Here we used single particle electron microscopy (EM) to determine a pseudoatomic model of the CaMKIIα holoenzyme in an extended and activation-competent conformation. The holoenzyme is organized by a rigid central hub complex, while positioning of the kinase domains is highly flexible, revealing dynamic holoenzymes ranging from 15–35 nm in diameter. While most kinase domains are ordered independently, ∼20% appear to form dimers and <3% are consistent with a compact conformation. An additional level of plasticity is revealed by a small fraction of bona-fide 14-mers (<4%) that may enable subunit exchange. Biochemical and cellular FRET studies confirm that the extended state of CaMKIIα resolved by EM is the predominant form of the holoenzyme, even under molecular crowding conditions.The Ca 2+/calmodulin-dependent protein kinase II (CaMKII) forms a 12 subunit holoenzyme central to synaptic plasticity. Here the authors report a 3D structure of the CaMKII holoenzyme in an activation-competent state obtained by single particle EM, and suggest a role for the intrinsically disordered linker domain in facilitating cooperative activation.Ca T286-phosphorylation has been described as a form of molecular memory, and is indeed required for normal learning and memory, as well as for two opposing forms of synaptic plasticity, long-term potentiation and depression4The CaBoth forms of molecular memory are enabled by the CaMKII holoenzyme structure, which is assembled by the association of 12 subunits via their C-terminal association domains . GluN2B binding is mediated by the kinase domain, but is dramatically more efficient for holoenzymes compared with monomers692+/CaM-stimulation. Thus, in order to make the holoenzyme competent for activation by Ca2+/CaM, it has been proposed that at least one alternative extended conformation must exist, even under basal-state conditions1182+/CaM-stimulation or be an intrinsic property of the activation-competent conformation of a holoenzyme that is present even in its basal state.Several crystal structures of individual CaMKII domains have been described17181920212+/CaM cooperative and thereby also fine-tune the frequency-dependent response of CaMKII to Ca2+-oscillation2+/CaM-stimulation has also been suggested. This alternative model is based on a previous crystal structure of isolated kinase/regulatory domains in which two kinase subunits dimerized via a coiled-coil interaction formed by their Ca2+/CaM-binding regulatory domains2+/CaM-binding sites are accessible for holoenzyme activation.For the compact CaMKII conformation, the biological occurrence and implications remains to be elucidated, especially in context of a naturally occurring full-length CaMKII that contains the linker region. However, it has been suggested that an equilibrium between the compact conformation and an additional activation-competent conformation would shape the CaMKII activation characteristics: it would make the stimulation by CaHere we set out to investigate the CaMKIIα holoenzyme structure and potential conformational equilibrium using a combination of single particle electron microscopy (EM) and functional mutation studies. Briefly, we describe a three-dimensional (3D) EM structure of an activation-competent CaMKIIα holoenzyme in an extended-state conformation at pseudo-atomic resolution. Single particle analysis revealed a high degree of flexibility for kinase domain positioning, with almost none of the individual particles completely matching the average structure. Within this continuum of conformational states, a compact conformation was found for <3% of the individual kinase domains, but various dimer-pair arrangements were suggested for as much as ∼20% of the kinase domains. Biochemical and cellular studies indicate that the extended state structures described by EM predominate even under conditions of molecular crowding, and point to an important role of the intrinsically disordered linker domain in facilitating cooperative activation for wild type holoenzymes.CaMKII holoenzymes were prepared for EM using negative stain . This prTwo-dimensional classification and projection averaging produced highly populated classes with well-defined hub assemblies and clear sixfold symmetry. However, the peripheral kinase densities were not resolved in these most populated classes. Rather, these domains appeared as a halo of density surrounding the central hub . The halFocused 2D classification of the hub domain was performed using a circular image mask (outer radius=75 Å). The vast majority of these masked particles classified as sixfold symmetric structures, with dimensions matching the crystallized dodecameric hub domain assembly2122A 3D EM density map was obtained at 20 Å resolution by using a combination of tilted-series tomographic imaging and 3D masking routines . A pseudThe model of the holoenzyme was completed by connecting the structured kinase/regulatory domains to the central hub complex by flexible linkers2+/CaM-stimulus. Therefore, the described structure represents an activation-competent state of the CaMKII holoenzyme.The pseudo-atomic model presented here provides a representative average view of the CaMKIIα holoenzyme in an extended (and activatable) conformation. In this form of the holoenzyme, the kinase domains are arranged independently from each other and removed from the central hub complex. The model implies that each kinase domain is tethered to the central hub complex by a flexible chain of amino acids (residues 301–344). Each kinase domain is separated from the central hub complex, extending the radius of the model to ∼135 Å. Neighbouring kinase domains are separated from each other by ∼60 Å (center-to-center distance). The co-planar arrangement of kinase domains is consistent with previous SAXS analysis19Based on single particle image analysis and 2D classification it is apparent that the flexible linker regions facilitate the formation of a variety of other conformational states that do not completely match the averaged 3D reconstruction. This conformational variability is appreciated when crystal structures of individual domains are fitted into the densities of individual particles images . To quanresiduesIn the first set of measurements, the radial extension for each kinase domain  was defined from the center of the hub complex to the outer radius of each kinase domain , inset. A prominent model of a linker-less CaMKII construct has been determined by X-ray crystallography, revealing a highly compact structure where all twelve kinase domains are packed tightly against the central hub complexC. elegansinset). This analysis yielded a Gaussian distribution of kinase domain separation distances, centered at ∼58 Å (±15 Å s.d.)  . This va Å s.d.) .In addition to these independent states, a significant population of neighbouring kinase domains (∼20% of the population) were found separated by <45 Å  . This clTo assess the various patterns of kinase domain pairing, we categorized the holoenzymes from our same individual image data set according to the number of putative kinase domain pairs, defined as having neighbouring kinase domains separated by <45 Å . This anC. elegans CaMKII kinase domain dimer fits well into the peripheral EM densities of this particular 2D class average  and a mildly increased Hill coefficient (∼1.5-fold) compared with full-length wild-type CaMKIIα, consistent with a compact conformation that decreases Ca2+/CaM binding and enhances cooperativity in solution50 was reported to increase equally by ∼3-fold for both wild type and I321E mutant, which was interpreted to indicate that the increase in the Hill coefficient (but not in the EC50) can be used as a readout of the compact conformationThe compact conformation CaMKII holoenzyme model is based on the crystal structure of a linker-less (LL) CaMKIIα mutant expressed in bacteria2+/CaM50. Thus, we first compared the effects of molecular crowding by 150 mg ml−1 lysozyme and the putatively more inert molecule bovine serum albumin (BSA)  . Consistr effect . Howeverr effect . Thus, f50 that was caused by crowding with BSA could be a reflection of a compact conformation, this experiment additionally compared the effects on the I321E mutant that is designed to be incompetent for the compact conformation. Again, the effects on human CaMKII and its I321E mutant were indistinguishable . Nonetheless, we decided to test if this one amino-acid difference could explain the different effect of molecular crowding on the Hill coefficient seen in our study with rodent CaMKII. However, the effects of molecular crowding with BSA on rodent versus human CaMKIIα were indistinguishable . In ordeuishable , indicat2+/CaM response for a linker-less CaMKIIα mutant (LL) that was described to be in the compact conformation even without crowding. We further compared this construct to a linker-less I321E combination mutant (LL+I321E) that has been shown to remain in the extended conformation2+/CaM sensitivity, both for activation and for T286 autophosphorylation  a pseudo-atomic model of the dodecameric CaMKII holoenzyme in its activation-competent extended conformation, (ii) a high flexibility of kinase domain positioning for this predominant extended state and (iii) an equilibrium with other conformational states that are activation-incompetent . The preOur 3D EM reconstruction shows a CaMKII holoenzyme in an extended activation-competent conformation at pseudo-atomic resolution, with each of the major domain components being well defined by high-resolution crystallographic structures. It should be noted that previous attempts at 3D CryoEM reconstructions have been made; however, the results were inconsistent with X-ray crystallographic structures of the organizing hub complex322+/CaM stimulation, or if it is already an intrinsic property of the holoenzyme in its basal state, as shown here. The presence of such dynamic behaviour even in the basal-state suggests an additional functional role, which we propose to be involved in shaping the activation properties of the holoenzyme.It was clear that kinase domain flexibility must exist in order to enable the inter-subunit autophosphorylation at T286 that generates autonomous CaMKII activity and is required for long-term synaptic plasticity. However, it was unclear if such flexibility needed to be induced by Ca2+-oscillations. Indeed, we found CaMKII activation with Hill Coefficients of 1.5–2. This is consistent with previous reports, and indicates that activation by Ca2+/CaM is a cooperative process2032in vitro or within cells). Indication for a compact conformation was found only for a linker-less CaMKII mutant, but not for full-length (wild type) CaMKIIα holoenzymes. Thus, we conclude that cooperativity of CaMKII activation must be based on additional mechanisms.It has been previously proposed that the cooperative activation profile of CaMKII is a result of a dynamic equilibrium between an activatable extended state and a non-activatable compact state2+/CaM-binding regulatory domains. Our results indicate that such dimeric-pairs may represent as much as 20% of the total subunit population; within single holoenzymes a variety of paired and unpaired arrangements appear to be present. For isolated kinase domains, such dimerization has been captured in a crystal structure of C. elegans CaMKIIKd 200–600 μM)  ref. . While tμM) ref. . Under mμM) ref. . One of Biochemical and EM studies have established a general consensus that the CaMKII holoenzyme is formed by 12 subunits33352+/CaM stimulation, and what organization takes place between local kinase domains to facilitate inter-subunit trans-activation and transition to autonomously active particles. Previous FRET studies suggest additional reorganization or expansion of the holoenzyme upon stimulation by Ca2+/CaM293969It remains unclear what other functional roles are enabled by the dynamic behaviour of CaMKII holoenzymes. For example, how are the various populations of conformational states affected by Caf9 cell expression and CaM was purified after bacterial expression. Briefly, CaM was purified by differential ammonium sulfate precipitation followed by phenyl-sepharose columnsg supernatants via two sequential column purification steps, a P11 phospho-cellulose column followed by a CaM-sepharose affinity column42Rat CaMKIIα wild type was purified after baculovirus/S2+-phosphate method44g for 20 min. The CaMKII concentration in the resulting supernatant was determined by quantitative western blot using a standard curve containing 250–1,250 fmole of purified recombinant CaMKIIα diluted in non-transfected HEK cell extract. Blots were probed for CaMKIIα expression using CBα2 antibody .Human CaMKIIα and CaMKIIα mutants were expressed in HEK-293 cells. The cells were grown to ∼50% confluence in 10 cm dishes, then transfected using the Ca32P incorporation into the peptide substrate Syntide-2. Reactions were started by adding purified CaMKIIα or HEK cell extract containing CaMKIIα to a final concentration of 2.5 nM in a mix of 50 mM PIPES pH 7.2, 0.1% BSA, 2 mM CaCl2, 10 mM MgCl2, 100 mM [γ-32P]ATP (∼1 Ci mmole−1) 1 μM microcystine-LR, 75 mM Syntide-2 and 3 nM to 20 μM calmodulin42−1 BSA or lysozyme, as indicated. Mixtures (50 μl) were reacted for 3 min at 30 °C. Reactions were stopped by adding 15 μl of ice cold 15% TCA, vortexing, and incubating on ice for 20 min. Reactions were then centrifuged at 16,000g for 20 min to remove precipitated protein. Thirty-five microlitres of the supernatant containing the peptide substrate was spotted onto P81 cation exchange chromatography paper (Whatman) squares. After extensive washes with water, phosphorylation of the substrate peptide bound to the P81 paper was measured by liquid scintillation counting. Results were plotted using Graph Pad Prism 5 and fit using a non-linear regression with variable slope.CaMKII activity was measured by 2, 10 mM MgCl2, 100 mM ATP, 1 μM microcystin-LR, 100 nM calmodulin. Kinases were reacted at 30 °C for 30 seconds, or as indicated. Reactions were stopped by addition of gel loading buffer  and boiling for 5 min. Samples were loaded on 10% SDS–polyacrylamide gel electrophoresis gels then transferred to polyvinylidene difluoride membranes424243To assess the ability of CaMKIIα WT and mutants to autophosphorylate at T286, the kinases were added at a final concentration of 20 nM to a reaction buffer containing 50 mM PIPES pH 7.2, 0.1% BSA, 2 mM CaCl2+/phosphate method, using a 4:1 ratio of mCherry over green fluorescent protein (GFP) constructs. Twenty-four hours after transfection, images of live cells were acquired at 32 °C on a climate controlled Zeiss Axiovert 200M microscope  at 100 × magnification using slide book 5.5 software (Intelligent Imaging Innovations) in imaging solution containing 0.87 X HBSS, 25 mM HEPES, 2 mM Glucose, 2 mM CaCl2, 1 mM MgCl2. FRET image acquisition and analysis were done by the three-filter ‘micro-FRET’ image subtraction methodC images, represented in pseudo-colour. The fractional subtraction coefficients are rounded up from average cross-bleed values determined in cells expressing GFP- or mCherry-tagged constructs alone. Thus, these coefficients result in a slight underestimation of FRETC signals for true FRET partners but limit false positive detection of FRET.HEK-293 cells (authenticated by short tandem repeat (STR) analysis and tested for mycoplams) were grown to 50% confluency on glass coverslips and then transfected using the CaC values depend both on FRET efficiency and on the amount of fluorophores present. Thus, in order to compare FRET among different cells, the FRETC values were divided by the intensity of the donor fluorophore. This simple normalization method is valid when the acceptor is in excess; here, only cells with 4–11-fold acceptor excess were included in the analysis. Acceptor/donor ratio was determined based on the detected GFP/mCherry signal combined with the 4.8 more efficient fluorescence detection of GFP in our setup (as determined in cells expressing a GFP-mCherry fusion protein).Absolute FRETImage acquisition was done based on the GFP and mCherry channels, that is, blind of the raw FRET channel. Image analysis was performed blind of the condition.f9 cell expression was prepared for negative stain EM by diluting the purified protein (1:40 vol vol−1) in EM buffer containing 50 mM HEPES (pH 7.4), 120 mM KCl and 0.5 mM EGTA. A 3 μl drop of sample (∼100 nM) was applied to a glow-discharged continuous carbon coated EM specimen grid (Ted Pella). Excess protein was removed by blotting with filter paper, and washing the grid two times with EM buffer. The specimen was then stained with freshly prepared 0.75% (wt vol−1) uranyl formate (SPI-Chem). Cryogenically prepared specimens were prepared by applying a 3 μl drop of sample (∼1 μM) to a negatively charged Quantifoil holey carbon specimen grid (Electron Microscopy Science). The sample was blotted with filter paper and plunged into liquid ethane using a vitrobot (FEI) and stored under liquid nitrogen.Full-length CaMKIIα purified from eukaryotic S−1 and a defocus of 1.5–2.5 μm. To overcome issues with particle orientation preference on the EM grid, serial tomographic images were collected at tilts angles of 0°–50° (Δ 10°)  at a nominal magnification of 49,000 × at the specimen level. Digital micrographs were recorded on a 2 K × 2 K CCD camera (FEI Eagle) with a calibrated pixel size of 4.37 Å pixel (Δ 10°) 47. Contr (Δ 10°)  and micr (Δ 10°)  and RELITo separate coexisting conformational states of the CaMKIIα hub domain (dodecamer or tetradecamer), a focused reference-free 2D classification was preformed in RELION using a subset of ∼8,000 un-tilted particles with a soft outer mask (75 Å radius) applied before classification. A set of 80 classes was produced and analysed to determine the relative populations of hub domains comprising of sixfold and sevenfold symmetry . The conCryogenically prepared CaMKIIα particles were imaged on a 300kV Titan Krios (FEI). Digital micrographs were recorded on a Falcon II direct electron detector (FEI) using low-dose imaging routines at a nominal magnification of 47,000 × and defocus of 3–5 μm. CTF correction and image processing routines were carried out in EMAN2, as described above. 2D class averages were obtained from a small dataset of ∼500 individual particle images.n=928) was obtained by measuring the distance from the center of the pore in the hub domain complex to the center of each peripheral density corresponding to the kinase domains. For each of these measurements, a distance of 22.5 Å was appended (corresponding to the average radius of the kinase domain) to yield a value that represents the full extension of the kinase domain. Inter-kinase domain separation distances were determined by measuring from the center of one peripheral kinase domain density to the center of the closest neighbouring density in the clockwise direction. Only densities that could be clearly resolved as individual kinase domains were incorporated into this analysis (n=883). The distribution of individual particle measurements were binned into 5 Å increments for histogram analysis . A standard Gaussian curve was fit to the distribution using the experimentally determined mean and s.d. Whisker plots were generated to represent the minimum, maximum and 25%, 50%, and 75% quartile values for each data set. A correlation map was obtained by scatter plot analysis of kinase radius versus kinase domain separation. All statistical analyses were performed in Microsoft Excel.Statistical analyses of individual particle dimensions were obtained by measuring particle lengths on un-binned micrographs using the measurement tool in EMAN2. A random set of 82 representative un-tilted individual particle images was inspected. A radius of extension for individual kinase subunits  was filmaxdiameter=∼125 Å). For the kinase domain refinement, the 3D mask was extended by 22 Å and inverted to remove density corresponding to the central hub domain during alignment. Both refinements proceeded with applied D6 symmetry. The hub-only refinement was determined to a resolution of ∼19 Å (gold standard FSC). For the kinase domain refinement, a subset of images was obtained from a combination of 2D and 3D classification performed in RELION. For 3D classification, six classes were generated with D6 symmetry imposed. Four of the six classes had clearly resolved kinase domains and the particles in these classes were selected for further refinement, resulting in a final resolution of ∼20 Å (gold standard FSC). These two maps were then combined using a spherical mask to remove overlapping densities (radius=75 Å). A final combined density map representing the full-length holoenzyme has been deposited to the EM Data Bank (EMD-8514).Separate refinements of the hub domain and kinase domains were performed in RELION by incorporating a 3D Gaussian mask based on the central hub feature produced by the initial 3D reconstruction in EMAN . For theA pseudo-atomic model of the full-length CaMKIIα holoenzyme was constructed by fitting the atomic coordinates of the dodecameric human CaMKIIα hub domain (PDBID 5IG3 ref.  residuesπr2) × (2πR)); where R and r represent the major and minor radius of the torus, respectively), as defined by the minimum and maximum kinase radius of extension determined in this work (providing R=126 Å and r=49 Å). This volume, representing the space occupied by kinase domains surrounding the central hub complex, was then used to estimate the local concentration of kinase domains for a dodecameric holoenzyme, resulting in ∼3.3 mM, which corresponds well to previously determined estimatesA model for calculating the local concentration of kinase domains within the CaMKII holoenzyme was based on calculating the volume of a torus . The pseudo-atomic model of the CaMKII holoenzyme fit to the EM density has been deposited with the Protein Data Bank (PDBID 5U6Y). The data that support the findings of this study are available from the corresponding authors upon request.How to cite this article: Myers, J. B. et al. The CaMKII holoenzyme structure in activation-competent conformations. Nat. Commun.8, 15742 doi: 10.1038/ncomms15742 (2017).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables and Supplementary References3D reconstruction and pseudo-atomic modeling of the CaMKIIα holoenzyme.


Apabetalone (RVX-208) is an epigenetic regulator developed to treat cardiovascular disease (CVD) that targets BET proteins. Through transcriptional regulation RVX-208 modulates pathways that underlie CVD including reverse cholesterol transport, vascular inflammation, coagulation, and complement. Using transcriptomics and proteomics we show that complement is one of the top pathways downregulated by RVX-208 in primary human hepatocytes (PHH) and in plasma from CVD patients. RVX-208 reduces basal and cytokine-driven expression of complement factors in PHH and in chimeric mice with humanized livers. Plasma proteomics of CVD patients shows that RVX-208 decreases complement proteins and regulators, including complement activators SAP and CRP. Circulating activated fragments C5a, C3b, and C5b-C6 are reduced by 51, 32, and 10%, respectively, indicating decreased activity of complement in patients. As complement components are linked to CVD and metabolic syndrome, including major acute cardiac events, modulating their levels and activity by RVX-208 may alleviate risks associated with these diseases.The online version of this article (doi:10.1007/s12265-017-9755-z) contains supplementary material, which is available to authorized users. The complement system is an essential component of the innate immunity and a major trigger of host inflammatory responses. As such, it helps immune cells fight infections and maintains homeostasis by eliminating pathogens, immune complexes, debris, and apoptotic cells. The complement system is composed of more than 30 proteins, some of which are secreted into the bloodstream by hepatocytes, while others are associated with plasma membrane of many cell types. Most of circulating complement proteins participate in activation of the complement cascade, serving as enzymes, enzyme cofactors, or precursors of biologically active fragments .Apabetalone (RVX-208) is an orally available small molecule developed for the treatment of cardiovascular disease (CVD) that targets epigenetic regulators bromodomain and extraterminal (BET) proteins BRD2, BRD3, and BRD4 . Mechaniv/v). Huh-7 cells (JCRB Cell Bank) were introduced to 96-well plates (∼2.5 × 105 per well) in 100 μL DMEM containing 10% (v/v) FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 5 μg/mL plasmocin . After 24 h, Huh-7 cells were treated with compounds dissolved in DMSO  and/or cytokines for 72 h. In a pre-treatment protocol, 24 h after plating, cells were pre-treated with cytokines for 24 h, before addition of compounds for 48 h. Fresh media containing cytokines and compounds were added to cells 24 h prior to collection of cells and media.Cryopreserved primary human hepatocytes (CellzDirect/Life Technologies) were plated and then overlaid with Matrigel™ as recommended by the supplier. Cells were treated with compounds dissolved in DMSO  and/or cytokines for the indicated time in media recommended by the supplier supplemented with 10% fetal bovine serum (www.qiagen.com/ingenuity). Activation z-scores were calculated using IPA and represent the bias in gene regulation that predicts whether the pathway exists in an activated or inactivated state.Cryopreserved primary human hepatocytes (CellzDirect™/ThermoFisher Scientific) were treated with DMSO (control) or RVX-208 at 30 μM for 48 h in triplicate. Total RNA was extracted with the mirVana™ kit (Ambion) and sent to Asuragen, Inc.  for microarray analysis using Affymetrix Human Genome U133 Plus 2.0 Array. Triplicate data was averaged to calculate fold change in gene expression relative to DMSO-treated controls. Data were analyzed through the use of QIAGEN’s Ingenuity® Pathway Analysis software . The level of the messenger RNA (mRNA) of interest was measured by TaqMan real-time PCR assays relative to the endogenous control cyclophilin A in a duplex reaction. Data were acquired using the ViiA-7 Real-Time PCR System (Applied Biosystems).ueous One Solution Cell Proliferation Assay (Promega)).Media from treated cell cultures were collected over the final 24 h of the experiment, flashed frozen in liquid nitrogen, and stored at −80 °C. C3, C4, C5, and C9 proteins were detected with ELISA kits as per the manufacturer’s instructions (AssayPro). Abundance of proteins secreted by Huh-7 and HepG2 were normalized to cell number (quantified with CellTiter 96® AQUrokinase-type plasminogen activator (uPA)/severe combined immunodeficient (SCID) mice with livers repopulated with human hepatocytes were generated as described previously  .The full design and rationale of the two studies, ASSERT NCT01058018) and ASSURE (NCT01067820), has been published previously 018 and A. The stuPreviously, RVX-208 was reported to downregulate the complement cascade in cultured PHH from a child donor . Here, wTo expand our understanding of BETi effects on the complement cascade, we assessed the effects of JQ1, a BET inhibitor with a chemical scaffold distinct from RVX-208, on PHH gene expression. Strikingly, JQ1 and RVX-208 showed the same pattern of complement gene repression between the two donors   mice with humanized livers . Using tMost complement proteins are synthesized by the liver and are classified as acute phase reactants, i.e., proteins of hepatic origin whose plasma concentrations increase following tissue injury or inflammation , 17. Thep < 0.05) or trending towards significant (p < 0.1) p value. Moreover, a substantial reduction of cleaved complement fragments that reflect complement activation was observed, including C3b , C5a , and C5b-C6  . Thus, Most complement factors participate in the acute phase response and are upregulated in response to infection and tissue injury as well as during acute and chronic inflammation . Those tProteomic analysis of plasma from RVX-208-treated patients showed a reduction in circulating levels of multiple complement components and regulators versus placebo, confirming that RVX-208 can modulate complement expression in humans. Although some factors were altered in both trials, several were identified in ASSERT but not ASSURE, and vice versa. This may be explained by differences in target population (stable versus unstable CAD) and trial duration (12 versus 26 weeks). In fact, patients enrolled in ASSURE displayed higher levels of CRP at baseline, indicating a higher inflammatory state as compared to patients from the ASSERT trial , again supporting the idea that RVX-208 modulates complement activation in vivo. C3b levels have not been previously correlated with CVD in clinical studies, but C3a and C3a-desArg, which are generated in the same proteolytic reaction, are linked to CVD risk factors both at early and late stages of the disease. Specifically, C3a is independently associated with markers of atherosclerosis  and predp = 0.01) in ASSERT and by 52% (p = 0.0001) in ASSURE, as compared to placebo. Further, APCS was reduced by 11% (p = 0.001) in ASSERT patients. Although CRP and APCS are not the sole complement activators, their decrease in RVX-208-treated patients may be linked to lower levels of downstream cleavage products. As an upstream activator of the complement cascade CRP correlates with circulating complement anaphylatoxins C3a and C5a [CRP and APCS are liver-derived, clinically relevant inflammatory markers from the pentraxin family that activate the complement system via the C1q complex . Inflamm and C5a . Convers and C5a . Thus, iIn addition to pentraxins, multiple other activators of the complement cascade were downregulated by RVX-208 in clinical samples including collectin-11, collectin-12, and properdin. Cascade inhibitors complement factor I, CD55, C1INH, and vitronectin were also reduced. Considering that complement activators and inhibitors form a complex network of interactions that involves multiple cell types [p = 0.01; data not shown). This observation is largely in agreement with gene microarray data from both PHH donors where the complement system was also ranked as a top canonical pathway downregulated by RVX-208 ESM 2(PDF 271 kb)


The p85α protein regulates flux through the PI3K/PTEN signaling pathway, and also controls receptor trafficking via regulation of Rab-family GTPases. In this report, we determined the impact of several cancer patient-derived p85α mutations located within the N-terminal domains of p85α previously shown to bind PTEN and Rab5, and regulate their respective functions. One p85α mutation, L30F, significantly reduced the steady state binding to PTEN, yet enhanced the stimulation of PTEN lipid phosphatase activity. Three other p85α mutations  also altered the regulation of PTEN catalytic activity. In contrast, many p85α mutations reduced the binding to Rab5 , and several impacted the GAP activity of p85α towards Rab5 . We determined the crystal structure of several of these p85α BH domain mutants  for bovine p85α BH and found that the mutations did not alter the overall domain structure. Thus, several p85α mutations found in human cancers may deregulate PTEN and/or Rab5 regulated pathways to contribute to oncogenesis. We also engineered several experimental mutations within the p85α BH domain and identified L191 and V263 as important for both binding and regulation of Rab5 activity. PI3K consists of a regulatory p85α protein and a catalytic p110 protein; their constitutive binding serves to stabilize the p110 protein and inhibit p110-PI3K activity2. The binding of p85α to newly formed phosphotyrosine sites on activated receptors, either directly or via adapter proteins, relieves the default inhibition of PI3K and also relocalizes p110-PI3K to the plasma membrane4. p110-PI3K phosphorylates phosphatidylinositol (PI) lipids such as PI4,5P2 converting it to PI3,4,5P3, an important lipid second messenger that recruits PH (pleckstrin homology) domain-containing proteins such as PDK1 (3-phosphoinositide-dependent protein kinase 1) and Akt to the plasma membrane5. Phosphorylation by PDK1 and mTORC2 activates Akt to phosphorylate numerous downstream target proteins that result in cell cycle progression, cell growth, migration and survival6.Phosphatidylinositol 3-kinase (PI3K) is an important signaling enzyme, acting downstream from many activated receptors, including receptor tyrosine kinases3 back to PI4,5P2. PTEN is differentially ubiquitinated, with monoubiquitination causing nuclear translocation, and polyubiquitination causing proteosomal degradation8. Nuclear PTEN has roles in chromosomal stability, DNA repair responses and cell cycle regulation9. Recent work has shown that in the absence of p110, the p85α protein homodimerizes10 and binds PTEN in response to growth factor stimulation to positively regulate PTEN lipid phosphatase activity11. Association with p85α has also been shown to reduce PTEN ubiquitination, thereby protecting PTEN from degradation and promoting PTEN protein stability12. Thus, p85α is a dual regulator of both the p110-PI3K and the PTEN-PI3 phosphatase controlling flux through the PI3K/PTEN pathway14.PTEN (phosphatase and tensin homologue deleted on chromosome 10) counteracts PI3K signaling by dephosphorylating PI3,4,5P15, and a BH (breakpoint cluster region homology) domain. The BH domain can bind to and regulate several GTPases including Rab5, Rac1 and Cdc4218. In addition, the p85α BH domain, either alone or together with the SH3 domain, can bind to and positively regulate PTEN activity11. The nSH2 and cSH2 domains of p85α bind to phosphotyrosine sites on upstream signaling proteins such as activated receptor tyrosine kinases (RTK)19. Between the SH2 domains is the p110 binding domain, also known as the inter-SH2 or iSH2 domain, and together with the nSH2 it binds p110 proteins20.The p85α protein has five domains that mediate interactions with different proteins Fig. . The N-t1. This occurs through gain-of-function mutations that cause activation of p110α, or through loss-of-function mutations or reduced levels of PTEN21. In addition, there are several cancer-associated p85α mutations in the C-terminal half of p85α that result in activated p110α24. These p85α mutations typically relieve the inhibitory effects yet retain the stabilizing interactions with p110α.The critical balance of PI3K/PTEN signaling controls the magnitude and duration of downstream Akt signaling and is frequently disrupted in many types of cancer11 and recent reports have now identified mutations within these regions in endometrial12 and bladder25 cancers. One endometrial cancer-associated mutation (I177N) was found to disrupt p85α homodimerization, resulting in reduced PTEN binding10. A second mutation (I133N) did not affect p85α homodimerization yet still showed reduced PTEN binding. Both mutations increased PTEN ubiquitination, and promoted downstream Akt phosphorylation10. Taken together, these results demonstrate that p85α directly binds to and blocks ubiquitination of PTEN to promote PTEN stability, and p85α also positively regulates the lipid phosphatase activity of PTEN. Mutations in p85α that reduce either of these effects could contribute to enhanced PI3K pathway activation and promote oncogenesis.Both the N-terminal SH3 and BH domains of p85α contribute to PTEN binding27. The BH/GAP domain of p85α is similar in sequence28 and structure30 to other GAPs, and mutation of the highly conserved arginine finger residue, R151, compromises its Rab5 GAP activity17. Overexpression of p85α in cells also results in decreased Rab5-GTP levels, consistent with its role as a Rab5 GAP in vivo31.The BH domain of p85α has GAP (GTPase activating protein) activity towards Rab GTPases Rab5 and Rab4, with key roles in vesicle tethering, which is important for receptor trafficking and down-regulation processes32, supporting its function as a Rab5 GAP. In addition, the p85α/p110β complex can compete with p85α for binding to Rab5-GTP, and this has been shown to block the Rab5 GAP activity of p85α32. A single point mutation within the BH domain (R274A) renders p85α Rab-GAP-defective and is oncogenic33 due to rapid trafficking of activated phosphorylated receptor complexes with reduced opportunities for sorting and lysosomal-mediated degradation34. Thus, mutations within the BH domain of p85α can be transforming through deregulation of Rab GTPase-mediated receptor trafficking.Free p85α can decrease the amount of Rab5-GTP in a dose-dependent mannerIn this report, we set out to determine if several p85α SH3 domain and BH domain mutations identified in human bladder and endometrial cancer tumors influence the binding and/or regulation of PTEN and Rab5.12 and bladder cancer samples25 identified a novel class of previously uncharacterized mutations within the N-terminal SH3 and BH domains of p85α  spectroscopy was used to evaluate possible changes in protein secondary structure , or GDP Fig. . Most of29 10. To determine if the patient-derived p85α BH domain mutations impacted the overall folded structure of the BH domain, several of these BH domain mutants were crystalized and their structures were determined using X-ray crystallography  and solved its structure to 2.25 Å resolution. X-ray diffraction data collection and structure refinement statistics for bovine p85α (105–319) wild type is provided in Supplementary Table 29 Fig. . These s29 Fig. . The burphy Fig. . X-ray d29. These residues included several that were highly conserved within the BH/GAP domain family , and the other that lacked GTPase activity and was thus locked in a GTP-bound conformation (Q79L)35. Several of the p85α BH domain mutations had little or no effect on Rab5 binding, including K187A, L267D (bovine residue corresponding to human I267), L271D (bovine residue corresponding to human M271)  caused a large reduction in binding to Rab5  Fig. . The p85ab5 Fig. . In eachab5 Fig. . These r11. To determine if p85α BH domain mutations that impact Rab5 binding and regulation  also influence PTEN binding, these p85α mutants were tested for their ability to bind to immobilized GST-PTEN in a pull-down assay; a double mutant p85α-L191D + V263D was also generated and tested 36. PIK3R1 mutations have been shown to occur at high frequency (25%) in endometrial cancer37 and at lower frequencies in several other cancers, including glioblastomas (7–10%38), colorectal cancers (8%23) and bladder cancer (6%25). Additional efforts have been made to determine the frequency and role of PIK3R1 mutations in cancer40. The most common p85α mutation sites include those within the nSH2 domain  and iSH2 domain 23. They are gain-of-function mutations resulting in a loss of p110α inhibition and enhanced PI3K signaling43.The 10. Other BH domain mutants stimulate PTEN activity either less (E137K) or more (K288Q) than wild type p85α, suggesting they could alter PI3K/PTEN pathway regulation. Consistent with the marked increase in the stimulation of PTEN activity for p85α-K288Q, cells expressing this mutant show reduced pAkt levels25.This study focused on characterizing several endometrial and bladder cancer patient-derived mutations located within the N-terminal half of p85α containing the SH3 and BH domains. The location of these mutations within each of these domain structures is shown in Fig. 29, but was in good agreement with a recent study suggesting that in solution BH domains do not typically interact44. Cheung et al. found decreased p85α-I177N dimer formation with wild type p85α, and decreased PTEN binding with a corresponding ~2–3 fold increase in pAkt in cells as compared to cells expressing wild type p85α10. It is possible that other protein partners or cellular factors may influence p85α dimerization and PTEN interactions in the context of these cell-based analyses that are not present when analyzing purified proteins.Our analysis using purified p85α and PTEN proteins showed that the p85α-I177N mutant retained its ability to both bind and regulate PTEN, similar to that of wild type p85α. This is in contrast to the proposed role for the I177 residue within the BH–BH dimer interface noted in the crystallized protein4, whereas dimeric p85α is required to bind, regulate and stabilize PTEN10. Given the importance of SH3 domain–proline-rich region interaction in mediating p85α dimerization, cancer patient-derived mutations within the SH3 domain might disrupt dimerization and thus the ability of the mutant protein to bind and/or regulate PTEN. Interestingly, the p85α-L30F mutant protein showed reduced steady state binding to PTEN, but this resulted in enhanced ability to stimulate PTEN lipid phosphatase activity. These contrasting results suggest that reductions in stable interactions between a p85α mutant and PTEN may increase the influence of the p85α mutant perhaps by allowing it to interact with more PTEN molecules in a given amount of time.Monomeric p85α has been shown to bind, regulate and stabilize p110α17. A G protein binding site was proposed within the human p85α BH domain29 that was used as a guide to identify the residues required for Rab5 binding and regulation. Subsequent studies on numerous G protein–GAP complexes confirmed that many of the residues are within the Rab5–BH domain interface28. Since there is no crystal structure of the p85α BH domain – Rab5 complex, we modeled this interaction after the known crystal structure of the Cdc42GAP – Cdc42 complex30 by aligning the structure of the human p85α BH domain with that of Cdc42GAP and the structure of Rab5 with Cdc42  or regulation  on one surface of the domain in close proximity to Rab5 near the bound GTP analogue , also observed for the R274A mutant17.Several bovine p85α BH domain mutations had little or no impact on Rab5 binding , suggesting that these residues are not required to mediate p85α BH domain–Rab5 contacts. Our previous mutational analysis of the p85α BH domain characterized the impact of R151A and R274A mutations on GAP activity and binding towards Rab530. The divergent residues are L191 (R in other GAP domains) and V263 (N in other GAP domains)  and yet are highly divergent from other GAP domains  and/or regulation  are shown in the modeled p85α BH domain: Rab5 complex Fig. . These mThe cancer-derived mutations within the SH3 domain of p85α  resulted in some reductions in Rab5 binding, with little impact on Rab5 regulation. Further, the L30F p85α mutation also affected PTEN binding. Given that the relative positions of the SH3 and BH domains within the ternary structure of the full-length p85α is not known, we can only speculate that the SH3 domain mutations may influence the interdomain interactions to impact the distinct PTEN and Rab5 binding sites within the p85α BH domain.45. Defects in Rab5 regulation can be oncogenic due to alterations in cell signaling pathways including that of PI3K33 and can contribute to changes in cell adhesion, migration, invasion and metastasis27. Some of the patient-derived mutations within either the N-terminal half of p85α (i.e. SH3 or BH domains) were able to alter Rab5 and/or PTEN regulation suggesting that they may contribute to oncogenesis and tumor progression, in addition to the more common p85α mutations within the C-terminal half that deregulate p110α-PI3K activity. Patients with N-terminal p85α mutations may require different therapies than those with C-terminal mutations as a result of their impact on Rab5 and/or PTEN instead of p110α-PI3K.In summary, we have defined key residues within the p85α BH domain that bind and/or regulate the Rab5 GTPase. Rab5 plays a key role in membrane tethering during receptor-endocytosis and trafficking46. The GST- p85α plasmid, encoding bovine residues 1–724, has been described47. The GST-p85α BH domain fragment was generated by PCR amplification of the GST-p85α cDNA and subcloned into pGEX6P1. After Prescission protease cleavage from GST, the p85α BH domain (residues 105–319) was 24 kDa. The GST- p85α plasmid contains full-length human p85α (residues 1–724), fused in-frame after GST. Site-directed mutagenesis of p85α was carried out using the QuikChange method (Stratagene), according to the manufacturer’s directions. DNA sequencing to ensure that no additional mutations had been introduced verified the entire p85α coding region. Generation of the GST-PTEN plasmid has been described11.The glutathione S-transferase (GST)-Rab5 mutants (S34N and Q79L) have been described11. Briefly, purified p85α protein  was precleared by incubating with GST (200 μg) immobilized on glutathione Sepharose beads overnight at 4 °C to remove non-specific binding proteins. Each pull-down experiment used GST or GST-PTEN (5 μg each) immobilized on glutathione Sepharose bead incubated with precleared p85α protein (5 μg) in 1% milk in a total volume of 500 μl PBS  for 30 minutes at room temperature. Beads were washed five times in 1 mL wash buffer . Each sample was resolved by SDS-PAGE and immunoblotted for p85α. Blots were quantified using the LI-COR Odyssey infrared imager, and the Odyssey v3.0 software was used to calculate the integrated intensity for quantification. Mutant binding values were normalized to that for wild type p85α binding on each blot by dividing the integrated intensity for the mutant by that for wild type p85α. Pull-down assays with immobilized GST or GST-Rab5 were carried out similarly after pre-loading the Rab5 with the indicated nucleotide according to the method detailed previously48. Bound p85α was detected after resolving samples by SDS-PAGE and transferring protein onto nitrocellulose, by immunoblotting with anti-p85α antibody . Secondary antibodies linked to Infrared-dyes  were used for detection and quantification using Odyssey software (v3.0). The results for all blots shown are representative of the results obtained from three independent experiments unless otherwise stated.Pull-down assays with immobilized GST or GST-PTEN were allowed to bind purified precleared p85α protein (after cleavage from GST)Circular dichroism spectra were recorded for the p85α BH domain mutants that showed reduced binding to PTEN as compared to the wild type p85α protein to ensure that protein folding was retained. Briefly, circular dichroism spectra from 200 nm to 280 nm  were obtained for p85α protein solutions (0.035–0.1 mg/ml) in 50 mM Tris pH 8, 150 mM NaCl, 100 μM TCEP (Tris(2-carboxyethyl) phosphine), 0.05% tween-20 using an Applied Photophysics Chirascan Plus CD Spectrometer at room temperature. At least four repeat scans were obtained for each sample and its buffer baseline. The averaged baseline spectrum was subtracted from the averaged sample spectrum and the net spectrum smoothed with Chirascan software.17. Rab5 (200 nM) was loaded with [α-32P]-GTP (0.89 pmol) in the presence of Mg2+ (10 mM) in GAP assay buffer . Rab5 was allowed to hydrolyze the bound nucleotide to [α-32P]-GDP for 20 min at room temperature either alone (no p85α) or in the presence of wild type or mutant p85α. A stop solution containing 1% SDS, 25 mM EDTA, 25 mM GDP, 25 mM GTP was added at a ratio of 1:6 to the reaction, which was then incubated at 65 °C for 2 minutes. Nucleotides were resolved by thin layer chromatography on PEI Cellulose F plates and quantified by the Typhoon FLA 7000 phosphorimager with ImageQuant TL software v8.1.0.0 . Each experiment was repeated three times. The amount of hydrolysis of GTP by Rab5 alone was used as a control and was subtracted from the experimental results. A range of concentrations of p85α was tested – 2 μM, 4 μM, 8 μM, 16 μM, and 32 μM – and the 8 μM experiment was used to compare p85α mutant GAP activity to wild type. The Rab5 GAP activity of wild type p85α was typically 0.23 ± 0.02 mmol GTP hydrolyzed/min/mol of p85α, similar to what has been reported previously17. The data is reported as relative GAP activity to wild type p85α.GAP assays were used to determine the enhanced rate of GTP-hydrolysis mediated by Rab5-GTP upon addition of 8 μM p85α protein6-PTEN (1 μM) with Di-C8-PI3,4,5P3 lipid  in the presence of wild type or mutant p85α (7.5 μM) in a final volume of 10 μl in phosphate release buffer . All incubations were performed at 37 °C for 20 min and reactions were stopped with the addition of 100 mM N-ethylmaleimide . For detection, 20 μl of the reaction was combined with 80 μl BIOMOL Green (Biorad) in a 96-well plate and the color was allowed to develop for 20 min at room temperature. The absorbance was measured at 620 nm with a microplate reader. Each data point was assayed in duplicate, and all experiments were repeated five times. Values for buffer controls were subtracted from those for experimental samples and reported relative to PTEN activity in the presence of wild type p85α.A phosphate release assay was used to determine the effects of wild type and mutant p85α on PTEN activity. Assays were performed by incubating HisE. coli (BL21 DE3) expression of the bovine GST- p85α BH domain (105–319) wild type and mutants were induced with isopropyl ß-D-1-thiogalactopyranoside  overnight at 25 °C17. Cell were pelleted and resuspended in lysis buffer benzenesulfonyl fluoride hydrochloride (AEBSF)) with 1 µg/mL lysozyme, incubated at 4 °C for 1 hour. Sample viscosity was reduced via sonication on ice. Cell debris was pelleted by centrifugation and the supernatant was filtered through 0.8 µm Nalgene syringe filters (Thermo Scientific). GST-p85α protein fragments in the supernatant were incubated with glutathione Sepharose high performance media  using an ÄKTA Purifier system. The sample was injected onto the column in phosphate buffered saline , using a flow rate of 1 mL/min, pressure limit of 0.5 MP. The column was washed until the absorbance at 280 nm was less than 20 milli absorbance units. GST-p85α proteins were eluted from the column using 50 mM Tris pH 8.0, 150 mM NaCl, and 10 mM reduced glutathione. Fractions containing protein were pooled and dialyzed for 16 hours in Spectra/Por molecular porous membrane tubing  against Prescission protease buffer . The p85α fragments were cleaved from GST by the addition of PreScission protease  for 72 hours at 4 °C, according to the manufacturer’s instructions. GST was removed from the samples by passing the sample through the HR 16/10 glutathione Sepharose column as before, collecting the desired p85α protein fragments in the flow through.The p85α samples were buffer exchanged using Millipore Amicon 10 kDa centrifugal filter units into buffer A  and loaded onto a Source Q column . Bound protein was eluted using buffer B  across 3 column volumes while collecting 1 mL fractions. Fractions were analyzed using SDS-PAGE analysis and Coomassie Blue staining to verify protein purity and yield. Fractions containing purified protein were pooled, concentrated and buffer exchanged into the desired storage buffer using Millipore Amicon 10 kDa centrifugal filter units. Proteins were stored at 4 °C.2SO4, and 4–8% Glycerol.Identification of initial crystal conditions was performed via preparation of hanging drop vapor diffusion screens of commercially available sparse matrix screening kits using 15 mg/mL p85α BH domain. These sparse matrix screens were set up using a GRYPHON robot (Art Robbins Instruments) which generated 0.4 µL drops by mixing 0.2 µL protein solution in crystal holding buffer  with 0.2 µL crystallization mother liquor in 96-well 2-well intelliplates (Art Robbins Instruments) with 100 µL of crystallization mother liquor deposited in the well reservoir. Conditions that yielded crystals for the p85α BH fragment and mutants were 0.1 M Sodium Cacodylate pH 6.0–6.2, 1.3–1.7 M Li49, or the Canadian Macromolecular Crystallography Facility 08ID beamline 50. Cryoprotectant solution  was added to the sample crystal drops prior to crystal harvesting and data collection.X-ray diffraction data were collected at the Canadian Light Source  using the Canadian Macromolecular Crystallography Facility 08B1-1 beamline (bending magnet)51 (Supplementary Table X-ray diffraction data (180°) were collected with a detector distance of 280 mm, in 0.5° or 1° wedges. X-ray detectors equipped at the beamlines were a Rayonix MX300 HE CCD for the CMCF-BM and a Rayonix MX300 CCD for the CMCF-ID. Processing of collected data was performed using HKL2000 softwarePHENIX software52. As the human p85α BH domain crystal structure has previously been solved29 we used molecular replacement with the human p85α BH domain (1PBW) as the initial model. Two molecules of the bovine p85α BH domain were located using PHASER. Starting atomic coordinates (with appropriate amino acid substitutions) were iteratively rebuilt using Coot and refined with PHENIX and Refine. Repeated cycles of phenix.refine and manual adjustments in Coot, including placement of solvent, were performed to further improve the structure. After refinement of the bovine wild type p85α BH structure, this was used as the starting structure for refinement of the p85α BH point mutant structures. Amino acids 113–297 of p85α were visible forming a homodimer within the crystal lattice. Residues 169–171 and 277–279 did not have visible electron density in either chain, and Pro 276 did not have visible electron density in Chain A. The root-mean-square difference on backbone atoms (RMSD) was determined for the overlay of the backbone atoms of the bovine and human p85α BH domain A chain monomers using Lsqkab in the CCP4 suite of programs. There was extra electron density at residue C146, consistent with the presence of either a sulfenic acid or S-nitrosocysteine residue. Hence the model contains sulfenic acid at this position. All B-plots were calculated using the CCP4 program “Baverage” using the final structures for the p85 BH Wild-Type, E137K, E217K, R262T, or E297K as input data.Structure determination was performed using Figures were generated using the PyMOL Molecular Graphics System .P < 0.05.Assay results are expressed as means ± standard errors from at least 3 independent experiments, unless otherwise indicated. One-way analysis of variance (ANOVA) with post hoc Bonferroni tests were used for multiple comparisons with differences considered as statistically significant if http://www.rcsb.org/pdb/explore.do): bovine p85α BH domain: wild type PDB ID# 6D81 and mutants E137K (PDB ID# 6D82), E217K (PDB ID# 6D85), R262T (PDB ID# 6D87) and E297K (PDB ID# 6D86). The materials generated and/or protocols used during the current study are available from the corresponding author on reasonable request.Atomic coordinates and structure factors have been deposited at the RCSB depository (Supplementary Information


Most two-dimensional (2D) covalent organic frameworks (COFs) are non-fluorescent in the solid state even when they are constructed from emissive building blocks. The fluorescence quenching is usually attributed to non-irradiative rotation-related or π–π stacking-caused thermal energy dissipation process. Currently there is a lack of guiding principle on how to design fluorescent, solid-state material made of COF. Herein, we demonstrate that the eclipsed stacking structure of 2D COFs can be used to turn on, and tune, the solid-state photoluminescence from non-emissive building blocks by the restriction of intramolecular bond rotation via intralayer and interlayer hydrogen bonds among highly organized layers in the eclipse-stacked COFs. Our COFs serve as a platform whereby the size of the conjugated linkers and side-chain functionalities can be varied, rendering the emission colour-tuneable from blue to yellow and even white. This work provides a guide to design new solid-state emitters using COFs. Encoding functionalities in covalent organic frameworks (COFs) is important for widening their application field but the development of fluorescent COFs is hampered by a lack of guiding design principles. Here the authors demonstrate tuning and switching of the photoluminescence in 2D COFs made of non-emissive building blocks. Guided by reticular chemistry, tremendous efforts have been directed towards developing highly crystalline COFs with different topologies such as two-dimensional (2D)13, three-dimensional (3D)15 and woven structures17. Meanwhile, the ease of encoding functionalities into building units makes COFs useful for wide-ranging applications such as sensing22, gas storage24, photocatalysis25, asymmetric catalysis29, chemical separation31, energy storage35 and photodetection36. However, most fields view COFs as merely a polymer platform and overlook the unique features of COFs such as their periodic layered structures, and this has given rise to the non-differentiated performance of COFs, amorphous polymers and even their building units.Covalent organic frameworks (COFs) allow molecular building blocks to be integrated into extended architectures via covalent bonds39. When an aggregation-induced emission (AIE) chromophore such as tetraphenylethene was connected via a boronate ester, a highly emissive 2D COF was obtained. Unfortunately, the solid-state PL was quenched when the AIE building unit was linked by imine bonds due to its rotationally labile nature under photoexcitation40. A similar phenomenon occurred when 2D COFs were constructed with non-AIE chromophores such as pyrene. In fact, the type of linker between the chromophores in the 2D COF strongly affects its ability to be fluorescent. Thermal dissipation via rotationally labile linkages, for example, the commonly used imine linkages, induces non-radiative decay of excited states40. Therefore, it is vital to restrict the bond rotation of the 2D COF linkages to induce solid-state PL.In layered materials, π–π stacking leads to aggregation, which causes photoluminescence (PL) quenching. Previously, highly emissive building units, such as pyrene and tetraphenylethene, have been used to construct solid-state photoluminescent 2D COFsOn the other hand, 2D COFs provide the equivalent of a high-viscosity microenvironment that limits rotational/vibrational relaxation. When properly designed, the eclipsed stacking structure of 2D COFs can block the non-radiative energy decay pathway through restriction of intramolecular bond rotation (RIR), thereby enhancing fluorescence. In addition, the molecular building blocks can be interconnected as donor–acceptor pairs (dyads) that are not conjugated, or the conjugation in the scaffold can be extended using aromatic linkers, leading to donor–acceptor excimer energy transfer and a large Stokes shift, respectively. The combination of these factors can be used to tune the intensity and energy of the fluorescence, but their effects have not been studied systematically in COFs.To date, there is a lack of guiding principles on how solid-state photoluminescent COFs with tuneable emissive properties can be constructed. Here, we demonstrate a molecular design strategy for obtaining solid-state PL from a hydrazone-based COF that allows strong emission intensity and wide colour tuneability. Starting from a non-emissive building unit Fig. , PL can 25, the interplay between intralayer and interlayer hydrogen bonding in eclipsed stacked COFs has never been studied. Based on density functional theory (DFT) simulation studies, the varying degrees of intralayer and interlayer hydrogen bonding affects whether the COFs is single or dual-emissive  and proton acceptor (C–O–C) (both intra- and interlayer) in close proximity, which means that the intra- and interlayer hydrogen bonds can form the basis of an excited-state proton shift mechanism. Third, a Stokes shift can be induced by incorporating a conjugated linker. Fourth, dual emission with a large Stokes shift is obtained by modulating the intra- and interlayer hydrogen bonding by adding side-chain functionalities Fig. . Last buive Fig.  and 32. The synthetic scheme for accessing the tuneable white-emissive COFs is illustrated in Fig. Rp, Rwp) values of , ,  and  for Tf-DHzDPr, Tf-DHzDAll, TFPB-DHzDPr and TFPB-DHzDAll, respectively. As shown in Fig. P3 space group with Pawley refined cell parameters of a = b = 29.37 Å, c = 3.44 Å for Tf-DHzDPr; a = b = 29.35 Å, c = 3.43 Å for Tf-DHzDAll; a = b = 42.28 Å, c = 3.56 Å for TFPB-DHzDPr; and a = b = 41.85 Å, c = 3.50 Å for TFPB-DHzDAll. Simulation of the staggered stacking mode of the same space group was also conducted for each COF, but the calculated PXRD pattern was not consistent with the experimental result , nitrogen sorption isotherms, FT-IR spectroscopy and elemental analysis  surface areas are 1035, 760, 432 and 569 m2 g−1 for Tf-DHzDPr, TFPB-DHzDPr, Tf-DHzDAll and TFPB-DHzDAll, respectively, suggesting that the four COFs have good to moderate porosity. In addition, the total pore volumes are estimated to be 0.77, 0.63, 0.49 and 0.45 cm3 g−1 at P/P0 = 0.99 for Tf-DHzDPr, TFPB-DHzDPr, Tf-DHzDAll and TFPB-DHzDAll, respectively. The pore distributions of the four COFs were calculated by non-local density functional theory (NLDFT), which gave a narrow pore width of 1.69 nm for Tf-DHzDPr and Tf-DHzDAll and a width of 2.77 nm for TFPB-DHzDPr and TFPB-DHzDAll. These values are consistent with the simulated values of 1.74, 1.85, 3.04 and 3.07 nm for Tf-DHzDPr, Tf-DHzDAll, TFPB-DHzDPr and TFPB-DHzDAll, respectively.The porous structures of the COFs were further investigated by nitrogen adsorption–desorption experiments at 77 K Fig. . The sorThe morphologies of each COFs were characterized by Scanning electron microscopy and Transmission electron microscopy  were conducted at this excitation wavelength unless otherwise specified. To elucidate the relationship between the structures of the COFs and their optical properties, model compounds were obtained from the condensation of hydrazide and benzaldehyde monomers  through proton transfer, and both tautomers contribute to the dual emission bands. However, in our COF building blocks, the proton acceptor (C–O–C) is not nucleophilic enough to fully capture the hydrogen from the proton donor (N–NH), which disfavours the ESIPT process COF Fig. . One reaing Fig. . Based oWhen the hydrazides are bridged with a larger spacer such as TFPB, the resulting COF, TFPB-DHzDR, show a redshift of 50–70 nm relative to the emission of the Tf-COFs, which are connected via a smaller spacer. This suggests that extending the conjugation size is effective to redshift PL emission in such COFs. Similarly, the TFPB-DHzDR COFs  also exhibit broad emission bands in the visible region. The TFPB-DHzDAll COF shows a very broad band with a shoulder at 490 nm instead of a dual-emissive band. This is probably because the first emission is redshifted into the ESIPS emission region due to the extended conjugation via TFPB. To further verify the role of the proton shift in modulating the emission properties, we synthesized two COFs incapable of hydrogen bonding, namely TFPB-THz and DFDM-THz. Although the condensation product formed between trialdehyde and 2,5-unsubstituted terephthalohydrazide (DHz) is more structurally related to DHzDR COFs, we could only obtain amorphous polymer instead of crystalline COF. Thus, we chose TFPB-THz COF and DFDM-THz COF for comparison with hydrogen bonded DHzDR COFs. As expected, they display much narrower single emission bands compared to the DHzDR COFs. Based on these results, the hydrogen bonding between the COF layers is vital to the broadening of the emission band. The interlayer proton shift in the excited state may create numerous energy states, which in turn give rise to the broadened band or even dual emission. The PL lifetime of each emissive COFs was also recorded as shown in Supplementary Figs. ΦPL = 11.9% and 3.9%) than their amorphous counterpart (ΦPL = 2.1% and 2.1%)  Fig. , which iThe CIE-1931 chromaticity diagram reveals the true colour of the emissions of the tuneable emissive COFs Fig. . By tuni3-hybridized alkyl chain to a π-conjugated allyl group, dual emission can be switched on with a large Stokes shift to give near white emission.Using these 2D COF platforms, the optical properties can be systematically tuned by modifying the spacer unit and side chains. By extending the conjugation length of the spacer unit used to link the hydrazides species, the emission of the COFs can be tuned from blue to green with a slight increase in the PLQY. Given the same spacer size, the length of the side chain has no effect on the emission colour; however, the PLQY tends to increase when the side chain is lengthened. Most significantly, when the functionality of the side chain is changed from an sp48. The design and synthesis are focused on two COFs, Tf-DHzDAll and TFPB-DHzDAll, with CIE coordinates of  and  and PLQYs of 3.9% and 3.6%, respectively. For Tf-derived COFs, the spacer is reacted with 2:1, 1:1 and 1:2 mole ratios of DHzDAll and DHzDPr, and the synthesized compounds are referred to as Tf-DHz(x)DAll(y)DPr. Similarly, TFPB-COFs are constructed between the spacer TFPB and 2:1 and 1:2 mole ratios of DHzDAll/DHzDPr or DHzDAll/DHzDS. The PXRD spectra confirm the successful synthesis of multicomponent COFs based on the characteristic peaks at 3.46 and 2.39° for the (100) facets of the Tf-COFs and TFPB-COFs, respectively. For Tf-derived multicomponent COFs, the PL emission exhibits a blueshift with an increasing ratio of DHzDPr, giving rise to CIE coordinates of ,  and , which are closer to the white point of . Meanwhile, the absolute PLQY improved from 3.9% to 7.3%. For the TFBP-derived multicomponent COFs, instead of giving two emission maxima, the emission from DHzDAll and DHzDS/DHzDPr merged into one peak at approximately 500 nm, which corresponds to an off-white, light-green colour with CIE coordinates of , ,  and . Similarly, compared to TFPB-DHzDAll, the PLQY increased from 3.6% to 11.8%.To achieve broadband PL emission with high PL efficiency, we propose the following strategy for constructing multicomponent COFs Fig. , 1648. TCompared to the RIR-emissive crystals of small molecules, a COF has several advantages: 1. Its extended conjugated structure offers COF materials greater colour tuneability than small emissive units. 2. COFs are more resistant to solvents than small-molecule crystals and are thus more stable. 3. Slip-packed crystals of small molecules can have only intramolecular hydrogen bond, while eclipsed-stacked COFs can have both intra- and interlayer hydrogen bonding in close proximity, which forms the basis for ESIPS. Therefore, COFs allow a wide chemical space for the design of new solid-state emitters for use as photosensitizers, sensors, lighting, displays and supramolecular encryption.We have demonstrated a strategy to enhance the solid-state PL of COFs by taking advantage of the RIR mechanism. The eclipsed stacking in 2D COFs containing hydrazone units is accompanied by interlayer hydrogen bonding, which provides additional channels for restricting RIR in addition to intralayer hydrogen bonding. Modulating the interlayer and intralayer hydrogen bonding allows access to excited states with different de-excitation pathways. Through this strategy, we observed dual emission in the blue and yellow wavelengths. By varying the spacer size and side-chain functionalities in this COF series, we can tune the intensity and colour of the emission and achieve near white PL emission. Through construction of multicomponent COFs, the emission colour can be further fine-tuned. This study provides new insights into how molecular building blocks in a 2D stacked framework, as well as their linkages and functional groups, can be tuned to achieve synergetic interactions for colour-tuneable PL emission, thereby providing the basis for designing new solid-state emitters.A mixture of 2,5-Bisterephthalohydrazide (DHzDAll)  and 1,3,5-triformylbenzene (Tf)  in mesitylene/1,4-dioxane  was sonicated for 10 min in a Schlenk tube (15 mm × 80 mm). Then the mixture was added with acetic acid , flash frozen at 77 K, and degassed under freeze–pump–thaw for three cycles. The tube was sealed and heated at 120 °C for 3 days. The precipitate was exchanged with anhydrous tetrahydrofuran (THF) (5 mL) for 10 times and dried at 100 °C under vacuum for 8 h to afford a beige colour solid .All data are available from the authors upon reasonable request.Supplementary InformationPeer Review File


Rg) and the fractions of native contacts (Q), the formation of the secondary structure of the I27-guest domain induces significant extending of the GL5-host domain; but the primary shrinking of the tertiary structure of the I27-guest domain causes insignificant extending of the GL5-host domain during the processes. The results indicate that only formation of the secondary structure in the I27-guest domain provides the main driving force for the mutually exclusive folding/unfolding between the I27-guest and GL5-host domains. A special structure as an intermediate with both host and guest domains being folded at the same time was found, which was suggested by the experiment. The analysis of hydrogen bonds and correlation motions supported the studied transition mechanism with the dynamical “tug-of-war” phenomenon.Mutually exclusive folding proteins are a class of multidomain proteins in which the host domain remains folded while the guest domain is unfolded, and both domains achieve exchange of their folding status by a mutual exclusive folding (MEF) process. We carried out conventional and targeted molecular dynamics simulations for the mutually exclusive folding protein of GL5/I27 to address the MEF transition mechanisms. We constructed two starting models and two targeted models, i.e., the starting models GL5/I27-S and GL5/I27-ST in which the first model involves the host domain GL5 and the secondary-structure unfolded guest domain I27-S, while the second model involves the host domain GL5 and the secondary/tertiary-structure extending guest domain I27-ST, and the target models GL5-S/I27 and GL5-ST/I27 in which GL5-S and GL5-ST represent the secondary-structure unfolding and the secondary/tertiary-structure extending, respectively. We investigated four MEF transition processes from both starting models to both target models. Based on structural changes and the variations of the radius of gyration ( The conformational transitions of some proteins play a crucial role in regulating protein function, and revealing fundamental biological processes of cell signaling and metabolism ,2,3. Man9k protein were investigated by using the targeted molecular dynamics simulations in our previous work [Mutually exclusive folding proteins are of particular interest in studies of protein folding/unfolding dynamics, and have been studied by many researchers ,22,23,24ous work . HoweverRg) and the fraction of native contacts (Q); (3) to explore the nature of the main driving force for mutually exclusive folding/unfolding processes.In order to explore the mutually exclusive folding mechanism at the atomic level, we carried out conventional molecular dynamics (CMD) and targeted molecular dynamics (TMD) simulations to investigate mutually exclusive folding transition processes for the GL5/I27 fusion protein. Our main objectives were (1) to address the structural property of transition processes with different unfolding degrees of GL5 and I27 domains; (2) to investigate the transition regularity for these processes by analyzing variation of the radius of gyration (i/I27i (i = S or ST), with the different unfolding degrees of the I27-guest domain or the GL5-host domain were designed from the Nuclear Magnetic Resonance (NMR) structures of I27 (PDB:1TIT) and GB1 (PDB:2GB1) proteins [vaI linker site in order to be inserted by the I27-guest domain (89 residues). Therefore, the constructed mutually exclusive folding protein model consists of the GL5-host domain , the I27-guest domain (Leu45–Leu133), and the linker  between the GL5-host domain and I27-guest domain  and the Discovery Studio visualizer (http://accelrys.com/). The details of the model construction are given in the The normal mutually exclusive folding protein contains a guest protein inserting into the certain loop region of a host protein with folded/unfolded structures. Based on the previous experimental studies, the mutually exclusive folding protein models, GL5proteins . The GL5main see . The twoFor constructing a special class of the mutually exclusive folding proteins GL5/I27 with the guest domain I27 inserting into the host domain GL5, four protein models of GL5/I27-S, GL5-S/I27, GL5/I27-ST, and GL5-ST/I27 with the different unfolding degrees of the guest and host domains were constructed. Based on these constructed models, the 50-ns CMD simulations were first carried out to understand the stability of these structures. The root mean square deviation (RMSD) values of all backbone atoms relative to the NMR data for the native domains except for the atoms in the loop regions over the corresponding trajectories for the four models were examined to determine the system equilibriums, and are shown in 1–2 (Gly9–Thr11), loop2–α , loopα (Gly38–Gly44), loop3 , and loop3–4 (Asp143–Thr145). Its structure presents one layer consisting of four parallel strands in the order of β2–β1–β4–β3, and the other layer consisting of the α1 helix [A (Leu45–Gly54), loopA–B (Phe58–Glu61), loopB–C (Leu69–Gly76), loopC–D (Leu80–Asp90), loopD–E (Asp96–Lys98), loopE–F (Asn106–Thr112), loopF–G , and loopG (Lys131–Leu133) [The four corresponding average structures of GL5/I27-S, GL5-S/I27, GL5/I27-ST, and GL5-ST/I27 models are shown in –Leu133) . The ext2). The plots for the RMSDs of all backbone and heavy atoms in the TMD-simulated structures relative to the corresponding target states are shown in Rg) and the fraction of native contacts (Q) in a protein are respectively used to measure the degrees of the unfolding and similarity of a protein to its native state (defined as Q = 0 or 1 at a fully unfolded state or a native state) [Rg) and the fraction of native contacts (Q) for both I27-guest and GL5-host domains along the two TMD simulations of both GL5/I27-S and GL5/I27-ST models to GL5-ST/I27 model were extracted from the corresponding trajectories by using the PTRAJ module of AMBER 9 program, and are shown in To address the mutually exclusive folding characteristics for the full extending of the GL5-host domain, we successfully gained the mutually exclusive folding processes from the starting GL5/I27-S and GL5/I27-ST models with the secondary-structure unfolding and the secondary/tertiary-structure extending of the I27-guest domains respectively to the target GL5-ST/I27 model with the full extending of the GL5-host domain by performing the 10-ns TMD simulations with the bias force constant of 3.0 kcal/(mol·Åe state) ,46,47,48Rg) in Rg values of the I27-guest domain decline slowly from 14.4 Å at 0 ns to 12.6 Å at ~7 ns due to the formation of secondary structures; simultaneously, those of the GL5-host domain increase rapidly from 10.8 Å at 0 ns to 18.6 Å at ~6 ns during this process. The large deviation of ΔRg of 7.8 Å reflects the significant extending of the GL5-host domain induced by the slow formation of secondary structure of the I27-guest domain with a small deviation of 1.8 Å. Furthermore, the uniform variation of Rg values for the two domains reveals qualitatively the complicated dynamic process of “tug-of-war” event under the mutually exclusive folding, suggested by the experiment [Q in Q values of the I27-guest domain from 0.54 at GL5/I27-S to 0.98 at GL5-ST/I27, but the decrease of that of the GL5-host domain from 0.97 to 0.34, which reflects the significant formation of the native I27-guest domain and the approximately full extending of the GL5-host domain due to the corresponding Q values approaching 1 and deviating from 1. The results illustrated that the formation of only secondary structures in the I27-guest domain may cause full extending of the GL5-host domain. Moreover, the analysis of correlation network for the dynamical mutually exclusive folding revealed the structural characteristics of the native guest and host domains, and the mutually exclusive communication between the formation of the I27-guest domain and the extending of the GL5-host domain during the transition process.For the process of the GL5/I27-S model with the secondary-structure unfolding of the I27-guest domain to the GL5-ST/I27 model with the full extending of the GL5-host domain, the time-average structures of the starting state (GL5/I27-S), the four transition conformations (I (~1.1 ns), II (~2.2 ns), III (~3.7 ns), IV (~6.5 ns)) and the target state (GL5-ST/I27) were extracted along with the CMD/TMD simulation, and are shown in periment . FurtherRg variations in Rg values of the I27-guest domain decline rapidly from 18.9 Å at 0 ns to 12.6 Å at ~6 ns due to the large size of the I27-guest domain in the starting GL5/I27-ST model and the primary shrinking of the tertiary structure followed by the secondary-structure formation, which is different from 14.4 Å at 0 ns in the starting GL5/I27-S model with the small size of the I27-guest domain, discussed above. However, those of the GL5-host domain remain at 10.8 Å in the native GL5 state during the first 1.3 ns, then increase rapidly from 10.8 Å to 18.6 Å during the remaining TMD simulation time. These data reflect that the primary decrease of Rg values in the I27-guest domain causes insignificant extending of the GL5-host domain due to the shrinking of only tertiary structure in the I27-guest domain. The further decrease of Rg values in the I27-guest domain induces significant extending of the GL5-host domain. Especially, it can be seen that the primary tertiary-structure shrinking of the I27-guest domain does not cause extending of the GL5-host domain during the period of the Rg values of the I27-guest domain decreasing from 18.9 Å at 0 ns to 15 Å at 1.3 ns. That is, the full extending of the GL5-host domain occurs only after the Rg values of the I27-guest domain decrease from 15 Å at 1.3 ns to 12.6 Å at 6 ns. It should be noted that the size of I27-guest domain Rg = 15 Å in the GL5/I27-ST to GL5-ST/I27 process almost equals that of Rg = 14.4 Å in the GL5/I27-S to GL5-ST/I27 process discussed above. The results indicate that the formation of the secondary structure provides the main driving force for extending the native GL5-host domain. The larger I27-guest domain with the secondary/tertiary-structure extending promotes insignificantly the extending of the GL5-host domain. That is, the primary tertiary-structure shrinking of the I27-guest domain has not yet induced the extending of the GL5-host domain. To further illustrate the nature of the driving force of secondary-structure folding, the superposition of variations of the Rg values at the TMD time of 0 ns for the process of GL5/I27-S to GL5-ST/I27 over that at 1.3 ns for GL5/I27-ST to GL5-ST/I27 is shown in Q for GL5/I27-ST to GL5-ST/I27 in Rg, that is, Q values of the I27-guest domain increase from 0.32 at GL5/I27-ST to 0.98 at GL5-ST/I27, while that of the GL5-host domain decrease from 0.98 to 0.35. Especially, Q = 0.32 at the GL5/I27-ST to GL5-ST/I27 process reveals the larger I27-guest domain with the secondary/tertiary-structure extending, which is compared to the Q = 0.54 in GL5/I27-S to GL5-ST/I27 process. Summarizing, the investigation of the two processes from the starting structures with the different guest-domain sizes to the full extending of the host domain reveals that the secondary-structure folding of the guest domain is the driving force for the mutually exclusive folding process.To address how the extended size of the I27-guest domain influences the full extending of the GL5-host domain, the time-average structures of the starting state (GL5/I27-ST) with the secondary/tertiary-structure extending of the I27-guest domain, the five transition conformations (I’ (~1.1 ns), II’ (~2.2 ns), III’ (~3.7 ns), IV’ (~4.8 ns), V’ (~6.8 ns)) and the target state (GL5-ST/I27) were extracted along with the TMD simulation of GL5/I27-ST to GL5-ST/I27, and are shown in Rg) and the fraction of native contacts (Q) for both the I27-guest and GL5-host domains along the two TMD simulations were also extracted from the corresponding trajectories, and are shown in Rg for the process of GL5/I27-S to GL5-S/I27 in ΔRg = 1.8 Å for the I27-guest domain, and the increase of ΔRg = 3.4 Å for the GL5-host domain, in which the increase of the ΔRg values in the GL5-host domain reflects only the secondary-structure extending of the GL5-host domain. However, the variation of the Rg values for the process of GL5/I27-ST to GL5-S/I27 shows the rapid decrease of ΔRg = 6.2 Å for the I27-guest domain, and the steady fluctuation followed by slow increase of ΔRg = 3.4 Å for the GL5-host domain. The Rg decrease in the I27-guest domain and its steady fluctuation in the GL5-host domain predict that the primary shrinking of only the tertiary structure in the I27-guest domain causes insignificant extending of the GL5-host domain. However, the continuous Rg decrease in the I27-guest domain and the following increase in the GL5-host domain reflect that the secondary-structure formation in the guest domain induces rapid extending of the host domain. These results still support the crucial function of the secondary-structure formation in the guest domain for the mutually exclusive folding process. It is worth noting that a special structure at the simulation time of ~3.3 ns during the process of GL5/I27-S to GL5-S/I27 was found with both I27-guest and GL5-host domains being folded at the same time, which has been suggested by the experiment as an intermediate occurring on rare occasions [To address further the effect of the extending size of the I27-guest domain on the secondary-structure unfolding of the GL5-host domain, the TMD simulations from the starting GL5/I27-S and GL5/I27-ST models, that are the same models in the processes discussed above, to another target GL5-S/I27 model with the secondary-structure unfolding of the GL5-host domain were performed, and similar mutually exclusive folding processes were also successfully attained. The plots for the RMSDs of all backbone and heavy atoms are shown in ccasions . The corRg for both I27-guest and GL5-host domains are shown in Rg of the GL5-host domains for both processes of GL5/I27-S to GL5-ST/I27 and GL5/I27-S to GL5-S/I27 are about 0.98 and 0.43 Å/ns, respectively, during the assigned simulation time of 8 ns, compared with actual variation rates of 1.56 and 0.68 Å/ns during the actual simulation time of 5 ns. The deviation between the expected and actual rates of Rg of the GL5-host domains for the process of GL5/I27-S to GL5-ST/I27 is larger by 0.33 Å/ns than that for GL5/I27-S to GL5-S/I27, which indicates that the folding for GL5/I27-S to GL5-ST/I27 with full extending of the GL5-host domain is faster than that for GL5/I27-S to GL5-S/I27 with secondary-structure unfolding. Moreover, the cross site of Rg curves of the GL5-host and I27-guest domains at Rg = 13.1 Å occurs firstly at the simulation time of 1.8 ns for GL5/I27-S to GL5-ST/I27 over that at the simulation time of 3.2 ns for GL5/I27-S to GL5-S/I27 due to the rapid variation rate for GL5/I27-S to GL5-ST/I27, which also supports the rapid folding of GL5/I27-S to GL5-ST/I27. The results indicate that the secondary-structure formation in the guest domain could generate enough driving force to induce full extending of the host domain.To address the mutually exclusive folding characteristics from the starting state with the small size of I27-guest domain involving only the secondary-structure unfolding to the target states with the large and small sizes of GL5-host domains, the comparison between the two processes of GL5/I27-S to GL5-ST/I27 and GL5/I27-S to GL5-S/I27 was analyzed, and the corresponding variations of Rg declining of the I27-guest domain and the rapid Rg increasing of the GL5-host domain, the rearrangement of the β sheets in the I27-guest domain causes tightness of the cage structure with decreases of the mass center distances between βA/βF, βC and βG, βF strands and the formation of several hydrogen bonds. Simultaneously, the GL5-host domain deforms at I, then the β1–β4, β1–β2, and β3–β4 sheets are partially unfolded at II, which causes decrease of the relative percentages of the total hydrogen bond occupancies of the β sheets occupancies by 42.6% from GL5/I27-S to II—the computational details are given in the The details of the structural changes for the starting state (GL5/I27-S), the four transition conformations (I (~1.1 ns), II (~2.2 ns), III (~3.7 ns), IV (~6.5 ns)) and the target state (GL5-ST/I27) in the mutually exclusive folding pathway from the GL5/I27-S model with the secondary-structure unfolding I27-guest domain to the GL5-ST/I27 model with the full extended GL5-host domain in Rg declining of the I27-guest domain and Rg invariableness of the GL5-host domain, the large extending I27-guest domain gradually shrinks with significant decreases of the mass center distances between βB, βC, βF and βE, βD, βG strands from ~21.5 Å, ~32.3 Å, ~14.0 Å at GL5/I27-ST to ~10.5 Å, ~17.1 Å, ~9.9 Å at II’, respectively, which mainly contributes to the rapid Rg declining. Especially, the large shrinking of I27-guest domain causes insignificant extending of the GL5-host domain verified by its Rg invariableness from GL5/I27-ST to II’. Furthermore, the formation of few inner-hydrogen bonds in the secondary structure of the I27-guest domain predicts its rearrangement of the tertiary structure , the four transition conformations (I’ (~1.1 ns), II’ (~2.2 ns), III’ (~3.7 ns), IV’ (~4.8 ns), V’(~6.8 ns)) and the target state (GL5-ST/I27) in the pathway from the GL5/I27-ST model with the larger extending of the I27-guest domain to the GL5-ST/I27 model with the full extended GL5-host domain were also analyzed see . The occture see . For theture see . TherefoTo explore the mutually exclusive communications of each residue in the I27-guest and GL5-host domains, we analyzed the motion correlations of all Cα atoms for the GL5/I27-S, GL5-ST/I27 models, and II, IV conformations in the process of the GL5/I27-S to GL5-ST/I27 models extracting from the simulation trajectories, which are displayed in All conventional molecular dynamics (CMD) simulations for the four models were performed by using the AMBER 9 package  togetherk is the harmonic force constant per atom, N is the number of the restrained atoms, RMSD(t) is the root-mean-square deviation of the simulated structure at time t relative to the target structure, and RMSD0(t) is the prescribed target RMSD value at time t that decreases to zero linearly with time to drive the system from an initial structure to the target structure. To determine appropriate values of the harmonic force constant k, we tested the use of various k values for these TMD simulations  as the lowest harmonic force constant to apply to all heavy atoms of four models to bias the trajectories toward the target structure. Several TMD simulations starting from different initial unfolded structures for the transition of GL5/I27-S to GL5-ST/I27 models were performed to test the result consistency that is shown in Generally, the structure transitions are beyond the reach of conventional simulations without biased potential constraint due to transition simulations on the scale of microseconds and longer. Nevertheless, we tried to obtain the mutually exclusive folding process from GL5/I27-S to GL5-ST/I27 models by using long time CMD simulation. Unfortunately, the process for the GL5/I27-S model did not succeed, which is consistent with the results of previous attempts by other researchers on large-scale conformational transition simulations. Targeted molecular dynamics (TMD) simulation is a method to observe large-scale conformational transition between two known end-point conformations of a molecule at ordinary temperature by applying a time-dependent with the help of a constraint. In TMD the root mean square deviation (RMSD) to a target structure as the offset parameter is constrained and continuously decreased during the simulation. The system is forced to find a path from its initial state to the final one. This time-dependent constraint in the TMD simulation introduces a bias which is minimal in the sense that only one degree of freedom is constrained . The TMDRg) and the fraction of native contacts (Q) for both I27-guest and GL5-host domains along the simulations trajectories were analyzed to investigate the nature of the mutually exclusive folding mechanism. For the process of GL5/I27-S model with the secondary-structure unfolding of the I27-guest domain to GL5-ST/I27 model with the full extending of the GL5-host domain, the slow formation of secondary structure of the I27-guest domain with a small deviation of radius of gyration Rg (ΔRg) of 1.8 Å causes significant extending of the GL5-host domain with ΔRg of 7.8 Å. However, for the process of the GL5/I27-ST model with the secondary/tertiary-structure extending of the I27-guest domain to the GL5-ST/I27 model, the rapid decline of the I27-guest domain size by the decrease of Rg values from 18.9 Å to 12.6 Å induces insignificant extending of the GL5-host domain due to primary shrinking of the tertiary structure in the I27-guest domain. Then, the rapid extending of the GL5-host domain occurs along with further decrease of the I27-guest domain size. The results indicate that the folding of only the secondary-structure induces significantly full extending of the native GL5-host domain, and provides the main driving force for “tug-of-war” event of mutually exclusive folding/unfolding between the I27-guest and GL5-host domains. The variation of the fractions of native contacts Q for these processes also supports similar results discussed from the variation of Rg values. Through the investigation of the process of GL5/I27-S to GL5-S/I27, a special structure with both host and guest domains being folded at the same time as an intermediate was found, which was suggested by the experiment. The correlation analysis for some conformations predicts the structural characteristics of the native guest and host domains, and the mutually exclusive communication between the formation of the β sheets of the I27-guest domain and the extending of the GL5-host domain during the transition processes. The mutually exclusive folding principle is a special bio-function and could be used in some fields. For example, it may be expanded to construct a biosensor, or as a driving force to engineer self-assembling protein hydrogels.Conventional molecular dynamics simulations were performed for the mutually exclusive folding protein of GL5/I27 to address the stable structure characteristics for the four mutually exclusive folding protein models with different unfolding degrees in the I27-guest or GL5-host domains. In the four models, the GL5/I27-S model involves the host domain GL5 and the secondary-structure unfolded guest domain I27-S, while the GL5/I27-ST model involves the host domain GL5 and the secondary/tertiary-structure extending guest domain I27-ST, and the other two GL5-S/I27 and GL5-ST/I27 models represent the secondary-structure unfolding and the secondary/tertiary-structure extending in the host domain GL5, respectively. Targeted molecular dynamics simulations were performed to address the mutually exclusive folding transition mechanisms from the two starting GL5/I27-S and GL5/I27-ST models with secondary-structure unfolding and secondary/tertiary-structure extending in the I27-guest domain to the two target GL5-ST/I27 and GL5-S/I27 models with the secondary-structure unfolding and full extending in the GL5-host domain. The variations of the radius of gyration (


Superplastic deformation behavior has been investigated at 1000 °C and 1050 °C with strain rates from 5 × 10−5 s−1 to 1 × 10−3 s−1. The effects of deformation on the microstructure and mechanical behaviors of high Nb containing TiAl alloy have been characterized and analyzed. The results showed that, the ultimate tensile strength of the alloy was 58.7 MPa at 1000 °C and 10.5 MPa at 1050 °C with a strain rate of 5 × 10−5 s−1, while the elongation was 121% and 233%, respectively. The alloy exhibited superplastic elongation at 1000 and 1050 °C with an exponent (m) of 0.48 and 0.45. The main softening mechanism was dynamic recrystallization of γ grains; the dislocation slip and γ/γ interface twinning were responsible for superplastic deformation. The orientation relationship of γ/γ interface twinning obeyed the classical one: (001)γ//(110)γ.In this paper, the equiaxed superfine/nanocrystalline duplex PM-TiAl-based alloy with (γ + α Additionally, 1/3 [111] Frank dislocations leave a dislocation step on the γ/γ interface. It is observed that 1/6<112] Shockley dislocations lead to the superlattice intrinsic stacking fault (SISF) at the tip of the deformed twin from the HREM images.The slipping of the 1/6 [11SISF on the twin interface, which results in the deviation of the twin interface. Therefore, the Shockley incomplete dislocation that produces the interface twin is not nucleated at the same location, but there are many nucleation points along the interface twins.Dynamic recrystallization is an important structural feature observed for PM-TiAl-based alloys. The microstructure and phase distribution of the alloy can be more uniform while maintaining the stability of the structure. Unlike ordinary thermal deformation, the internal grain is no longer the nucleation position of dynamic recrystallization during superplastic deformation. The main nucleation positions of dynamic recrystallization occur mainly at the junctions and grain boundaries where the stress is highly concentrated, and near the dislocations and twins. As shown in the TEM micrographs of σ is proportional to the a power of the grain size d (0.7 < a < 2) [In the process of superplastic deformation, the deformation stress < a < 2) . Therefo2) microstructure of the PM Ti-45Al-5Nb alloy has favourable superplasticity. The ultimate tensile strength of the alloy was 58.7 MPa at 1000 °C and 10.5 MPa at 1050 °C with a strain rate of 5 × 10−5 s−1, while the elongation was 121% and 233%, respectively.The equiaxed superfine/nanocrystalline duplex (γ + αγ//(110)γ. The main softening mechanism is the dynamic recrystallization of γ grains. Dynamic recrystallization nucleates mainly at grain junctions and grain boundaries, where the stress is highly concentrated, and near dislocations and twins.The mechanism of superplastic deformation is dislocation slipping and twinning of the γ/γ phase. The orientation relationship between the γ/γ phase interface is (001)2 phase plays a promoting role. The α2 grains undergo rotation with the deformation of the γ phase in the process of superplastic deformation because of the fine grains and less slip of the system.The plastic deformation mainly occurs in the matrix γ phase, and the dispersion of the equiaxed nanocrystalline α