation to outlier) or human sequences (conservation within the group). The homology pair distributions were plotted onto box plots , which interact with Ubc9 Arg13 and Arg17, are highly conserved in all species analyzed. Similarly, the hydrophobic interaction of the human UFD Ile489, which interacts with Ubc9 Met36 and Leu38, is also conserved but can be occasionally substituted by valine in some species. The major differences in the UFD interface in metazoa are located in the β22-β23 connecting loop, formed in human by Gly485 and Gly487, which interact with Ubc9 Leu6. Whereas in chordate this connecting loop is highly conserved, in arthropod and particularly in nematode, this loop displays different lengths and amino acid composition. It is worth mentioning here that the extension and composition of this particular loop in UFD domains is highly divergent between E1-activating enzymes specific to different UbL modifiers, suggesting that this domain has evolved to interact with its cognate E2-enzyme.Therefore, all major specific UFD contacts with Ubc9 are basically conserved in metazoa, sequence comparison of the E2 binding region of the yeast UFD domain with members of the Fungi kingdom is highly diverse, and further structural analyses will be required for establishing the molecular basis of E1-E2 interactions in those divergent groups. In the order of Saccharomycetales, the major specific contacts described in the structure of S. cerevisiae UFD-Ubc9 complexSaccharomycetales is completely different to metazoan. Yeast UFD residues Asp488, Asp490 and Asp493, which interact with the basic patch of Ubc9, are highly conserved in Saccharomycetales  or in the case of the Fungi kingdom, in the same order . Despite the low level of sequence homology in the UFD domains among these distant species of different kingdoms, these two types of interfaces maintain the structural and chemical properties necessary to interact with a conserved E2 binding surface. Structural and sequential comparisons have also revealed at least two different types of consensus sequences in the E1 UFD domain, which we named LHEB2 sequences, that can complement the conserved surface in the E2 enzyme. Interestingly, sequence conservation in the E2 binding region is higher than in the overall UFD domain, suggesting the presence of an evolutionary pressure to maintain the contacts with the E2-conjugating enzyme, which are essential for the correct function of each UbL pathway.In summary, structural comparison of yeast and human SUMO UFD-E2 complexes and sequence alignment of SUMO UFD domains, reveal the presence of at least two complementary types of interfaces, which are conserved across species from the same kingdom (Escherichia coli BL21(DE3) plysS containing the expression vector were grown in Luria Bertani medium with chloramphenicol (17 μg/mL) and kanamycine (50 μg/mL) at 37 °C until the OD600 reached to 0.8. Expression was induced by 0.1 mM IPTG, followed by overnight culturing at 28 °C. Recombinant proteins were purified by nickel-nitrilotriacetic acid agarose resin (Qiagen) and dialyzed against 250 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM β-mercaptoethanol in the presence of thrombin protease overnight at 4 °C to remove the 6x His tag. Proteins were further purified by gel filtration chromatography on a Superdex75 column , which was pre-equilibrated in 250 mM NaCl, 20 mM Tris-HCl pH 7.5, 1 mM β-mercaptoethanol.Expression constructs were generated by a standard PCR-based cloning method. The full length human Ubc9 and E1 UFD domain (residues 447–547) were cloned to pET28a tagged with 6x His at the N-terminal. Ubc9 and UFD complex was made by mixing equimolar amounts of proteins and purified by gel filtration chromatography using a Superdex75 column. Ubc9 and UFD were co-eluted in a single peak and confirmed by SDS-PAGE. After gel filtration, the complex was dialyzed against 250 mM NaCl, 50 mM HEPES pH 7.5, 1 mM β-mercaptoethanol for lysine methylation based on a published strategy3212 and the unit cell has a dimension of a = 129.61 Å, b = 129.61 Å, and c = 66.60 Å. Data were processed with XDSCrystals were grown by the sitting-drop vapor diffusion method by mixing the protein complex 30 g/L) with an equal volume of reservoir solution containing 16% PEG6000 (w/v), 100 mM MES pH 6.5, and 5% MPD (v/v), at 18 °C. Crystals appeared after 24 hours and continued to grow to full size in one week. Big crystals were soaked in mother liquor supplemented with gradually increasing concentration of 5%, 10%, 20% (v/v) MPD for 60 seconds each time and flash frozen in liquid nitrogen. Diffraction data were collected to 2.20 Å resolution at ALBA synchrotron in Barcelona (BL13-XALOC beamline). The crystals belong to the space group P4 g/L withThe structure was determined by molecular replacement method using the full length human Ubc9 (protein data bank code 1U9B) as a search model for one molecule in the asymmetric unit in PHASERhttp://eggnogdb.embl.de/#/app/home) and sequences from Metazoa and Saccharomycetales were selected for homology analysis. Detailed information about sequence homology analysis methods is indicated in figure legends. Briefly, when sequences were highly divergent, multiple sequence alignments were performed using Muscle tool (http://www.ebi.ac.uk/Tools/msa/muscle/). When sequence displayed higher conservation level, homology analyses were performed using Clustal Omega program . Phylogenetic distances were calculated by the maximum likelihood method and the JTT model included in the Seaview v4 software packagehttp://itol.embl.de/). Consensus sequences were calculated using WebLogo software (http://weblogo.berkeley.edu/)http://iubio.bio.indiana.edu/soft/molbio/ibmpc/genedoc-readme.html). Data distribution was plotted on box plots using “BoxPlotR: a web-tool for generation of box plots”.Human Sae2 orthologs were search in EggNOG database .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


NLRP12 is a NOD-like receptor that plays multiple roles in both inflammation and tumorigenesis. Despite the importance, little is known about its mechanism of action at the molecular level. Here, we report the crystal structure of NLRP12 PYD domain at 1.70 Å fused with an maltose-binding protein (MBP) tag. Interestingly, the PYD domain forms a dimeric configuration through a disulfide bond in the crystal. The possible biological significance is discussed in the context of ROS induced NF-κB activation. NOD-like receptors (NLRs) belong to a large multi-domain protein family and have prominent biological functions. Many NLRs have been characterized as pattern recognition receptors that sense microbial products in the cytoplasm of cells. Among them, NLRP1, NLRP3, NLRC4, NLRP6, NLRP10, NLRP12, NOD1 and NOD2 are best-studied members, and many of them function through a caspase-1 activating protein complex, called the inflammasomes [Yersinia pestis, the causative agent of plague, through controlling interleukin-18 (IL-18) and IL-1β production [NLRP12 is a NLR family member that received much attention recently. NLRP12 is mainly expressed by the immune cells, and its expression is down-regulated in response to pathogen products and inflammatory cytokines. It has been predicted to play a role as a negative regulator of the inflammatory response . Mutatiooduction . In addioduction . A recenoduction .The significance of NLRP12 in both chronic inflammation and cancer warrant extensive structure-function characterization of this protein and its mutants. Most of the NLRPs proteins contain an N-terminal Pyrin domain (PYD), a central nucleotide oligomerization domain (NOD) and a C-terminal Leucine-rich repeats (LRR). The PYD domain belongs to a large protein superfamily called death domain superfamily together with the Death Domain (DD), the Death Effector Domain (DED) and the Caspase Recruitment Domain (CARD). Death domain fold containing proteins play pivotal roles in apoptosis, necrosis and inflammation. Many of these members function through homotypic interactions that mediate the formation of oligomeric structures , 12.Despite the report of an NMR structure of the NLRP12 PYD , its homCoding sequence for residues L10-S105 of the human NLRP12 (NCBI accession # NP_653288) was cloned into a pET30a expression vector  with a non-cleavable N-terminal MBP tag. The MBP tag contains surface entropy reducing mutations D82A/K83A/E172A/N173A/K239A to enhance crystallization probability , 15. A cPurified MBP-PYD protein in buffer B was concentrated by Amicon centrifugal concentrators  to 20 mg/ml before setting up hanging drop vapor diffusion method for crystallization. Multiple commercially available crystal screens were tested using the Mosquito crystallization robot . Single crystals grew after one week with a well solution containing 3.5 M sodium formate, 0.1 M sodium acetate, pH 4.6. 20% glucose (w/v) was added to the reservoir solutions as the cryoprotectant to flash cool the crystals in liquid nitrogen for X-ray diffraction data collection.1212 with unit cell parameters a = 103.62 Å, b = 186.74 Å, c = 52.72 Å . Data were processed with HKL2000 program suite  and XDS also see . The Math Phaser . The MBPh Phaser  was usedh Phaser . The cryh Phaser  and RCSBh Phaser . Most ofh Phaser  with PDBh Phaser  using thh Phaser  with manThe coordinates and structural factors of MBP-PYD fusion protein have been deposited in the Protein Data Bank with accession code 4XHS.Our initial effort to crystallize the human NLRP12-PYD domain did not identify any promising conditions of crystallization that could yield crystals suitable for X-ray data collection. As a result, we tried an alternative strategy of using the bacterial MBP as a crystallization tag to facilitate the crystallization of the PYD domain. This approach of using MBP as a crystallization chaperone has led to structural determination of many proteins from the death fold superfamily and other protein families , 28–30.1212 space group . The NMR structure can be superimposed over the PYD region of chain A and chain B with rmsd of 1.15 Å to 1.28 Å.The PYD structures of four other NLRP proteins have been solved previously including that of NLRP1 , NLRP3 , NLRP7  and NLRPInterestingly, in the asymmetric unit, we identified a ‘dimer’ of fusion proteins. The dimer was mediated by an intermolecular disulfide bond formed between the Cys11 residues of the PYD domains . There aThe current study identifies an exposed cysteine residue Cys11 at the N-terminal PYD domain of the human NLRP12 protein that can mediate disulfide bond formation. Although this intriguing finding is preliminary, we speculate that this PYD domain can interacte with other PYD domains or other proteins/domains through this exposed cysteine. Indeed, this Cys11 of human NLRP12 is conserved in NLRP12 and NLRP3 proteins . InteresEven though the mechanism for such disulfide formation in vivo is not well defined, it was proposed that reactive oxygen species (ROS) is a cancer inducing agent, although its mechanism is less clear . There a


Mechanical metamaterials are engineered materials whose structures give them novel mechanical properties, including negative Poisson's ratios, negative compressibilities and phononic bandgaps. Of particular interest are systems near the point of mechanical instability, which recently have been shown to distribute force and motion in robust ways determined by a nontrivial topological state. Here we discuss the classification of and propose a design principle for mechanical metamaterials that can be easily and reversibly transformed between states with dramatically different mechanical and acoustic properties via a soft strain. Remarkably, despite the low energetic cost of this transition, quantities such as the edge stiffness and speed of sound can change by orders of magnitude. We show that the existence and form of a soft deformation directly determines floppy edge modes and phonon dispersion. Finally, we generalize the soft strain to generate domain structures that allow further tuning of the material. et al. propose a design principle using operations that cost little energy and realize mechanical metamaterials that can be easily and reversibly transformed between states with different mechanical and acoustic properties.Here Rocklin  Because each hinge as a point-like object in the model contributes d degrees of freedom and each strut contributes one constraint, structures satisfying 〈z〉=2d, now called Maxwell latticesThe emergence of mechanical stability is a central theme in many branches of condensed matter physics, ranging from jamming of granular matter1347z〉<2d and lead to instabilities, for example, flow of granular matter. A particularly interesting case is the generation of floppy modes by cutting a finite piece from an infinite Maxwell lattice. Because the hinges on the boundary have z<2d, the finite system must have as many floppy modes as the length of the boundary. Recent studies reveal that these floppy modes exhibit very rich physics. They can either appear as plane wave modes that penetrate through the bulk121415161721What is central to the understanding of mechanical stability is the concept of floppy modes, which are normal modes of deformation that do not stretch or compress any strut and only involve rotations at the hinges in the frame. In general these modes emerge when 〈Here we show that, interestingly, simple operations that cost little energy can be utilized to induce transitions that change the topological polarization in a structure, analogous to the change of topological states in the quantum Hall effect through the change of the magnetic field. This leads to a design principle for mechanical metamaterials that can be easily and reversibly transformed between states with dramatically different properties, and we use example lattices to illustrate this see . Recentl262728293031323334z=2d. The lattice in We start our discussion of the topological transitions using the example of the deformed kagome lattice, which is the first two-dimensional lattice that was shown to exhibit topological polarizationRT, as introduced in ref. RT=0 the lattice has floppy modes on all edges, but when RT≠0, the direction it points towards is the edge that gains extra floppy modes, whereas the opposite edge lacks floppy modes.The topological polarization RT can change, which dictates that the edge floppy modes have to move from one edge to the opposite edge at the transition. In the following discussion, we call such uniform floppy deformations uniform soft twistings, and we use a bond angle θ →a2→0, so the two regimes θ<θ<RT and are topologically polarized. The transitions at RT changes its value across them. At a transition, edges of the triangles form straight lines along a particular direction, which is intimately related to the rise of bulk floppy modes at the transition. As discussed in refs As shown in These transitions lead to a dramatic change in the edge stiffness because an edge becomes rigid as it loses floppy modes. We perform conjugate-gradient minimization calculations of the response to a point force on one edge of a lattice with other edges held fixed  can be written in terms of the left Cauchy–Green strain tensorθ. As proved in which is homogeneous in space and is a function of r= is the coordinate, f±(w) are two arbitrary scalar functions and λ± are two constants determined by where where The characteristics of these floppy modes are dictated by the sign of f± into Fourier series In the dilation dominant regime k), these floppy edge modes have decay rate κ∼k2 and penetrate much deeper into the bulk, in comparison with the floppy edge modes in the dilation dominant regime discussed above, which have κ∼k.For the shear dominant regime OOz〉>2d has a uniform soft twisting  at small k, but deviate at larger k , either slightly increasing or slightly decreasing the twisting angle θ results in the decrease of a1. The same primitive vector a1 can be reached by two choices of twisting angle θt and θb which are above and below θt and θb may be joined together at a domain wall interface as shown in Ny rows separately one twisting direction or the other, resulting in a domain wall structure that then passes on a fixed course . We emphasize that this response is not set by the material but tuned by the domain structure it is placed in at the critical point.By choosing the fraction of the system subject to one twisting or the other, one can tune the effect on the average lattice primitive vectors, resulting in a new nonlinear soft strain, which we call domain soft twisting. The domain soft twistings can be used to generate a desired mechanical response. For example, choosing a path such that |φi of zero modes i's two degrees of freedom isChanging the domain structure through the domain soft twistings has very interesting effects on the edge floppy modes of the Maxwell lattices. To demonstrate this, we show the spatial extent of zero modes, obtained by directly calculating the zero modes of a finite Maxwell lattice and measuring the amplitude of zero modes projected into every lattice site. The total amplitude j labels zero modes, and φi varies from 2 for completely free particles in two dimensions to 0 for completely pinned particles. The spatial extent of zero modes φi is an indicator of the local ‘floppiness', because stress-free spatially localized deformations such as the edge indentation considered in φi.where φi and thus higher floppiness.We show the effect of the domain soft twisting on the edge floppy modes in a1 direction, as shown in For a homogeneous unpolarized state, the zero modes are distributed roughly evenly around the edge, as shown in The domain structure can also be used to enlarge the crystal basis of a cell and reduce its symmetry, similar to the ‘multiple folding mechanisms' considered previously in regular kagome latticesθt, θb as one or the other passes through topological transitions (dashed lines). In this way, one may create topological polarizations pointing towards domain walls, resulting in domain walls that contain zero modes and hence may be used as channels to convey motion across an otherwise rigid bulk, as shown in self stresses along such domain boundaries, which we may similarly create as shown in Furthermore, different domains can undergo additional topological polarizations separately, as shown in This method of creating domains should be fully extendable to analogous three-dimensional (3D) structures. A domain plane in such a structure would require that that the amplitudes of and the angle between the two primitive vectors in the plane be equal across the domain. However, 3D Maxwell lattices have three Guest modes, and hence these three requirements should result in a unique path through configuration space for a given set of domains. It has recently been shown that 3D Maxwell lattices can be topologically polarized and have other nontrivial propertiesIn summary, we have examined TTMMs, a broad class of systems characterized by their uniform soft elastic deformations. As we have shown, this soft deformation can be activated to reversibly tune, without the application of significant stress, between states with dramatically different edge stiffnesses, bulk speeds of sound and location and spatial extent of floppy edge modes. All of these properties are robust, with some protected by the system's topological state and others guaranteed by the form of the soft elastic deformation. Domain structures may be implemented at certain lattice configurations, allowing generalized nonlinear modes and localized control of elastic properties through the change of topological states in domains. These materials reveal a deep connection that endures between structure and function even as both are altered, and have broad potential applications. Realization of the full phenomenology, including inserting domain wall structures, requires flexible hinging motions capable of undergoing large deformations without significant resistance and fine manipulation of the edge or bulk.In the 163941Examples of future applications of TTMMs include materials with tunable ability to conform to a surface, a quality that affects adhesion and grip in systems as varied as rubber tires that can be switched to perform on hard or soft surfacesOur primary methods were analytical theories aided by computer-aided symbolic analytical and numerical computations in the program Mathematica. The prototype was generated from assembling commercially available plastic parts, known as K'Nex and other simple load-bearing elements .Further details are available in the How to cite this article: Rocklin, D. Z. et al. Transformable topological mechanical metamaterials. Nat. Commun.8, 14201 doi: 10.1038/ncomms14201 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Notes and Supplementary ReferencesThis video contains 3 demonstrations: (1) A prototype of the Transformable Topological Mechanical Metamaterials (TTMM) in its non-polarized state where both edges are soft, (2) transforming the TTMM prototype using its uniform soft twisting, and (3) the TTMM prototype in its polarized state where the two edges have dramatically different stiffness.


Bacillus subtilis (Uniprot: P39695) were characterized. DNA‐protein interactions were investigated in silico by analyzing models for structural conservation, surface electrostatics and structure‐based DNA binding propensity; and by data‐driven macromolecular docking of DNA to models. Our findings suggest that the DUF4131 domain contains a cryptic DNA‐binding OB fold domain and that the β‐lactamase‐like domain is the hitherto cryptic competence nuclease. Proteins 2016; 84:1431–1442. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.Bacterial competence, which can be natural or induced, allows the uptake of exogenous double stranded DNA (dsDNA) into a competent bacterium. This process is known as transformation. A multiprotein assembly binds and processes the dsDNA to import one strand and degrade another yet the underlying molecular mechanisms are relatively poorly understood. Here distant relationships of domains in Competence protein EC (ComEC) of  Natural transformation is the uptake of free environmental double stranded DNA (dsDNA) that has been secreted by other bacteria or is the product of cell lysis.Helicobacter pylori depends on its natural transformation,The composition of the competence system varies from species to species.B. subtilis protein, key for competence yet poorly understood, is ComEC. ComEC is composed of 776 residues and matches three domains in the Pfam databaseStreptococcus pneumoniae,B. subtilisT. thermophilus.B. subtilis: its deletion renders the bacterium incapable of transformation.Haemophilus influenzaeNeisseria gonorrhoeaeListeria where it is required for the escape of infecting bacteria from host cell phagosomes.One in silico using a variety of homology, structural and covariance‐based bioinformatics methods. We can strongly assert that the β‐lactamase‐like domain in B. subtilis ComEC supports a nuclease function. We discover and describe a presumed single‐stranded DNA‐binding OB fold within DUF4131. The results enhance our understanding of bacterial competence machinery and will guide experimental structural biology efforts.Here we probe ComEC structure and function 2+ ions found in the template. Modeling of the OB fold of ComEC DUF4131 (V60‐H160) used two templates; subunit E of Replication Protein A  was used to align and visualize protein structures. 500 models of the OB‐fold and 1000 models of the lactamase‐like domain were constructed using MODELLER 9.12The webserver HHpred was used to search for distant homologues of ComEC domainset al. 2013, 2014) results from eight alignments, derived by JackhmmerE‐value cutoffs of 10−40, 10−10, 10−4, and 1. bbcontacts was applied to results from CCMpred,E‐value cutoff of 10−3 and filtering to 90% sequence identity using HHfilter v2.0.15Using the ComEC DUF4131 sequence obtained from UniProt, evolutionary covariance analysis was used to predict residue‐residue contacts with PconsC2ab initio fragment‐based folding of regions mapping to the OB fold region in Rosettaab initio modelling. The resulting 1000 models were clustered using Spicker.The resulting contact maps informed the definition of two distinct domains within DUF4131. TMHMME‐value of 0.0001. The models, along with comparator crystal structures of known function were submitted to the structure‐based nucleic acid binding prediction servers DNABIND,Electrostatic analysis was carried out using the APBS Tools plugin of PyMOL.B‐form double‐stranded (ds)DNA (default sequence CCCTGTGGAGCCACACCCTAG and its complementary strand) was generated at the make‐NA server.B. subtilis ComEC DUF4131 sequence and proteins of the OB fold superfamily . The relationship was supported by a good match between the predicted secondary structure of the former and the actual secondary structure of the latter  with probabilities reaching 100% for the closest structural match teichoic acid phosphorylcholine esterase . Asp residues are commonly found at Zn‐binding sitesModel analysis strongly suggested that the β‐lactamase‐like domain is catalytically active since Zn‐ligating residues present in the template structure are almost entirely conserved in ComEC sequences [Fig. Methanosarcina mazei cleavage and polyadenylation specificity factor  clearly distinguishable from Pce with −1.67. In agreement with these predictions and known trends among DNA‐binding proteins,B. subtilis . Although DUF4131 domains most commonly precede Competence domains, as in ComEC  of S. pneumoniae EndA and a ComEC protein which retains all the key metal‐binding residues. Adding a further layer of complexity, no EndA homologue is apparent in H. pylori suggesting that this bacterium, anomalous in other ways too,Although requiring experimental validation, this predicted nuclease domain appears to be a strong candidate for the currently unassigned activity in The limited experimental characterization of ComEC largely owes to the work of Draskovic and DubnauB. subtilis ComEC. The key role of ComEC in competence has been known for 20 years,In summary, we provide important insights into ComEC domain structure and function predicting DNA binding and cleavage roles to the DUF4131 and β‐lactamase‐like domains, respectively, of DJR conceived and supervised the study. HMCT and FS carried out the covariance analysis and the contact‐assisted fragment assembly modeling. JAB carried out all other experiments. All authors contributed to writing the manuscript.Supporting InformationClick here for additional data file.


Arabidopsis thaliana has two type 4 seed-specific metallothionein homologues, MT4a and MT4b, with likely roles in zinc(II) homeostasis. These two metallothioneins are 84% identical, with full conservation of all metal-binding cysteine and histidine residues. Yet, differences in their spatial and temporal expression patterns suggested divergence in their biological roles. To investigate whether biological functions are reflected in molecular properties, we compare aspects of zinc(II)-binding dynamics of full-length MT4a and MT4b, namely the pH dependence of zinc(II) binding and protein folding, and zinc(II) transfer to the chelator EDTA. UV–Vis and NMR spectroscopies as well as native electrospray ionisation mass spectrometry consistently showed that transfer from Zn6MT4a is considerably faster than from Zn6MT4b, with pseudo-first-order rate constants for the fastest observed step of kobs = 2.8 × 10−4 s−1 (MT4b) and kobs = 7.5 × 10−4 s−1 (MT4a) . 2D heteronuclear NMR experiments allowed locating the most labile zinc(II) ions in domain II for both proteins. 3D homology models suggest that reactivity of this domain is governed by the local environment around the mononuclear Cys2His2 site that is unique to type 4 MTs. Non-conservative amino acid substitutions in this region affect local electrostatics as well as whole-domain dynamics, with both effects rendering zinc(II) ions bound to MT4a more reactive in metal transfer reactions. Therefore, domain II of MT4a is well suited to rapidly release its bound zinc(II) ions, in broad agreement with a previously suggested role of MT4a in zinc(II) transport and delivery to other proteins.The dynamics of metal binding to and transfer from metalloproteins involved in metal homeostasis are important for understanding cellular distribution of metal ions. The dicotyledonous plant The online version of this article (10.1007/s00775-017-1516-6) contains supplementary material, which is available to authorized users. The unique importance of the essential micronutrient zinc(II) to the health of all organisms is increasingly recognised , with thAlthough raising awareness of the prevalence and far-reaching consequences of (human) zinc deficiency has been a long and slow process , zinc deIt has been argued recently that enzyme-bound zinc(II) is now reasonably well understood, but that the nature of “mobile” zinc(II) in biological systems has remained largely mysterious . In partC-I/II from wheat  was used to study the metal transfer dynamics of MT4a and MT4b by UV–Vis spectroscopy. Zn4HCO3, pH 7.8) using PD-10 columns . The protein eluates were concentrated using Amicon Ultra-4  filter devices.The proteins were desalted and buffer-exchanged  were acidified with small volumes of different concentrations of formic acid (0.1–5 M). Mass spectra of the samples  were recorded. To avoid salt contamination from the KCl solution of the glass electrode (Hamilton Biotrode pH electrode), each sample was divided into two aliquots and the pH was measured before  and after  acquisition of mass spectra. The pH values given are not corrected for the 10% CH3OH content.To observe speciation at different pH values, desalted aliquots of Zn6MT4a or Zn6MT4b  were reacted with six molar equivalents of EDTA  for up to 22 h. At different time intervals, aliquots were taken and diluted  prior to recording mass spectra. The pH value after the reaction was checked and found to have not changed significantly.For observation of speciation during reaction with EDTA, desalted solutions of Zn−1. Other parameters were fixed as: temperature 195 °C, nebulizer 0.6 bar, dry gas 4.5 L min−1, capillary exit 100 V, skimmer1 50 V, skimmer2 25.2 V, hexapole1 24.2 V, hexapole2 22.4 V, hexapole RF 450 V, transfer time 81 μs, and detector TOF 2300 V. The data were recorded over 0.4–2 min for a range of 500–5000 m/z. The experimental data were then smoothed and deconvoluted using Bruker Compass DataAnalysis v. 4 .All mass spectra were recorded on a Bruker Daltonics MicroTOF mass spectrometer fitted with an electrospray ionisation source operating in positive mode. The samples were injected into the spectrometer by a syringe pump with a flow rate of 240 μL h11, 10% D2O, 50 mM NaCl) at different pH values (vide infra). Homonuclear 1H spectra were acquired on unlabelled samples for Zn6MT4a and Zn6MT4b. Heteronuclear 2D and 3D  spectra were obtained for 15N-labelled samples for Zn6MT4a and Zn6MT4b, and a 13C- and 15N-labelled sample of Zn6MT4b was used to record triple-resonance data (HNCA and HN(CO)CA) to facilitate sequential assignment.All protein samples (various concentrations) were prepared in a standard buffer, in the following referred to as “NMR buffer”  and 15NH4Cl, respectively. All NMR spectra were acquired at 298 K. Recorded spectra were processed using Bruker Topspin v. 2.1 software. For 2D and 3D spectra, phase- and baseline-corrected Topspin files were transferred to Sparky v. 3.106  Total Correlation  and Nuclear Overhauser Enhancement  spectra were recorded with 16 scans, 4k data points in F2 and 512 or 400 increments in F1 with a spectral width of 16 ppm centred at the water peak at ~ 4.7 ppm. A squared sine-bell function was used to apodize the raw data prior to Fourier transformation with 2k × 2k data points. 2D heteronuclear single quantum coherence  HSQC spectra for Zn6MT4a and Zn6MT4b used for assignment purposes were recorded with 4k data points in F2 (1H) and 128 increments in F1 (15N). Spectra were acquired with a spectral width of 16 ppm in F2 and 40 ppm in F1 over 16 scans with a 1JNH coupling constant of 90 Hz. The raw data were apodized using a squared sine-bell function and Fourier transformed into 4k × 512 data points in the F2 × F1 dimensions, respectively.1D 1H, 1H, 15N] TOCSY-HSQC (mixing time of 60 ms) and NOESY-HSQC (mixing time of 100 ms) spectra were acquired with spectral widths of 14 ppm for F3 (1H), 32 ppm for F2 (15N) and 14 ppm for F1 (1H) with 16 scans. Spectra were acquired with 2048 data points in F3 (1H), 36 increments for F2 (15N), and 128 increments for F1 (1H). HNCA and HN(CO)CA spectra were obtained with 2048 data points in F3 (1H), 40 increments for F2 (15N), and 64 increments for F1 (13C) with 16 scans. Spectral widths were 14.0 ppm for F3 (1H), and 32 ppm for both F2 (15N) and F1 (13C). The data were apodized and Fourier transformed into 2k(F3) × 64(F2) × 512(F1) data points for  TOCSY-HSQC and 2k(F3) × 64(F2) × 256(F1) data points for  NOESY-HSQC, HNCA and HN(CO)CA.3D . Immediately after mixing a series of 1D, 1H-NMR spectra were recorded. The pH value after the reaction was checked and found to have not changed significantly.For experiments to study the pH dependence of protein folding, the pH of Zn15N) samples of Zn6MT4a or Zn6MT4b  were mixed with EDTA . Immediately after mixing, a series of 2D  HSQC spectra  were recorded. The pH value after the reaction was checked and found to have not changed significantly.Homogeneously labelled  were usl v. 4.0 ; howeverF server . Electrov. 2.0.0 .A. thaliana  HSQC NMR spectroscopy to correlate the folding state of individual domains in different metalloforms. By necessity, the reaction conditions differ from those employed for UV spectrophotometry, because NMR spectroscopy requires higher protein concentrations. Hence, all MS and NMR timecourse experiments were carried out with 250–280 µM solutions of proteins, but only equimolar amounts of EDTA with respect to [Zn2+], to bring the reaction times within a manageable range  proceeded more slowly than those observed by NMR .Native ESI-MS has been used previously to monitor the speciation of MTs during their interactions with other metal acceptors including proteins , 96–101.ge Figs. . We alsoC  HSQC NMR spectroscopy  confirmed that equimolar EDTA was insufficient for complete demetallation.To shed some light on the likely locations of the most reactive Zn sites and remaining Zn ions, and whether the two domains in each protein displayed different reactivities that may help explain the observations from UV–Vis spectroscopy and ESI-MS, 1D opy Fig.  was empl2 (and Zn3) species observed by ESI-MS correspond to fully metallated domain I.The 2D NMR data show that for both proteins, domain I was initially less reactive than domain II, as in both cases, domain I residues were visible at the 10 and 17 min time points—whereas this was not the case for domain II (see below). In fact, a proportion of the domain I resonances persisted throughout the reaction for both proteins, albeit at clearly diminished intensities . The decreasing intensities for both domains indicate that in principle, both domains of both proteins are able to react with EDTA simultaneously, albeit at different rates—at least when EDTA is supplied in equimolar amounts. Interestingly, the intensity of the domain I resonances for MT4a even increased slightly after the 25 min time point, perhaps an indication for slow metal redistribution after the initial removal from domain I. This observation highlights that the kinetic product(s) of the reaction with EDTA may not necessarily be identical to the thermodynamic products. It is somewhat surprising that this redistribution/refolding seems to be a slow process, as it has been pointed out that MT metallospecies, due to fast on/off rates, usually equilibrate within milliseconds with each other . Either C mutant proteins had indicated that the Zn-bound status of the equivalent of His51 was critical to structuring the entire domain II via crosstalk to these four residues [6MT4b occurred not from the mononuclear site, but preferentially from the three-metal cluster, although we would caution that resonances for the three residues Cys71, Thr72, and Cys73 were already weaker in the unreacted protein.Significant differences between the two homologues were observed for domain II. For MT4a, all domain II resonances had already been lost after 10 min Fig. a. A corrresidues . ResidueIn summary, the likely origin of the different metal transfer dynamics of MT4a and MT4b resides predominantly in domain II, with MT4a showing more rapid and more cooperative metal transfer to EDTA. In the following section, we will seek to identify the cause for this difference.C [C produced a prominent Zn4 species at moderately acidic pH that coexisted with either the Zn6 species (pH 5.5) or the Zn2 and apo species (pH 4.0), whilst Zn5, Zn3 or Zn1 were much less abundant; yet, the loss of two zinc(II) ions from Zn6EC did not correspond to cooperative demetallation of the Zn2 domain I as might have been concluded, as the NMR signals from this domain persisted at lower pH than those for domain II [C nor A. thaliana MT4b lose their domain II-bound zinc(II) ions cooperatively as far as proton-driven metal loss is concerned. There is some indication for moderately cooperative loss of zinc(II) from domain II of MT4a, where the Zn2 species that most likely corresponds to fully metallated domain I was most abundant at pH 4.9. Furthermore, since the backbone amide 1H NMR signals for the Zn6 forms of either protein neither shift nor broaden, the species observed are unlikely to be in chemical exchange with other species. Hence, the decreases in intensity correspond to the decrease in the concentration of the fully metallated domains in both cases. This also means that the Zn4 or Zn5 species for MT4b, which are present at significant amounts above the pH(1/2), do not seem to give rise to new dispersed NMR signals. Hence, partially metallated domains are likely to be structurally disordered.Studying metallospecies and protein folding as affected by proton-driven demetallation has revealed that for both MT4a and MT4b, domain II is more pH-labile than domain I. A re-analysis of published data for wheat EC  indicateomain II . Therefo6EC from wheat appeared to display triphasic reaction kinetics, although the fastest step was only discernible at lower EDTA:protein ratios, and could hence not be quantified under appropriate pseudo-first-order conditions [−4 s−1, between the rates for the fastest observed step for MT4a and for the only step seen for MT4b. In the case of wheat EC, it was not possible to determine whether the most reactive Zn ions originated from domain I or II. The slowest third phase observed for wheat EC was attributed to protein degradation. This did not appear to be a major contribution within the timeframe of the present experiments. Furthermore, monitoring the reaction at lower EDTA:protein ratios for MT4a or MT4b gave no clear indication of whether a faster step for either MT4a or MT4b exists , but comparison of the time required to completely deplete the Zn6 form  suggests that Zn6MT4b reacted considerably more slowly than either wheat Zn6EC or Zn6MT4a; thus, it is most likely that the fast component observed for wheat EC does not occur for MT4b.Regarding metal transfer dynamics, we will first attempt to contextualise and integrate the results from the three techniques employed to monitor the reactions with EDTA. The UV–Vis timecourse data revealed monophasic reaction kinetics for MT4b, and biphasic kinetics for MT4a, with the first phase of the latter being considerably faster than the single phase for MT4b. For full-length mammalian MTs, different phases were ascribed to differences in the reactivity of their two domains , 93, 95.nditions . This fa5MT4a species points to a degree of cooperativity—but the NMR results indicate that the observed Zn4 species do not correspond to fully loaded domain II, but instead include species with fully loaded domain I. This also means that these Zn4 species harbour domain II with two Zn ions bound—reminiscent of the Zn4 species observed for wheat EC at moderately acidic pH. Although these under-metallated species have been generated in different ways, it is clear that the three cluster sites are not equivalent in either MT4a or wheat EC—irrespective of at which sites the remaining two ions are bound. In any case, the clear occurrence of the Zn4MT4a species indicates that removal of the first ion did not lead to “cluster collapse” for MT4a either, although it is clear that the four zinc(II) ions bound to domain II are more prone to transfer than the two in domain I.In the case of mammalian MTs, kinetic data were compatible with removal of the first metal ion being the rate-determining step for the reactivity of the entire cluster, with the loss of the subsequent cluster ions being much faster than the first—in essence, metal loss was cooperative within the individual domains , 94. In C [6MT4a.Finally, the NMR results clearly indicate that in both proteins, both domains are capable of metal transfer, a conclusion that was also reached for wheat EC . Thus, iThe differences observed between MT4a and MT4b must ultimately arise from differences in their sequences . Taken together, these two substitutions do not alter the overall charge of the domain. To discern whether one might expect an effect of these substitutions on the immediate environment of the zinc(II) ions, we have generated homology models of the two domains I, based on the published structure of the wheat Eγ domain . InspectC [3Cys9 clusters are surrounded by substantial negative charge. This is mostly a consequence of the formal − 3 charge of such clusters, which is not fully compensated by the presence of the two lysine residues in this domain. It should be noted that the side-chain conformations of lysine residues in our models are variable, and in solution would be quite flexible as well. It is, therefore, possible that their positive charges are at least occasionally closer to the negatively charged clusters in the vicinity, and this may in principle reduce average negative charge in certain areas to some degree, and thus facilitate nucleophilic attack.Within domain II, the most remarkable dissimilarities occur around the first histidine residue (His42/His43). MT4a harbours a GHMV 41–44) motif, whilst for MT4b, the sequence is EHNT (42–45). Indeed, many type 4 MTs have the equivalent of Glu42 . In contrast, such stabilising H-bonds are not possible for Met43 or Val44. These two residues together form a small hydrophobic patch on the protein surface that is rather unusual for an MT. Whilst the interaction between these two residues may be mutually stabilising, hydrophobic sidechains on a protein surface are inherently unfavourable, and thus, we hypothesise that these two residues are significant contributors to the higher structural flexibility of domain II in MT4a. Such increased flexibility is likely to increase the lability of bound metal ions, and this may be a further contribution to the faster metal transfer dynamics of MT4a—and this may not only affect transfer from the mononuclear site, but also from the Zn3Cys9 cluster.The above considerations are based on the hypothesis that bimolecular assemblies are required for efficient metal transfer. Indeed, such assemblies were observed in this work by ESI-MS, and electrostatics are undoubtedly important for understanding the likelihood for their formation. However, another critical contributor to metal lability in MTs is protein dynamics. Although we have not carried out extensive NMR-based backbone dynamics measurements, the HSQC spectra shown in Figs. A. thaliana that demonstrated MT4 protein localisation in the embryo [A. thaliana MT4a and MT4b principally fulfil essential criteria to act as cellular zinc(II) donors: at least some of their bound metals are bound in a fairly labile fashion, allowing zinc(II) transfer to EDTA to occur with rates comparable to those observed for other MTs. We note that another important aspect in this scenario is metal affinity, and that in the case of wheat EC, it has been found that the average apparent binding constant for zinc(II) was particularly low at higher ionic strength (logK = 8.6 at pH 7.4 and I = 105 mM) [The suggested biological function of type 4 MTs includes zinc(II) donation to zinc(II)-requiring proteins, peptides, or small molecules during seed maturation and/or germination , 50, 55.e embryo , 104. Th 105 mM) . It is cC [A. thaliana, these are characterised by high thermodynamic stability, as judged from both pH-dependent studies  via the vasculature and MT4b functions as a store for zinc(II) throughout the embryo [2His2 site and its surroundings as the lynchpin that governs the reactivity of these two type 4 MTs, with non-conservative substitutions around the first histidine residue playing a crucial role for electrostatics and overall backbone dynamics.However, the most intriguing finding of the current study concerns the differences observed between two closely similar homologues that both occur in the seeds of a single plant. In planta observations had suggested that e embryo . Althouge embryo , the fasC [Ultimately, it is clear that also zinc(II) bound to MT4b will need to be mobilised; this may or may not occur through direct transfer to other proteins. Several other options for zinc(II) release from MTs exist; these include oxidative reactions with electrophiles such as peroxide or oxidised glutathione , but mayC . In addiSupplementary material 1 (PDF 768 kb)Below is the link to the electronic supplementary material.


Bcl2-associated athanogene 2 (BAG2) shares a similar molecular structure and function with other BAG family members. Functioning as a co-chaperone, it interacts with the ATPase domain of the heat shock protein 70 (dHsp70) through its BAG domain. It also interacts with many other molecules and regulates various cellular functions. An increasing number of studies have indicated that BAG2 is involved in the pathogenesis of various diseases, including cancers and neurodegenerative diseases. This paper is a comprehensive review of the structure, functions, and protein interactions of BAG2. We also discuss its roles in diseases, including cancer, Alzheimer’s disease, Parkinson’s disease and spinocerebellar ataxia type-3. Further research on BAG2 could lead to an understanding of the pathogenesis of these disorders or even to novel therapeutic approaches. The BAG (Bcl-2-associated athanogene) family was first identified as a group of proteins that prevent cell death through their interaction with Bcl-2 , 2. TheyTo date, six members of the family have been identified in humans: BAG1 RAP46/HAP46), BAG2, BAG3 (CAIR-1/Bis), BAG4 (SODD), BAG5 and BAG6 (BAT3/Scythe) [6/HAP46, In humans, BAG2 is widely expressed in many tissues, including brown adipose, heart and lung tissue. BAG2 has also recently been in various types of tumor cells, including renal cell carcinoma, glioblastoma and thyroid carcinoma cells –11. ImmuStudies on the crystal structure of BAG family proteins show that the BAG domain contains between 110 and 124 amino acids forming three anti-parallel α-helices with a relatively conserved C-terminus. Hsc70/Hsp70 binds with the second and third α-helix . AdditioBAG2 is an Hsp70/Hsc70 molecular chaperone-interacting protein originally discovered using yeast two-hybrid screening. It is composed of 211 amino acids. Although the C-terminus of BAG2 is classified as a BAG domain, researchers have found that it shares a low level of homology with other BAG domains. So, does the widely accepted functional model of BAG domain apply to BAG2?Using crystallographic technology, Xu et al.  successfMeanwhile, crystallization and preliminary X-ray crystallographic analysis of the BAG2 N-terminal domain (NTD) suggested that it forms a dimer and adopts a folded conformation distinct from any other domains annotated in the Pfam or SMART domain databases .The BAG2 BNB domain harbors dual functions of nucleotide exchange and client binding. When binding with ATP, Hsp70 exhibits low substrate affinity, whereas in the ADP-bound state, it has high affinity for its substrates. As Hsp70-mediated protein refolding is limited by an inherently low rate of nucleotide exchange, efficient protein refolding requires interaction between Hsp70 and NEFs. Previous studies have indicated that BAG2 binds with high affinity to the ATPase domain of Hsp70 and inhibits its chaperone activity in a Hip-repressible manner. The NEF function of BAG2, which accelerates the ATPase cycle, can affect folding, aggregation and degradation reactions in different ways depending on the associated client and the cooperation with other chaperones and co-chaperones , 5, 14. Aside from the formation of the BAG–Hsp70 complex, BAG proteins functionally interact with a variety of binding partners and coordinate diverse cellular processes, such as stress signaling, cell division, cell death and cell differentiation , 6, 18. Molecular chaperones are believed to play a prominent role in protein homeostasis. They regulate the balance between protein folding, translocation, assembly, disassembly, differentiation and degradation. In cells, molecular chaperones help peptides fold correctly, prevent protein misfolding and aggregation, and promote refolding of denatured polypeptides. If promoting refolding of a misfolded protein is not possible, chaperons can promote protein degradation via the ubiquitin–proteasome pathway. In eukaryotic cells, two distinctly regulated chaperone networks perform these specific functions. One includes chaperones linked to protein synthesis (CLIPS), which are functionally and physically linked to translation machinery and assist in the folding of newly translated proteins. The other is the heat shock proteins (HSPs), which can be induced by HSF and serve to protect the proteasome from stress –23. The Two mechanisms are involved in protecting cells from damage induced by misfolded proteins, including molecular chaperones and the ubiquitin–proteasome system (UPS). Molecular chaperones help proteins fold correctly, while the UPS mainly facilitates misfolded protein degradation.CHIP is an Hsp70-associated ubiquitin ligase that participates in the UPS by ubiquitinating misfolded proteins associated with cytoplasmic chaperones. There are at least two domains in CHIP: a u-box domain, which interacts with the ubiquitin-conjugating enzyme E2, and a TPR domain, which links to Hsp70 and Hsp90 –26. PrevIn vivo, BAG2 colocalizes with CHIP, especially within the endoplasmic reticulum . Using bBAG2 is phosphorylated by MAPK-activated protein (MAPKAP) kinase 2, which is known as a primary p38 MAPK substrate that mediates several p38 MAPK-dependent processes. This phosphorylation is part of a novel signaling pathway involved in the response to extracellular stresses.MAPK cascades integrate and process various extracellular signals by phosphorylating substrates. Various stresses, endotoxins and cytokines can activate p38, whereupon it phosphorylates and regulates downstream protein kinases to modulate various intracellular processes. Using enrichment of phosphorylated proteins, fluorescent two-dimensional difference gel electrophoresis and mass spectrometric identification of proteins, Ueda et al.  identifiThe BAG2 coiled coil domain functions in protein–protein interactions. Further investigation is required to determine if BAG2 interaction with other unidentified proteins through the coiled coil domain is regulated by phosphorylation of BAG2 within the complex.BAG2 is involved in numerous diseases. Most previous studies on BAG2 emphasized its important role in cancer cell metastasis –11. RecePrevious studies demonstrated that most of the BAG family proteins play important roles in the regulation of apoptosis, cell survival and the stress response. BAG1, BAG3, BAG4 and BAG6 have anti-apoptotic activities –35.Using reverse transcription-polymerase chain reaction (RT-PCR), Wang et al.  found thTau inclusions are classic hallmarks of many neurodegenerative diseases including Alzheimer’s disease . Though Interestingly, BAG2 also mediates cold-induced tau hyperphosphorylation , 38. It It has been suggested that polyphenol compounds, including curcumin, (−)-epigallocatechin-3-gallate (EGCG) and resveratrol, have the potential to prevent Alzheimer’s disease pathology due to their anti-amyloidogenic, anti-oxidative and anti-inflammatory properties . Recentl1–42 from neurotrophic to neurotoxic [Recent studies indicate that BAG2 expression is negatively regulated by NF-kB signaling  and c-Myurotoxic . FurtherParkinson’s disease, a progressive neurodegenerative disorder, is characterized by progressive degeneration of dopaminergic neurons and the formation of Lewy bodies, mainly in the substantia nigra pars compacta (SNpc) , 48. WitPINK1 is the second most common pathogenic gene associated with autosomal recessive, early onset Parkinson’s disease [ disease . Previou disease , DJ-1 [5 disease , Cdc37/H disease , HtrA2 [ disease , TRAP1 [ disease  and BAG5 disease  might co disease  revealedDrosophila models of Parkinson’s disease demonstrated that Parkin and PINK1 function in a common pathway that regulates mitochondrial quality control [Studies in  control , 58. Fur control .BAG5 was identified as a gene that is upregulated in dopaminergic neurons following medial forebrain bundle transfection. BAG5 was also found to function as a pro-apoptotic factor enhancing dopaminergic neuron degeneration via suppression of the E3 ligase activity of parkin , 61. It ATXN3 [Spinocerebellar ataxia type-3 (SCA3) is the most common dominantly inherited ataxia in the world. It is characterized by a pathological, unstable cytosine–adenine–guanine trinucleotide repeat expansion in the translated region of ATXN3 , 63. TheATXN3 . In the ATXN3 .Overall, BAG2 shares similar molecular structure and function with other BAG family members, but it possesses some unique features. It interacts with several proteins that are involved in many molecular pathways regulating diverse biological and pathological processes Fig. . Though A number of studies of tumor cell lines of different origin have shown that BAG2 influences cell survival and exhibits a pro-apoptotic property. More importantly, recent findings suggest that BAG2 takes part in the aggregation of aberrant proteins through chaperone-mediated refolding and ubiquitin–proteasome degradation pathways. It has potentially significant roles in neurodegenerative disease pathogenesis. For example, BAG2 delivers tau to the proteasome for ubiquitin-independent degradation, and stabilizes both PINK1 and pathogenic ataxin3-80Q by decreasing its ubiquitination. Further research on BAG2-related molecular pathways may give additional insight into the pathogenesis of numerous diseases and help to devise future strategies for diagnosis and treatment.


GJA1) is a cell-cell communication gap junction protein expressed in all organ systems. It was recently found that GJA1 mRNA undergoes alternative translation to generate N-terminal truncated isoforms, of which GJA1-20k is the most abundant. Here we report a surprising finding that, unlike full length GJA1-43k, GJA1-20k has a strong tropism for mitochondria. Exploring function, we found that GJA1-20k appears to be an organelle chaperone and that overexpression of GJA1-20k is sufficient to rescue mitochondrial localization to the cell periphery upon exposure to hydrogen peroxide, which effectively limits the network fragmentation that occurs with oxidative stress. By high-resolution fluorescent imaging and electron microscopy, we determined that GJA1-20k is enriched at the interface between mitochondria and microtubules, appearing to load organelles for transport. Mutagenesis experiments revealed that although the microtubule-binding domain (MTBD) in GJA1-20k is not necessary for protein localization to mitochondria, the MTBD is essential for GJA1-20k to facilitate mitochondrial transport and maintain mitochondrial localization at the periphery. These results reveal an unexpected role for the alternatively translated isoform of the Cx43 gap junction protein, GJA1-20k, which is to facilitate microtubule-based mitochondrial transport and to maintain mitochondrial network integrity during cellular stress.Connexin 43 (Cx43, encoded by  Connexin 43 (Cx43) is the most widely expressed of the 21 human connexins, occurring in all organ systems and is best recognized as a cell-cell communication protein  segments of Cx43 protein. These products of alternative translation are N-terminal truncations of the full length Cx43 protein. Alternative translation of GJA1 occurs widely in cell lines  encoding full-length Cx43 and small isoforms were first cloned into pDONR/221 to generate Gateway entry clones (Thermo Fisher Scientific) as previously described. Destination vectors (pDEST) encoding C-terminal GFP-, mCherry-, and HA-tagged proteins were subsequently made for mammalian cell expression. All constructs are driven by the cytomegalovirus (CMV) promoter, and contain internal methionine start sites mutagenized to leucine to ensure single isoform expression. Mutagenesis was carried out with the Quick Change Lightning Mutagenesis Kit (Agilent) according to manufacturer's instructions. Mutagenesis primers used to remove all six tubulin-interacting residues , nonessential amino acids, sodium pyruvate (Thermo Fisher Scientific), and Mycozap-CL (Lonza). Primary cardiac fibroblasts were isolated from neonatal mouse hearts as described  were maintained at 37°C in a humidified atmosphere of 5% CO2, 2 K2HPO4, 1 DTT, 1 NaF, 0.1 Na3VO4, 0.5% NP40, pH7.4 with halt protease inhibitor). Cells were rotated for 30 min and spun down at 10,000 × g for 20 min at 4°C to remove insoluble debris. Following protein normalization, cell lysates were precleared using Dynabeads protein G (Thermo Fisher Scientific) for 30 min with rotation at 4°C. Beads were discarded, and 2 mg of precleared lysate was used for each reaction. Immunoprecipitation was undertaken using 5 μg of either mouse anti-HA , or mouse anti-GST  as negative isotype control at 4°C for 4 h with rotation. Dynabeads protein G (20 μl) was added to each reaction, and the tubes were rotated for an additional 45 min at room temperature. Protein complexes were washed 3 times with lysis buffer using a Dynamag-2 magnet. Proteins were then eluted with 30 μl 2X NuPAGE sample buffer containing 100 mM DTT, incubated at 37° for 20 min, and subjected to SDS-PAGE electrophoresis and Western blotting. Membranes were imaged using the ChemiDoc MP detection system (Bio-Rad). The experiments were repeated three times.Co-immunoprecipitation was carried out using HeLa cells expressing HA-tagged GJA1-20k. Cytoskeletal-protein interactions were stabilized by the addition of phalloidin  during cell lysis in 0.5% NP40 buffer  in a fixed angle rotor at 50,000 × g at 4°C for 45 min. After ultracentrifugation, three fractions were collected and labeled as F1, F2, and F3 from cell samples. Fractions were pelleted at 12,000 × g for 5 min at 4°C for Western blotting. Three independent experiments were conducted and the representative blots were shown.Mitochondria fractionation of HeLa cells transfected with HA-tagged GJA1-43k or GJA1-20k, was performed as previously described  with 486, 561, and 647-nm diode-pumped solid state lasers, and an ORCA-Flash 4.0 Hamamatsu camera (C11440), controlled by NIS Elements software. Live cell imaging was carried out in transiently transfected HeLa cells as previously described . At 16–24 h post transfection, cells were immersed in imaging solution containing HBSS supplemented with 10% FBS, and 1x Mycozap-CL. Images were acquired at 37°C using our Nikon Eclipse Ti imaging system described above. Active mitochondria were labeled by loading cells for 30 min with imaging solution containing 2.5 μM TMRM (Thermo Fisher Scientific). Pearson's coefficient was calculated using the ImageJ JACoP plugin  or PBS in fully supplemented medium for 4 h. Cells were fixed in 4% PFA (Electron Microscopy Services) in PBS for 20 min at RT. Immunolabeling: rabbit anti-Tom20  and chicken anti-GFP . Alexa Fluors (Thermo Fisher Scientific) were used for secondary antibody detection for 1 h at RT. ProLong Gold Antifade containing DAPI was used to mount samples. Using ImageJ (NIH), the peripheral/central fluorescence ratio was determined by dividing peripheral Tom20 signal by that of the perinuclear and Golgi regions. First, each image is background-subtracted using a rolling ball radius of 50 pixels. Thresholding for the inner region of interest (ROI) encompassing the nucleus was set based on 1/6 of the maximal Tom20 intensity of the entire cell. Signal from the nuclear ROI was removed from the cell. The middle ROI (perinuclear/Golgi) boundary was set by expanding the inner ROI by 4 micrometers. Finally, signal density from the remaining outer ROI (periphery) is divided by the sum of that of the inner and middle ROIs to obtain the ratio , and sectioned using an ultramicrotome . Sections were then mounted on EM grids and stained with uranyl acetate and lead citrate. Images were acquired using the JEM1200-EX, JEOL microscope equipped with a digital camera . For immunogold labeling, cells were fixed in 4% formaldehyde and 0.1% glutaraldehyde in PBS at RT for 10 min, scraped, and pelleted as described above. Cells were fixed overnight at 4°C in 4% formaldehyde and 0.1% glutaraldehyde in PBS and thereafter mixed with 1.5% low melting temperature agarose. Small pieces of agarose with embedded cells were incubated overnight in 1.85 M sucrose/20% PVP-10/50 mM Hepes pH 7.4. Each piece was mounted on an aluminum pin and snap frozen in liquid nitrogen. Ultra-thin sections were prepared using a cryo-ultramicrotome  with an F6 cryo-attachment and a Diatome cryoimmuno (35°) diamond knife. The sections were incubated with rabbit anti-GFP primary antibody  at 4°C overnight. The sections were then incubated with secondary antibody conjugated with 10 nm gold particles  and imaged as described above. Electron microscopy work was done by the Electron Imaging Center at California NanoSystems Institute, University of California Los Angeles.2 ROI. Four separate experiments were performed.Live cell imaging was performed 24 h post transfection of GST-GFP, GJA1-20k-GFP, or GJA1-20k-del6-GFP together with mito-BFP. Fresh imaging solution, containing either 25 μM nocodazole (to disrupt microtubules) or 0.08% DMSO, was added to cells for 45 min prior to image acquisition. Mito-BFP signal was tracked every 5 s for 5 min. To determine mitochondrial velocity and displacement, the MTrackJ plugin  to assess the goodness of fit: 0.93 (GST control), 0.95 (GST treated with nocodazole), 0.78 (GJA1-20k), and 0.85 (GJA1-20k-del6). Based on these values, data for GJA1-20k and GJA1-20k-del6 were fitted with the sum of two Gaussian distributions to yield R2-values of 0.91 and 0.97, respectively.All quantitative data were expressed as mean ± SEM and analyzed using Prism 6 software (GraphPad). For comparison between two groups, unpaired two-tail Student's r = 0.722). However, the more intense clusters do not appear to share ER localization. The larger GJA1-20k clusters either overlap with ER (arrowhead) or exist in shapes independent of ER (arrow). These results prompted us to determine the identity of the structures associated with GJA1-20k.In addition to the full-length Cx43 protein, endogenous GJA1-20k , HEK293 cells, isolated primary neonatal mouse glial cells and cardiac fibroblasts. As seen in Figure We have previously reported that GJA1-20k can organize the cytoskeleton trafficking pathways of Cx43  together with immunogold labeling of exogenous GJA1-20k to explore in high resolution the spatial relationship among GJA1-20k, mitochondria, and microtubules. Consistent with the confocal imaging results, mitochondria in GJA1-20k-expressing cells are closely associated with microtubules, stretching along them Figure . Moreove239RV240 and 247YHAT250) in MTBD have been found critical for microtubule/Cx43 interaction (Saidi Brikci-Nigassa et al., GJA1-20k, beginning with amino acid 213, contains a portion of the fourth transmembrane domain of Cx43, and the entire cytoplasmic C-terminal tail Figure . Like itBased on the association between GJA1-20k with mitochondria Figures . GJA1-20R2 fit was less than 0.9 and then double Gaussian curves were used to fit the data, Figure We next examined how the mutation of MTBD affects dynamics of mitochondrial transport in transfected cells. Mitochondrial movements were assessed by tracking individual mitochondrion over a 5-min period. Representative tracks are presented in Figure 2O2 for 4 h. Confocal imaging of cells expressing GFP-tagged GST, GJA1-43k, or GJA1-20k revealed a distributed mitochondrial network in all groups treated with PBS (Figure 2O2 treatment however, mitochondria became centralized and fragmented in cells expressing GST and GJA1-43k, indicating a damaged mitochondrial network. Interestingly, the majority of GJA1-20k transfected cells retained well distributed network and elongated mitochondrial morphology (Figure 2O2 stress (Figure 2O2 (Figure Since our results indicate that GJA1-20k mediates mitochondrial movement along microtubules Figures , we nextS Figure . Upon H2s Figure . We then2 Figure . These d2O2 stress. The results, in Figures In Figure The present study identified that GJA1-20k, produced via alternative translation from the same mRNA encoding gap junction protein Cx43, preferentially localizes to mitochondria and mitochondria/microtubule interfaces. We find that GJA1-20k facilitates microtubule-dependent trafficking of mitochondria to cell periphery, thereby preserving distributed mitochondrial network and preventing mitochondrial fragmentation induced by oxidative stress (depicted in the cartoon in Figure GJA1 mRNA in the heart (Smyth and Shaw, It is commonly accepted that, in eukaryotic cells, mature mRNA molecules encode and correspond to single protein products. In recent years, accumulating evidence has suggested that alternative translation start sites are highly conserved in the mammalian genome and that proteome diversity produced via alternative translation may be more important than previously thought (Kochetov, Alternative translation is a highly regulated process. In this study, we took advantage of HeLa cell lines with little endogenous expression of Cx43 which provides a clear background to study function of alternatively translated smaller isoforms. However, it is known that cellular stress may act as a trigger for alternative translation to generate isoforms that increase survival. For instance, the protein level of GJA1-20k in mouse primary astrocytes nearly doubled following exposure to intermittent hypoxia for 120 min (Ul-Hussain et al., Mitochondria form an extensively regulated and highly dynamic organelle network that is responsible for energy production, calcium homeostasis, cell signaling, cell growth and survival (Frederick and Shaw, 2O2 (Figure In multicellular eukaryotes, mitochondria move along microtubule tracks by attaching to motors such as kinesin for anterograde transport and dynein for retrograde transport (Hollenbeck and Saxton, 2 Figure . Through2 Figure  which reOf note and in additional to spatial mobility, mitochondria engage in repeating cycles of fusion and fission to intermix membrane lipids and matrix contents to ensure organelle network health and function (Ong et al., We have identified that GJA1-20k enriches at mitochondria and facilitates microtubule-based mitochondrial transport which preserves organelle network integrity upon oxidative stress. GJA1-20k, a protein that was only recently recognized to exist by alternative translation (Smyth and Shaw, YF, S-SZ, and SX contribute equally to the experimental design, acquisition, interpretation of data and drafting the manuscript. WB, RB, and IE contribute to experimental design, data acquisition and drafting the manuscript. TH and RS contributed to all aspects of this work. All authors agree to be accountable for accuracy and integrity of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We have studied the mobility of the multidomain folding catalyst, protein disulfide isomerase (PDI), by a coarse‐graining approach based on flexibility. We analyze our simulations of yeast PDI (yPDI) using measures of backbone movement, relative positions and orientations of domains, and distances between functional sites. We find that there is interdomain flexibility at every interdomain junction but these show very different characteristics. The extent of interdomain flexibility is such that yPDI's two active sites can approach much more closely than is found in crystal structures—and indeed hinge motion to bring these sites into proximity is the lowest energy normal mode of motion of the protein. The flexibility predicted for yPDI (based on one structure) includes the other known conformation of yPDI and is consistent with (i) the mobility observed experimentally for mammalian PDI and (ii) molecular dynamics. We also observe intradomain flexibility and clear differences between the domains in their propensity for internal motion. Our results suggest that PDI flexibility enables it to interact with many different partner molecules of widely different sizes and shapes, and highlights considerable similarities of yPDI and mammalian PDI. Proteins 2016; 84:1776–1785. © 2016 Wiley Periodicals, Inc. Proteins show internal mobility over a wide range of time‐ and length‐scales, from the rapid local motions that define the conformational entropy of a given protein conformation (<ns), to the functionally significant, concerted, slower motions (>ms) of loops or whole domains that interconvert different conformations.a‐b‐b′‐x‐a′‐c, where a and a′ are redox‐active trx‐fold domains, b and b′ are structurally similar domains lacking redox activity, x is an extended linker and c is a highly acidic C‐terminal extensiona′ and b′ domains, facilitated by the x‐linker.Saccharomyces cerevisiae) PDI (yPDI) at different temperatures produced two crystal structures which differ markedly in the relative orientation of the a and b domains.One of the large proteins studied intensely is protein disulfide isomerase (PDI), an abundant catalyst of oxidative protein folding in the endoplasmic reticulum of secretory cells Fig. . PDI is In this article, we have two purposes: (i) to explore and describe in quantitative detail the motions of PDI and (ii) to compare our predictions to the diverse experimental data on PDI flexibility. Our results give quantitative predictions of interdomain mobilitystochastic motion in a thermal bath as a function of time, but rather the possibility of motion along the most relevant low‐frequency elastic modes. Each trajectory leads to a gradual shift of the protein from the starting structure, as measured by Cα‐RMSD between original and derived structures, and this shift may reach an asymptote, where no further motion is possible along the initial vector, as a result of steric constraints.We use a recent protein flexibility modeling approach,+ ions have been added to neutralize the net charges on yPDI using the tleap module in Amber9. The MD simulations are performed under NPT conditions using the Berendsen thermostat and periodic boundary conditions. Particle Mesh Ewald summation is used for long‐range electrostatics with van der Waals cut‐off of 10 Å. The pressure and temperature relaxations are set to 0.5 ps−1. SHAKE constraints are applied to all bonds involving H atoms. A time step of 2 fs is employed with the integration algorithm and the structures are stored every 1 ps.As a consistency check of our flexibility approach, we have also undertaken short, all‐atom MD simulations of the intramolecular motion of yPDI at 300 K, employing the Amber9 suite of programsccd between Cα atoms of active site residues located in domains a (Cys61 in the full‐length sequence) and a′ (Cys406), respectively. This distance is 27 Å in 2B5E. In addition to ccd, we construct the following convenient measures of interdomain motion. Each of the a, b, b′, a′ domains contains a central β‐sheet, characteristic of the trx‐fold. The orientation of the domain can be represented by a vector normal to a central part of the β‐sheet. The variation of this vector between different β‐sheets can be described by interdomain tilt and twist angles. The normal vectors on each sheet are generated by selecting the Cα atoms of four “central” residues, namely alternating central residues in each of the adjacent antiparallel strands β2 and β4 . These four atoms define a quadrilateral and from its two diagonals we construct a normal vector n→. When we now consider two adjacent domains in the yPDI structure—labeled as domains 1 and 2, with central positions r→1, r→2 and plane normals n→1, n→2—we can define the tilt angle θ in the range 0 to 180 degrees as cos(θ)=n→1·n→2. The dihedral twist δ, with range from −180 to + 180 degrees, is obtained by constructing an interplane vector r→12=r→2−r→1 and considering the dihedral δ between the plane containing n→1, r→12 and the plane containing n→2, r→12 (see Supporting Information).We use a variety of quantitative structural measures to describe the intra‐ and interdomain motion in yPDI as observed in the flexibility and MD simulations. To indicate the scale of the relative motion of domains in our simulations, we measure the distance intradomain motion, we generate the ipseudodihedral ξi at Cα‐atom position r→i. We consider the interresidue pseudo‐bonds q→i−1,q→i,q→i+1 defined such that q→i=r→i+1−r→i for all residues i along the protein main chain (except for the first residue in the sequence and for the last two residues). The dihedral angle iξ formed by q→i−1 and q→i+1 about the axis of q→i then characterizes the orientation of the residues and their variation and flexibility. When the protein main chain is in a fully extended (β‐strand) state, iξ is near zero (cos(ξi)≈1). When the main chain is in a coiled or turned conformation (α‐helix or β‐hairpin), iξ has magnitude above 90°, and cos(ξi) is negative, typically around −0.7 for α‐helices. We extract cos(ξi) for each residue i and each conformer generated during a motion. We then use the root‐mean‐square‐deviation of all such cos(ξi) values as our measure of flexibility at residue i.To describe the a, b, b′, a′ domains as well as the x‐linker and the c terminal.m7–m11) which are the five lowest‐frequency nontrivial normal modes . We find that the motions are primarily motions of a and  a′ domains relative to a b‐b′ base. Figure m7 by showing superimposed structures representing the conformers computed at steps 0, ±2500 and ±5000. The central domains b and b′ form an essentially invariant base, while in comparison the N‐ and C‐terminal domains a and a′ show coordinated movement toward and away from each other, relative to their positions in the crystal structure; movement in the positive sense closes the structure, while that in the negative sense opens it. Figure m8 where the motions are essentially rotations of domains a and a′ around intradomain axes. For the next low‐frequency modes m9–m11, the motion can be roughly characterized as comprising combinations of hinge motions and rotations of domains .The starting point for our approach to mobility simulation of full‐length yPDI is the high‐resolution structure (pdb: 2B5E), which is used to generate a representation of the molecule as a set of rigid clusters and flexible linkers. The four‐domain structure thus obtained via First [cp. Fig. β‐sheet geometry for each domain and asked how constant these measures remained through the simulations of motion. For each domain, we selected a number of Cα atoms to define the sheet and extracted angles between sets of three atoms as measures of sheet geometry. We then derived the mean and standard deviation of these angles through the trajectories of motion obtained both by MD simulation and flexibility analysis. Figure β‐sheets of each domain retain essentially constant geometry throughout both sets of simulation .We next generated a relatively short molecular MD trajectory >30 ns  and interrogated the trajectory in a number of ways to compare it with the outputs from the computationally significantly less demanding flexibility approach. Both the flexibility and MD simulations generate trajectories comprising a series of all‐atom protein structures and hence both can be subjected to the same analyses. Initially, we asked whether we could confirm that the major features of motion were motions of whole domains. To test if the domains remained essentially intact as structural units, we obtained measures of Let us emphasize that, here and in the following, the aim of the relatively short MD runs is just to provide a consistency check on the flexibility results. Clearly, we would expect that the full range of motion found in, for example, Figure β‐sheet and calculated the relative orientations of these planes, tilt (θ) and dihedral twist (δ), through simulations of motion. Figure a‐b domain pair and shows that the MD simulation explores a considerable range around the 2B5E starting structure, but that all orientations fall close to a single line in twist/tilt space. Flexibility analyses in modes m7–m9 predict a similar pattern of motion, but modes m10  begin to explore other regions of twist/tilt space. In particular, motion along the positive direction of m10, converts the relative orientation of the a and b domains into precisely that found in the alternative “high‐temperature” crystal structure of yPDI ; this structure lies far away from the region explored in the MD simulation. The correspondence to the 3BOA structure provides experimental crystallographic evidence that our identification of the character of motion that is possible at the a‐b domain interface is correct. As the 2B5E and 3BOA structures are connected by flexible motion, we suggest that they are not separated by a major structural transition. Rather, they are both members of one wide‐ranging flexible ensemble in solution, from which the two crystal structures are selected by slight differences in crystallization conditions. A very similar phenomenon was recently identified in the domain orientation of ERp27.To provide a quantitative account of the large‐scale interdomain motion of this multidomain protein, we have represented each domain as a plane based on its core b‐b′ domain pair and indicates that MD and flexibility both explore the same limited region of twist/tilt space but with the flexibility simulation extending slightly further away from the orientations defined by the crystal structures. The data in the lower panel refer to the b′‐a′ domain pair and show different characteristics. The MD simulation covers an extended area of twist/tilt space while the normal mode trajectories form various paths through this area and also extend well beyond it; both simulations predict very extensive relative motion of this domain pair .The central panel shows comparable data for the ccd between active site residues located in domains a (Cys61) and a′ (Cys406). It demonstrates that movement along m7 allows ccd to vary freely from 15 to 80 Å due to the coordinated hinge‐like motion of domains a and a′ relative to b‐b′. Figure Figure ξm7–m10 and then derived the RMS variation of cosξ as a measure of the net flexibility at each residue. As shown in Figure x‐linker, with local maxima also shown at the a‐b and b‐b′ domain boundaries. This confirms that the major flexibility of the protein arises from the relative motion of domains but it also indicates that there is considerable intradomain motion. Figure ξ, as derived from the MD analysis. MD detects much more local and higher frequency motion and hence it is not surprising that the MD‐derived plot shows much greater scatter. Taking a running average >5 residues smooths some of this and again shows that the most marked motion is at the termini and at the x‐linker; motion at the other domain boundaries is not more marked than at several intradomain sites. Interestingly, both methods indicate much greater intradomain motion within the a′ domain than within the other domains.To focus more on local, intradomain motion, we have determined the pseudodihedral angle α‐Root mean square deviation between each of the domains and the structure of that domain in the initial full‐length protein [Fig. a′ showing the greatest RMSD and domains a and b showing the smallest. What also emerges from Figure b (in contrast to the other domains) begins a further phase of motion, which considerably increases its RMSD from the starting structure. For comparison, we analyzed the change in RMSD for each domain through the flexibility trajectories of modes m7–m11 .To obtain an alternative measure of the extent of intradomain motion through our simulations, we monitored the evolution of the MD trajectory >30 ns in terms of the Cm7 suggested that the molecule was able both to “open” and “close” relative to the starting structure, generating values for the intersite distance in the range 15–80 Å [Fig. m7 and used it as the starting point for the MD simulation. Figure Our analysis of motion along normal mode a and a′ domains show considerable freedom of motion with respect to a relatively fixed base provided by the b and b′ domains. This motion includes both rotations and hinge‐like opening and closing about the a‐b and b′‐a′ hinges. These findings on the scope for flexibility of yPDI are valuable in two respects: (i) they suggest that differences in the literature data on the mobilities of PDIs from yeast and other sources can easily be reconciled and (ii) they provide insight into PDI's cellular function as a catalyst of oxidative protein folding and its interactions with substrates and partner proteins.The main feature of PDI molecular motion predicted by both flexibility and MD approaches is that the redox‐active x linker region is a site of substantial flexibility and that its motion relative to the adjacent b′ domain can modulate access to the major noncovalent ligand binding site on b′ and alter the orientation of this domain relative to its neighboring catalytic a′ domain. A major change in relative orientation of these two domains was also inferred from NMR, X‐ray crystallographic, and Small‐angle X‐ray scattering studies on the reduced and oxidized states of the fungus H. insolensa and a′ domains, including between residues that are >50 Å apart in the crystal structure, providing dramatic evidence for extensive interdomain mobility in solution giving rise to structures much more compact than those observed in crystals.a and a′ domains were in close proximity.b′ and a′ domains, but show clear differences in the N‐terminal half of the protein, in the relative orientation of the a and b domains.a and a′ domains, with the motion of the a′ domain being more marked, but that the mode 10 trajectory involves a rotation of the a domain relative to the b domain which results in the orientation found in the higher temperature crystal structure. Our modeling of yPDI flexibility indicates that yPDI shows extensive mobility both in the a‐b and in the b′‐x′‐a′ regions, comparable to that observed experimentally in solution for other PDIs, but that the nonstatistical sampling of conformations provided by the crystallization has highlighted only the a‐b motion.A substantial body of work in human PDI and its fragments in solution, using techniques such as partial proteolysis, intrinsic fluorescence and high‐field NMRb′ domain facing in to the volume defined by the abb′xa′ “horseshoe.”a and a′ domains and the site on the b′ domain where protein ligands bind noncovalently. We have used the distance between redox active sites as the major indicator of this flexibility, but could also have used the volume enclosed within the PDI “horseshoe” or the distances between other functional sites. It is reasonable to infer that such a promiscuous protein as PDI will exploit its flexibility to interact with its wide range of partners that differ considerably in size and shape. Furthermore, the core of the ligand‐binding site is an exposed hydrophobic region on the b′ domain located on the inner surface of the “horseshoe.”b′ domain, we observe extensive intradomain flexibility [Fig. The extensive flexibility observed in our work also throws light on the most striking of PDI's functional properties, namely its wide substrate range—its ability to interact with diverse newly synthesized proteins destined for secretion. These substrates for oxidative protein folding  can differ widely in size, shape, and charge. Using the small model protein, basic pancreatic trypsin inhibitor, we recently showed that PDI interacts with this folding substrate at every stage along its oxidative folding pathway.b′‐a′, constrained to a specific twist mode at a‐b, and with no freedom to twist at b‐b′. There is also intradomain flexibility and clear differences between the domains in their tendency for such internal motion. The extent of the interdomain flexibility is such that the two active sites can approach much more closely than is found in crystal structures. Indeed, we find that hinge motion to bring these sites into proximity is the lowest energy normal mode of motion of the protein. Furthermore, the flexibility predicted for yPDI (2B5E) includes the other known conformation (3BOA) and is consistent with the mobility observed experimentally for mammalian PDI.The wide‐ranging and disparate experimental data that indicate the extensive and functionally significant flexible motion of PDI have now been combined with structurally detailed simulations which confirm the character and extent of this flexibility. Our results show that there is interdomain flexibility at every interdomain junction but showing very different characteristics, that is, extensive freedom to tilt and twist at Our flexibility‐based method provides quantitative measures, such as distances and angle variations, to be tested in the future by, for example, crosslinking and FRET experiments. In addition, as demonstrated here, the generated conformers can serve as starting points for more in‐depth MD runs, combining the remarkable computational economy of the flexibility approach with the biologically more detailed MD dynamics. Such a combination should be most interesting, for example, when applied to a study of the whole family of PDIMore details on the flexibility analysis and the MD approach, as well as the structural measures used, can be found in the Supporting Information.References Supporting Information 1Click here for additional data file.