The COMMD proteins are a conserved family of proteins with central roles in intracellular membrane trafficking and transcription. They form oligomeric complexes with each other and act as components of a larger assembly called the CCC complex, which is localized to endosomal compartments and mediates the transport of several transmembrane cargos. How these complexes are formed however is completely unknown. Here, we have systematically characterised the interactions between human COMMD proteins, and determined structures of COMMD proteins using X-ray crystallography and X-ray scattering to provide insights into the underlying mechanisms of homo- and heteromeric assembly. All COMMD proteins possess an α-helical N-terminal domain, and a highly conserved C-terminal domain that forms a tightly interlocked dimeric structure responsible for COMMD-COMMD interactions. The COMM domains also bind directly to components of CCC and mediate non-specific membrane association. Overall these studies show that COMMD proteins function as obligatory dimers with conserved domain architectures. The COMMD (Copper Metabolism Murr1 (Mouse U2af1-rs1 region 1) Domain) proteins are highly conserved in metazoans and unicellular protozoa . There aCOMMD1 gene was first identified in Bedlington terrier dogs with hepatic copper toxicosis  are expressed broadly in many tissues . Much ofoxicosis . In accooxicosis , althougoxicosis . In humaoxicosis  and are oxicosis , suggestoxicosis . Althougoxicosis . Membersoxicosis .COMMD proteins have been found to exist in an endosome-associated complex with the coiled-coil domain-containing proteins CCDC22 and CCDC93, which has been termed the CCC  complex. In addition, functional proteomics and bioinformatics have independently shown that the CCC complex can associate with a stable heterotrimeric assembly of VPS29, C16orf62 and DSCR3, to form a larger macromolecular complex dubbed the ‘Commander’ or CCC/Retreiver complex . Other pIn this study we provide a comprehensive and systematic analysis of the structural, biophysical and biochemical properties of the COMMD family of proteins, and their recruitment to the CCC complex. These analyses show that COMMD proteins form family-wide homo- and heterodimers through their C-terminal COMM domains. The crystal structure of the COMM domain from Commd9 reveals an intimately interlocked α/β homodimeric structure, which involves formation of a large and conserved hydrophobic interface. The crystal structure of the N-terminal domain of Commd9 shows an all α-helical structure, and using small angle X-ray scattering we establish that homodimers of full-length Commd1, Commd7 and Commd9 adopt similar tertiary architectures. Intriguingly, the COMMD proteins show clear structural homology to a poorly characterised protein found in chlamydial species of bacteria. Further biochemical studies show that C-terminal COMM domains have pleiotropic roles in both recruitment of CCDC22 and CCDC93 via their N-terminal calponin homology-like (NN-CH) domains, and in binding non-specifically to negatively charged phospholipids.As a first step towards assessing the self-assembly activities of COMMD proteins, we studied the biophysical properties of recombinant full-length COMMD proteins in solution. Of all COMMD proteins we tested Commd1, Commd7 and Commd9  were reaWe next sought to determine the X-ray crystallographic structures of COMMD proteins. However, although crystals of various full-length COMMD proteins grew rapidly, their diffraction quality was not sufficient for structure determination. Therefore a divide-and-conquer approach was taken to determine structures of the two individual domains of the protein, the conserved C-terminal COMM domain, and the variable N-terminal HN domain.2 (nearly 1/3rd of the monomer surface area) supporting the notion that the native state of COMMD proteins is to form dimers via the COMM domain.We determined the crystal structure of the Commd9 COMM domain to 2.2 Å resolution by single-wavelength anomalous dispersion (SAD). The overall structure of the Commd9 COMM domain is composed of two cone-shaped chains that are tightly intertwined with each other to form a globular dimeric module , Table 1In Helical N-terminal) domain. The overall structure of the HN domain has a similar all α-helical fold to the equivalent domain of Commd1, which was previously determined by NMR s) both signify a relatively globular structure. Ab initio structures of these calculated from the SAXS data reveal compact but elongated molecules. We next performed rigid body fitting of the SAXS data with the program SASREF  using DALI  did idenChlamydia trachomatis (CT584) and Chlamydia pneumoniae (Cpn0803)   (DALI Z-Cpn0803) . The onlCpn0803) . The N-tCpn0803) . Like thCpn0803) . In addiCpn0803)   followed by affinity purification using glutathione sepharose beads  to obtain a 1:1 stoichiometric complex. The SEC fractions under the peak clearly show reconstitution of a 1:1 Commd5-Commd10 complex .Since the COMM domain singularly mediates the formation of homo- and heteromeric complexes of COMMD proteins, we also attempted to make the complex using the full-length Commd5 and COMM domain of Commd10 and COMM domains of Commd5 and Commd10 respectively. Indeed,highly pure and stoichiometric species were isolated confirming the role of the COMM domain in COMMD complexes . SeveralCentral to the endosomal trafficking function of COMMD proteins is their assembly into the CCC complex . CCDC22 Kd) was calculated to be 10.3 and 14.6 µM respectively  in combination with pull down experiments. Non-deuterated BS3 cross-linker was used to crosslink full-length Commd9 with the NN-CH domain of CCDC93. Due to the availability of HN and COMM domain structures, Commd9 was chosen for these experiments. The NN-CH domain of CCDC22 could not be used as it does not contain any lysine residues. Upon crosslinking, three major bands  in the Commd9 and NN-CH domain of CCDC93 mixture were observed, which were excised for MS analysis . Lysozymd family .To minimize the possibility that the cross-linked peptides captured were non-specific interactions caused by BS3, we also performed a cross-linking reaction using the unrelated VPS26 – VPS29 – VPS35 retromer complex from zebrafish (hereafter designated as zfRetromer) as a positive control. Inspection of the xQuest database and the MS spectra reveal a total of 18 cross-linked peptides . VPS26 aCOMMD proteins are peripheral membrane proteins commonly associated with endosomal compartments . To examIn contrast to the other family members, Commd9 appears to associate weakly if at all with the membranes tested . A compaP and PIP2 compared to Folch and PS containing membranes, Commd9 showed little association with any of the lipids tested, in line with the liposome-pelleting assay (Using BLiTz we next quantified the interactions of Commd1 and Commd9 with different phosphoinositide-containing membranes. While Commd1 possesses a higher level of binding response for phosphoinositides PI(3)ng assay . We perfng assay . ConfirmP-enriched endosomal compartments.We next assessed the importance of the Commd1 membrane-binding surface for its cellular localization. GFP-tagged Commd1 wild type and mutant were expressed in HeLa cells following lentiviral transduction . As seenThe COMMD proteins were recently identified as conserved and central components of the CCC complex , a largeChlamydia-specific proteins from C. trachomatis and C. pneumonia respectively. The function of these proteins is unknown, although there is evidence that Cpn0803 can interact with phospholipids as well as components of the Type III secretion system responsible for injection of bacterial effectors into the host cytoplasm  was originally identified in dogs with copper toxicosis, which has been mechanistically linked to interactions with the Wilson disease ATPase protein ATP7B . Most prThe CCC complex and associated COMMD proteins are predominantly localised to early endosomal membranes . The mecThe ability to self-assemble and form heteromeric complexes is a core property of the COMMD proteins identified in some of the very first studies . Our wor2 ) , PIP2 ) , PIP2 )  and PIP3 )  lipids were obtained from Avanti Polar Lipids, Inc.The monoclonal mouse anti-His antibody was purchased from Genscript Corporation . Goat anti-mouse IgG HRP was from Life Technologies . POPC  , POPE  , DOPS   and biotinylated POPE )  were purchased from Avanti Polar Lipids. PI ) , PI(3)P ) , PI(4)P ) , PI(5)P ) , PIPAll the constructs cloned into bacterial expression plasmids are listed in Escherichia coli BL21-CodonPlus (DE3)-RIPL competent cells (Agilent). The bacterial cultures were grown in LB until OD600nm reached 0.6. The cultures were cooled to 18˚C before inducing protein expression by adding 0.5 mM isopropylthio-β-galactoside (IPTG) and allowed to grow for 16 h  gravity column and eluted with 500 mM imidazole in buffer containing 150 mM NaCl, 20 mM Tris (pH 8.0) and 1 mM β-mercaptoethanol. GST-tagged proteins were purified on a glutathione-Sepharose  gravity column and eluted with 10 mM glutathione, 150 mM NaCl, 20 mM Tris (pH 8.0) and 1 mM β-mercaptoethanol or the GST tag was cleaved with the addition of thrombin or precision protease cleavage on to the beads with overnight incubation at room temperature. Finally, proteins were subjected to size exclusion chromatography using a superdex-200 16/60 Hiload column or superdex-75 16/60 Hiload column attached to an AKTA pure .Escherichia coli BL21 (DE3) competent cells (Agilent). The cells were cultured, harvested and lysed as described above. The complexes were first purified using glutathione sepharose beads and the GST tag was cleaved using prescission protease. The eluted fractions were then mixed with equilibrated TALON beads to obtain a 1:1 stoichiometric complex. The complexes were eluted by supplementation of 200 mM Imidazole (pH 8.0) in the wash buffer. The eluted proteins were subjected to size exclusion chromatography using a superdex-200 16/60 Hiload column.The Commd5-Commd10 complexes were reconstituted by co-transformation of GST-Commd5 or GST-tagged COMM domain of Commd5 with Commd10-His or His-tagged COMM domain of Commd10 into E. coli BL21 (DE3) cells grown in LB at 37°C, and protein expression was induced at an OD600 of 0.7–0.8 by the addition of 1 mM IPTG. Cells were harvested after 18 hr of growth at 18°C. Cell pellets of overexpressed zfVPS26, zfVSP29 and zfVPS35 were mixed and purified using standard metal affinity, glutathione affinity and size-exclusion chromatography techniques to obtain the purified retromer complex. The size-exclusion buffer contains 50 mM HEPES (pH 7.5), 150 mM NaCl, 2 mM DTT.For crystallization, SAXS, and MALLS experiments, proteins were buffer exchanged into 10 mM Tris (pH 8.0), 100 mM NaCl and 2 mM DTT using SEC. For the structure determination of Commd9 COMM domain, the protein was labeled with selenomethionine using the method described by Van Duyne et al . zfVPS26W characterization. Measurements of the COMMD COMM and HN domains were made using a superdex-75 5/150 column  at a flow rate of 0.25 ml/min. UV, MALLS and RI data were collected and analysed using the ASTRATM software (Wyatt Technology)  connected to a multi angle laser light scattering and, differential refractive index (RI) detector. The protein samples were gel-filtered in a buffer containing 25 mM Tris (pH 8.0), 300 mM NaCl and 2 mM DTT that had been filtered (0.22 μm) and degassed. Measurements of full-length COMMD proteins were made using a superdex-200 increase 5/150 column  at a flow rate of 0.25 ml/min with in-line UV, MALLS, and RI detectros  for Mhnology)  to compuThe Commd9 COMM domain was buffer-exchanged into 10 mM Tris (pH 8.0), 100 mM NaCl, 2 mM DTT, and concentrated to 8 mg/ml for crystallisation at 20˚C. The protein was supplemented with 10 mM DTT before setting up hanging-drop crystallization screens using a mosquito liquid handling robot (TTP LabTech). Commd9 COMM was crystallised in 0.1 M HEPES (pH 7.0), 6% Jeffamine M-600.In the case of the Commd9 HN domain, protein was buffer exchanged into 50 mM HEPES (pH 8.0), 200 mM NaCl, 1 mM tris (2-carboxyethyl) phosphine (TCEP) and initial crystallisation screens were set up at 12 mg/ml at 18 ˚C. Crystals of the HN domain were obtained in 0.2 M citric acid (pH 4.9), 28% MME-PEG5000 in a hanging drop setup respectively. Data were collected at the Australian Synchrotron MX1 and MX2 Beamlines. iMOSFLM  was usedhttp://www.synchrotron.org.au). The buffer frames were averaged after assessing the statistical equivalence using CorMap p values with a significance threshold (α) of 0.01. The averaged buffer scattering was subtracted from statistically similar data from Commd proteins elution peak. gR was evaluated using the Guinier approximation and was found to be consistent under the elution peak. Primary data processing was performed in Primus using the ATSAS suite (version 2.6) (P(r) of Commd proteins was determined using GNOM. C2 symmetry was assumed in generating low-resolution three-dimensional Ab initio envelopes using the program GASBOR (ab initio models were superimposed on to the rigid body modeled structures using SUPCOMB (In line SEC-SAXS measurements on homogeneous protein samples (assessed using MALLS) were performed at the SAXS/WAXS beamline at the Australian Synchrotron using a superdex-200 increase 5/150 column , and Pilatus 1M detector (Dectris). The scattering data were measured in a q range of 0.011 to 0.4 Å at 12 keV using a 1.6 m camera length. Samples were loaded on to the size exclusion column that was equilibrated with 10 mM Tris (pH 8.0), 100 mM NaCl, 2 mM DTT and 5% Glycerol. Data reduction was performed using the ScatterBrain program  . Pair dim GASBOR . DAMAVERm GASBOR  was usedm GASBOR  and the m GASBOR . The ab  SUPCOMB .BL21-CodonPlus (DE3)-RIPL competent cells were co-transformed with either His6-tagged Commd1, Commd1 COMM domain, Commd1 HN domain, Commd9, Commd9 COMM domain or Commd10 with each of the GST-tagged proteins of interest , CCDC22_C (194-627), CCDC93_N (17-155), CCDC93_C (239-630) and empty pGEX4T-2 vector (expressing GST). Transformants were selected via overnight growth using triple antibiotic agar plates. A single colony was picked to initiate the culture and proteins were co-expressed using the standard protein expression protocol as described above. Proteins were purified by affinity chromatography using glutathione sepharose beads  and SDS-PAGE was run to visualize GST-tagged bait proteins. Binding of His-tagged proteins (prey) to GST-tagged (bait) was observed by Western blotting using mouse anti-His antibody (Genscript). Genscript generated all the mutants used in this study.1 nmol GST-tagged CCDC22_N (1-139) and CCDC93_N (17-155) were mixed with 1 nmol of His-tagged Commd1 and Commd9, and COMM domains of Commd1 and Commd9 and Commd1 HN domain, for 1 hr at 4˚C. Protein mixture was then centrifuged at high speed to remove any precipitated proteins. The supernatant was then added to pre-equilibrated (20 mM Tris (pH 8.0), 300 mM NaCl, 1 mM DTT) glutathione sepharose and allowed to mix for a further 30 min at 4˚C. Beads were washed five times in the above buffer supplemented with 0.5% triton X100 (Sigma Aldrich). Bound proteins were analysed by Western blots using mouse anti-His antibody (Genscript).For cross-linking, the purified full-length Commd9 and NN-CH domain of CCDC93 mixture at 50 μM in 50 mM Hepes (pH 7.5), 150 mM NaCl were incubated with 100 molar excess of BS3-d0 cross-linker (Sigma-Aldrich) for 30 min at room temperature. The reaction was quenched by addition of 100 mM Tris-HCl (pH 8.5), and the cross-linked products were analysed by SDS-PAGE and subjected to MS analysis. A negative control cross-linking reaction was performed between the full-length Commd9 and lysozyme using the same condition described above. For the positive control cross-linking reaction, the purified retromer complex at 15 μM was reacted with 100 molar excess of BS3-d0 cross-linker. BS3-d0 was purchased from Sigma Aldrich . The gel band that corresponds to the molecular weight of monomeric retromer complex was subjected to MS analysis. The bands from the SDS-PAGE gels were excised and reduced with dithioerythritol followed by alkylation with iodoacetamide. Alkylated samples were digested with trypsin (Promega) in 50 mM ammonium bicarbonate pH 8.0 overnight using an enzyme-to-substrate ratio of 1:100 (w/w) at 37°C. The digested samples were extracted using extraction buffer containing 5% formic acid and 50% acetonitrile followed by sonication for 1 min. The supernatant was then dried down in a vacuum centrifuge and redissolved in 0.1% formic acid prior to analyse by LC-MS/MS. The extracted peptides were analysed by uHPLC-MS/MS on an Eksigent, Ekspert nano LC400 uHPLC  coupled to a Triple Tof 6600 mass spectrometer  equipped with a duo microelectrospray ion source. In brief, samples were injected onto a 300 µm x 150 mm ChromXP C18 CL 3 µm column  at 5 µl/min. The bound peptides were eluted with a gradient using solvent containing 0.1% formic acid in acetonitrile. 250 ms full scan TOF-MS data was acquired followed by up to 30 50 ms full scan product ion data in an Information Dependant Acquisition, IDA, mode. TOFMS data was acquired over the mass range 350–2000 and for product ion ms/ms 100–1600. Ions observed in the TOF-MS scan exceeding a threshold of 100 counts and a charge state of +2 to +5 were set to trigger the acquisition of product ion, ms/ms spectra of the resultant 30 most intense ions. Acquisition of all MS/MS samples was performed using Analyst TF 1.7 software . Inspection of the raw MS data was done using ProteinPilot software . The assignment of cross-linked peptides was made based on xQuest database search engine . Trypsin3) and dried under argon. Dried lipids were then left in a desiccator for 1 hr to remove any remaining moisture. Dried lipids were resuspended in CHCl3:Methanol (MeOH):1N hydrochloric acid in a 2:1:0.01 molar ratio, lipids were dried once again and allowed to desiccate. Lipids were then resuspended in CHCl3:MeOH in a 3:1 ratio dried once again under argon. Finally, dried lipids were resuspended in CHCl3 and stored at −20°C.All the phosphoinositides were protonated prior to usage. In brief, powdered lipids were resuspended in chloroform , and 220 mM sucrose to obtain a suspension of multilamellar liposomes containing sucrose. This solution was then freeze-thawed five times to produce unilamellar liposomes. Liposomes were then diluted 1:5 in 25 mM HEPES (pH 7.2), and 125 mM NaCl solution. The solution was then centrifuged at 250,000 g to remove sucrose from the medium and maintain osmolarity. The pelleted liposomes were resuspended in 25 mM HEPES (pH 7.2), and 125 mM NaCl solution to the desired concentration of 0.5 mM. All liposomes were used within 1 day of preparation.10 µM of the protein of interest was added to a final volume of 200 µl of the liposome solution. This solution was left at room temperature for 25 min to allow for protein-liposome interaction. After incubation, the solution was centrifuged at 400,000 g for 30 min. Supernatant and pellet fractions were separated and the pellet was resuspended in 200 µl of 25 mM HEPES (pH 7.2), and 125 mM NaCl, samples were then collected for analysis on a precast 4–12% bis-tris gel (Novex) by coomassie staining.Protein-lipid and protein-protein interactions were determined using the bio-layer interferometry from the BLiTz system. Protein-lipid interactions were observed by immobilizing 500 µM of biotinylated liposomes on a streptavidin biosensor. After immobilization, the sensor was washed with buffer containing 10 mM Tris (pH 8.0), 150 mM NaCl and 0.1% BSA to prevent non-specific association. Increasing concentrations  of protein were added to the sensor and the change in binding (nm) was measured. Proteins were then allowed to disassociate from the probe in the buffer previously mentioned. The kinetics of the protein-protein interactions were determined in the same fashion using 5 µM of His-tagged COMMD1 COMM domain immobilized on a nickel-NTA probe and increasing protein concentrations of 62.5, 125, 250 and 500 µM. The data was processed and plotted using the Sigmaplot package (Systat Software Inc.).RPE1, HEK293T cells were maintained in DMEM  plus 10% fetal calf serum  under standard conditions. These cell lines were obtained from America Type Culture Collection (ATCC). Parental and stable cells lines were negative for mycoplasma by DAPI staining, and authenticated by STR profiling. Lentivirus particles for producing stably expressing cell lines were generated in HEK293T cells using the pXLG3 vector to carry the GFP tagged Commd1 WT and Commd1 . Cells were transfected with DNA using polyethylenimine (Sigma-Aldrich). Virus was harvested from the growth media 72 hr post transfection.For stable transduction with lentivirus, cells were seeded at 75,000 per well in six well plates. The cells were then incubated under normal conditions with titrations of viral supernatant for 72 hr. Cells were then passaged and expression of the GFP tagged protein of interest assessed by western analysis. Cell lines that displayed similar expression levels were selected for comparison and those closest to endogenous levels of protein.RPE1 cells grown on 13 mm coverslips were washed with PBS before being fixed in ice cold 4% formaldehyde in PBS for 25 min. Cells were permeabilised in 0.1% Triton X-100 (Sigma) for 6 min. The cells were then blocked with 1% bovine serum albumin (BSA) in 0.01% Triton for 15 min at room temperature. Primary antibodies were diluted in 1% BSA and samples were incubated for 1 hr at room temperature. The samples were then incubated with Alexa Fluor conjugated secondary antibody and 0.2 µM DAPI for 30 min at room temperature. Coverslips were mounted in Mowiol-DABCO mounting medium (Sigma). Cells were visualised using a Leica TCS SP5 X confocal microscope (Leica Biosystems).Coordinates and structure factors for the COMM and HN domain of Commd9 have been deposited at the Protein Data Bank (PDB) with accession codes 6BP6 (COMM domain Commd9) and 4OE9 (HN domain of Commd9). In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Vivek Malhotra as the Senior Editor.Thank you for submitting your article "Structural insights into the architecture and membrane interactions of the conserved COMMD proteins" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife. The reviewers have suggested that this could be addressed by using the structure to identify COMMD residues that are involved in binding CCDC22 and/or CCDC93.The reviewers are in broad agreement that the COMMD proteins have an important, if enigmatic, role in endosome function and that insight into their structure will help underpin studies aimed an better understanding this role. However, there is a concern that the biological significance of the structural data presented is not fully established. Such insight would be expected for a broad interest journal such as If you feel that you can add this further experimental work, or carry out related experiments to address the same point, then we would be happy to consider a revised version. If you submit a revision, please tend to other aspects of the experimental work that need attention.Summary:COMMD proteins comprise a highly conserved eukaryotic protein family. Its 10 members in human are implicated in cellular trafficking and the regulation of transcription. The proteins consists of an N-terminal calponin homology-like domain (HN) and a C-terminal COMM domain. To understand the molecular basis for its function, the authors of this study determined the crystal structure of the isolated COMM and HN domain of COMMD9. They found that COMM domains form homo- and heteromeric assemblies via a highly conserved interface, whereas the HN domain is monomeric. SAXS measurements with the full-length constructs of Commd1, Commd7 and Commd9 were used to extract information on the quaternary assembly. Using pull-down studies, gel filtration analysis and Blitz measurements, they further characterized heteroassembly of COMMDs within the family and with the CCC components CCDC22 and 93. Finally, they found unspecific membrane-binding of Commd 10 to liposomes of various compositions. Mutations of a basic patch in Commd10 reduced membrane binding but not its cellular localization on endosomal structures.Essential revisions:1) COMMD homo- and heterodimers:E. coli, Commd10 binds strongly to Commd2. Is heterotetramerization also applicable to Commd10- Commd2 complex?. Is tetramerization a property of heterodimers?.Homo- and heterodimerization seems to be promiscuous among Commd proteins. While Commd1, Commd7 and Commd9 behave as 1/1 homodimers , the Commd5-Commd10 complex behaves as a 2/2 heterotetramer . From Figure 7A, it seems that by co-expression in 2) COMMD-CCC interactions:COMMD domains of Commd1, Commd7 and Commd10 interact directly with the NN-CH domain of CCD22 and CCDC93 whereas Commd9 binds specifically to CCDC93 . The crystal structure of COMMD9 presented in this work and the homology model of COMMD1  should facilitate the identification of a similar surface patch, distinct from the conserved dimerization core, responsible for binding to the NN-CH domain of CCDC93. Authors could exploit this structural information together with site-directed mutagenesis and bio-layer interferometry as shown in Figure 8C, D to identify and confirm the binding interface. This would add a great value to the present work.3) Membrane association:The basic patch  identified in COMM1 that is important for interaction with model membranes, is not conserved in Commd7 or Commd10, which also interact with membranes . Furthermore, this patch is neither conserved in other Commd members  suggesting a singular and partial mechanism only applicable to Comm1. How other Commd complexes, dimers or heterodimers, are recruited to the membrane remain unknown. One possibility for membrane recruitment might be through interaction with the CCC complex but exploring this possibility would require the identification of the conserved patch in COMMD proteins for binding to CCDC22 and/or CCDC93.4) Figure 5: The graphs on top are too small to recognize anything. More importantly, the scattering curves and the resulting envelopes look very similar. Given the ambiguities in fitting such low resolution envelopes, the authors should not make any firm conclusions on the details of the assemblies, since they are most likely wrong. The firm conclusions of these measurements appear to be: Homomeric COMMDs form a dimer in SAXS. In some likelihood, the COMMD domains are in the center whereas the HN domains are at the periphery.. the authors are recommended to tone down the conclusions of different assemblies and maybe only show data and the model of Commd9 in the main manuscript.5) Figure 7A: Commd8: There is hardly any protein of the correct size in the Commassie gel, and it appears even unclear whether the proposed band is at all related to COMM8. Strangely enough, this protein still interacts with high efficiency with all Commd proteins. That does not make much sense: Either improve or remove.6) Figure 8A: Again, hardly any CCDC22 full length is expressed and a strong interaction with COMM1 is observed . Furthermore, COMM1 also appears to be partly pulled down by GST. This is worrisome, there seems to be an unspecific binding of COMM1 to almost anything and the data for CCDC22 are uninterpretable. These experiments should be improved, for example with more stringent washing conditions or finding better expression conditions.7) The structural homology analysis of COMMD proteins to Pur-α, CT584 and Cpn0803, do not contribute much to the overall story and are distractive. In this sense, the homology only hints at a shared fold for dimerization but there is no support for functional similarities to Pur-α, or mimicking function by the chlamydial proteins. This section might be shortened and/or incorporated into supplementary material.8) The liposome binding experiments appear to lack a negative control – i.e. liposomes that are not acidic such as those of pure PC.eLife. Your revised article has been favorably evaluated by a Senior Editor and a Reviewing Editor.Thank you for resubmitting your work entitled "Structural insights into the architecture and membrane interactions of the conserved COMMD proteins" for further consideration at The manuscript has been improved but there are some remaining issues about the text that the reviewers agree need to be addressed before acceptance, as outlined below:1) In the summary the authors make a claim on defining the core principles of Commd homo- and heteromeric assembly. This is clearly an overstated claim as the interaction between Commd proteins seems to be quite promiscuous and still there are no clear principles of how homodimer-versus-heterodimer preference is achieved.2) Crosslinking data: The WRVD motif should be better introduced. The cross-linked results are interesting and narrow down the potential interface between Commd9 and CCDC93, yet the proposed WRVD motif seems to be part of the conserved dimerisation core . Based on the calponin homology domain of CCDC93, the structure of Commd9, the BS3 cross linking distance, and conserved residues on the surface, authors should be able to propose a plausible contact area in Commd9 despite the NMR unfruitful results.3) Figure 4: I am still not happy to see three different molecular models for full length Commd1, Commd7 and Commd9. It's just misleading, the envelopes look all the same. One representative model showing that the COMMD and HN domain fit into all envelopes would be completely sufficient and less confusing.4) Following our initial advice, Figure 5 should go to the supplement.5) Typo: subsection “COMM domain binds to CCDC22 and CCDC93 through a conserved motif”, first paragraph – should be K133, not K131 (K131 is the residue in CCDC93).6) Subsection “COMM domain binds to CCDC22 and CCDC93 through a conserved motif” – is missing a full stop.7) Figure 3—figure supplement 1: aa numbers 151 to 200 are missing.8) Figure 7 legend: Description for panel D is missing (CCDC93 interaction).I hope that addressing these points will not be difficult for you. If you can provide a point-by-point summary of the changes you have made I should be able to make a final decision without having to consult the reviewers again. Essential revisions:1) COMMD homo- and heterodimers:Homo- and heterodimerization seems to be promiscuous among Commd proteins. While Commd1, Commd7 and Commd9 behave as 1/1 homodimers , the Commd5-Commd10 complex behaves as a 2/2 heterotetramer . From Figure 7A, it seems that by co-expression in E. coli, Commd10 binds strongly to Commd2. Is heterotetramerization also applicable to Commd10- Commd2 complex?. Is tetramerization a property of heterodimers?.The reviewers correctly observed that among the promiscuous Commd-Commd interactions we detect, Commd10 appears to bind strongly to Commd2. We have been able to reconstitute this complex see , however2) COMMD-CCC interactions:COMMD domains of Commd1, Commd7 and Commd10 interact directly with the NN-CH domain of CCD22 and CCDC93 whereas Commd9 binds specifically to CCDC93 . The crystal structure of COMMD9 presented in this work and the homology model of COMMD1  should facilitate the identification of a similar surface patch, distinct from the conserved dimerization core, responsible for binding to the NN-CH domain of CCDC93. Authors could exploit this structural information together with site-directed mutagenesis and bio-layer interferometry as shown in Figure 8C, D to identify and confirm the binding interface. This would add a great value to the present work.This is an excellent suggestion and we have attempted to define the interface on Commd9 that is coordinating the interaction with NN-CH domain of CCDC93 using cross-linking mass spectrometry. This new data together with text has been included in the revised manuscript. While this data now identifies a region on the Commd9 COMM domain that is involved in binding to the CCDC93 CC-NH, we have not been able to discover a point mutant that blocks this interaction based on this information. Until we are able to determine a high-resolution structure of the complex between the two proteins we would argue that this is likely just because XL-MS is unable to define the process side-chains required for binding.15N HSQC spectra that can be used for such experiments.We also attempted to use NMR to test whether it is possible to assign the backbone of the Commd9 COMM domain for titration mapping experiments with NN-CH domains of CCDC22 and CCDC93. However, the NMR spectra in the conditions tested did not give These data are included in the new Figure 8, the previous Figure 8 is now Figure 7.3) Membrane association:The basic patch  identified in COMM1 that is important for interaction with model membranes, is not conserved in Commd7 or Commd10, which also interact with membranes . Furthermore, this patch is neither conserved in other Commd members  suggesting a singular and partial mechanism only applicable to Comm1. How other Commd complexes, dimers or heterodimers, are recruited to the membrane remain unknown. One possibility for membrane recruitment might be through interaction with the CCC complex but exploring this possibility would require the identification of the conserved patch in COMMD proteins for binding to CCDC22 and/or CCDC93.We agree that it is currently not clear how various members of the COMMD family tether to the membranes. Indeed as our data for Commd1 shows, although it can bind membranes this doesn’t appear to be essential for specific endosomal localization. We actually debated showing this negative data, but eventually felt it was important to show this for completeness. Although speculation, we think that the avidity effect provided by all of the COMMD members and perhaps through direct interaction of other CCC components with membranes and membrane-associated proteins will prove to be key determinants for endosomal recruitment. As discussed above although we now have used XL-MS to gain an insight into the interaction region of Commd9 with CCDC proteins, we are not yet able to generate specific mutants that perturb these interactions to determine the importance in membrane recruitment.4) Figure 5: The graphs on top are too small to recognize anything. More importantly, the scattering curves and the resulting envelopes look very similar. Given the ambiguities in fitting such low resolution envelopes, the authors should not make any firm conclusions on the details of the assemblies, since they are most likely wrong. The firm conclusions of these measurements appear to be: Homomeric COMMDs form a dimer in SAXS. In some likelihood, the COMMD domains are in the center whereas the HN domains are at the periphery.. the authors are recommended to tone down the conclusions of different assemblies and maybe only show data and the model of Commd9 in the main manuscript.This figure has been made larger to more clearly show the data. We have also changed the language in the text to suggest that SAXS data confirms the MALLS and crystallography data that COMMD proteins are homodimers in solution. We agree that bold interpretations cannot be made from the scattering experiments, however at the least the data clearly shows the overall shape of various COMMD proteins, and confirms their homodimeric nature. Therefore, we have for now opted to keep all of the SAXS data in this revised version.The old Figure 5 is now replaced with Figure 4.5) Figure 7A: Commd8: There is hardly any protein of the correct size in the Commassie gel, and it appears even unclear whether the proposed band is at all related to COMM8. Strangely enough, this protein still interacts with high efficiency with all Commd proteins. That does not make much sense: Either improve or remove.GST-Commd8 has a tendency to degrade over time, but we have identified through time course experiments that the marked band is indeed GST fused Commd8. The protein appears to be binding other Commd proteins and fragments, as seen in the western blotting-based detection method we have employed that is quite sensitive. However, on the advice of reviewers we have removed this data for Commd8.Figure 7 has been changed to Figure 6 due to merging of Figure 1 and 2.6) Figure 8A: Again, hardly any CCDC22 full length is expressed and a strong interaction with COMM1 is observed . Furthermore, COMM1 also appears to be partly pulled down by GST. This is worrisome, there seems to be an unspecific binding of COMM1 to almost anything and the data for CCDC22 are uninterpretable. These experiments should be improved, for example with more stringent washing conditions or finding better expression conditions.New text describing the interactions of COMM domains with both HN and COMM domain has been included in the manuscript. With regards to the full length CCDC22 protein band, like Commd8, CCDC22 also has a tendency to degrade in to smaller fragments. However, there is still some fraction of intact GST fused CCDC22 visible on the Coomassie stained gel as shown by the asterisk and since the detection method is very sensitive, we can still detect an interaction band.We have also repeated the pull down again and have included the new blot in the revised manuscript. In this new round, the non-specific GST binding has been reduced and comparison with other bands on the gel clearly shows that the interaction to CCDC proteins is specific. We also note that GST is expressed heavily compared to the other bait proteins.Please note, Figure 8 is now Figure 7.7) The structural homology analysis of COMMD proteins to Pur-α, CT584 and Cpn0803, do not contribute much to the overall story and are distractive. In this sense, the homology only hints at a shared fold for dimerization but there is no support for functional similarities to Pur-α, or mimicking function by the chlamydial proteins. This section might be shortened and/or incorporated into supplementary material.We have truncated this section in the text.8) The liposome binding experiments appear to lack a negative control – i.e. liposomes that are not acidic such as those of pure PC.We have now included the data containing PC as a negative control.The manuscript has been improved but there are some remaining issues about the text that the reviewers agree need to be addressed before acceptance, as outlined below:1) In the summary the authors make a claim on defining the core principles of Commd homo- and heteromeric assembly. This is clearly an overstated claim as the interaction between Commd proteins seems to be quite promiscuous and still there are no clear principles of how homodimer-versus-heterodimer preference is achieved.We have toned down this statement in the summary to “Here, we have systematically characterised the interactions between several human COMMD proteins, and determined structures of COMMD proteins using X-ray crystallography and X-ray scattering to provide insights into the underlying mechanisms of homo- and heteromeric assembly.” We agree that our data still cannot provide a concise explanation for why specific homo and heterodimers are formed, but it does reveal new insights into how these interactions occur.2) Crosslinking data: The WRVD motif should be better introduced. The cross-linked results are interesting and narrow down the potential interface between Commd9 and CCDC93, yet the proposed WRVD motif seems to be part of the conserved dimerisation core . Based on the calponin homology domain of CCDC93, the structure of Commd9, the BS3 cross linking distance, and conserved residues on the surface, authors should be able to propose a plausible contact area in Commd9 despite the NMR unfruitful results.128WRVD131. The strictly conserved Trp128 in particular does make key contacts that appear to be required for COMM domain dimerization. But, unusually for a Trp side-chain, it is also surface exposed, suggesting it may be part of a binding surface for the CCDC93 NN-CH domain. However, we did not want to speculate too much based on our data and have therefore modified the text as follows:We apologise that this was not described clearly enough. We have now modified the text and also Figure 7 showing the sites of lysine cross-linking to better explain our finding. What we observe is that the two major sites of lysine cross-linking in the Commd9 COMM domain are part of a contiguous surface, and that this surface also includes the residues from the conserved sequence 128WRVD131, with Trp128 in particular being strictly conserved across the entire Commd family .”“Notably, K133 and K152 are located on contiguous surfaces of the β-sheets of the COMM domains , suggesting a likely binding surface for the CCDC93 NN-CH domain. This surface also includes the side-chains of the conserved residues 3) Figure 4: I am still not happy to see three different molecular models for full length Commd1, Commd7 and Commd9. It's just misleading, the envelopes look all the same. One representative model showing that the COMMD and HN domain fit into all envelopes would be completely sufficient and less confusing.We have removed the data showing the SAXS-derived solution structures of Commd1 and Commd7 into Figure 4—figure supplement 2, and retained only the SAXS-derived solution structure of Commd9 in the main text for clarity. We hope this satisfies the reviewer’s concerns.4) Following our initial advice, Figure 5 should go to the supplement.Figure 5 has now been moved to Figure 4—figure supplement 4. Subsequent figures have been renumbered accordingly, and figure citations in the text have been modified to reflect the changes.5) Typo: subsection “COMM domain binds to CCDC22 and CCDC93 through a conserved motif”, first paragraph – should be K133, not K131 (K131 is the residue in CCDC93).This has been corrected.6) Subsection “COMM domain binds to CCDC22 and CCDC93 through a conserved motif” – is missing a full stop.This has been corrected.7) Figure 3—figure supplement 1: aa numbers 151 to 200 are missing.This has been corrected.8) Figure 7 legend: Description for panel D is missing (CCDC93 interaction).This has been corrected.


The article presents the influence of structure formation on the properties of 321 metastable austenitic stainless steel in the process of cold radial forging (CRF). The steel under study after austenitization was subjected to CRF at room temperature with degrees of true strain (e) 0.26, 0.56, 1.00, 1.71 and 2.14. It has been shown that structure formation of the studied steel during CRF consists of three stages: formation of the lamellar structure of austenite, formation of the trapezoidal structure, and formation of the equiaxial grain structure. The kinetics of the strain-induced α′-martensitic transformation is related to the stages of structure evolution. Hardness, ultimate tensile strength and yield strength uniformly increase in all stages of structure formation with a significant decrease of elongation to fracture during the first stage of structure formation while the value of elongation to fracture remains constant in the subsequent stages of deformation. Impact strength of fatigue cracked specimens (KCT) decreases sharply at the first stage of structure formation and smoothly increases at the second and third stages. However, the impact strength of V-notch specimens (KCV) continuously decreases when deformation degree increases in the overall investigated deformation range. Metastable austenitic stainless steels (MASS), along with high corrosion resistance, impact strength and plasticity, have a rather low yield strength, which significantly limits the scope of their application. One of the most common ways to increase the yield strength of MASS is cold plastic deformation. It should be noted that significant structural changes occur as a part of this process. These changes are associated with the glide of dislocations, strain-induced twinning, formation of shear bands, precipitation or dissolution of particles, strain-induced martensitic transformation (SIMT), rotation of substructure elements and recrystallization ,2,3. FeaAt present, the processes of formation of structure and properties during cold plastic deformation of MASS by rolling ,10,11, tIt is known that during radial forging, the plastic flow develops inhomogeneously, and at the same time a fairly soft plastic deformation pattern is formed, which is close to uniform triaxial compression ,21. ThisWithin the present research, the commercial MASS was studied, which chemical composition is given in The initial rod ø33 mm of the 321 MASS being studied was obtained by hot forging within the temperature range of 1220–900 °C with subsequent cooling in the open air to the ambient temperature. Preliminary thermal treatment consisted of heating to 1050 °C, holding within one hour and subsequent cooling in water. After such treatment, the steel was in an austenitic state with the average grain size of 10 ± 1 µm. In addition, the structure showed a certain amount of large irregular-shaped particles of primary titanium carbonitride and rod axis oriented fields of δ-ferrite, the content of which did not exceed 2%. Then, the rod of the steel observed was deformed at room temperature in a radial forging machine SXP-16 with four radially moving hammers in the following mode: workpiece feeding rate 180 mm/min, stroke frequency 1000 blows per minute, workpiece rotation speed 25 rpm. The deformation center was water-cooled during forging. Forging was performed in five steps: deformation from ø33 to ø29 mm, from ø29 to ø25 mm, from ø25 to ø20 mm, from ø20 to ø14 mm and from ø14 to ø11.3 mm, which made up 0.26, 0.56, 1.00, 1.71 and 2.14 of true strain, respectively. The true strain (e) was calculated using the following equation [3COOH) and 5% of chloric acid (HClO4).Transmission electron microscopy (TEM) was performed on thin foils with the use of the FEI Tecnai 20 G2 TWIN microscope  at the accelerating voltage of 200 kV. Thin foils were obtained by means of preliminary cutting of flat templates with the thickness of 200 µm on aspark erosion machine from the central part of rods in cross and longitudinal axial section. Then, double-sided thinning was performed with the use of abrasive paper with P360, P800, P1200, P2000 grit size. Subsequent jet electropolishing of discs of 3 mm in diameter and thickness of 100 µm was performed with TenuPol-5 until perforation with electrolyte cooled down to −40 °C and consisting of 95% of acetic acid , yield strength (σ0.2), elongation to fracture (δ) and area reduction (ψ) were determined.Tensile tests were conducted by the Instron 5882 electromechanical testing system  at room temperature at the strain rate of 1 × 10ttest = −20, −60 °C) or in the air . Temperature control was performed with the use of the TM-8N thermometer and a L-type thermocouple with accuracy 1 °C. After testing precracked specimens (KCT) in the central part of fractures near the mouth of the fatigue crack, microfractographic analysis was performed with the use of the Hitachi S-3400N Christmas Edition scanning electron microscope  at the accelerating voltage of 20 kV. With the use of the obtained images of the fracture microsurface, the size of ductile fracture dimples was determined in two mutually perpendicular directions with the use of the Olympus Stream Motion software 1.8 .Impact strength testing for evaluation of the impact strength were performed on the Instron 9350  test bench with the dropped weight (hammer displacement speed at the moment of impact −4.3 m/s) and in the KM-30 impact testing machine (hammer displacement speed at the moment of impact 5.5 m/s) at room and reduced temperatures  on V-notch specimens (KCV) with the dimensions of 5 mm × 10 mm × 55 mm and on precracked specimens (KCT) with the dimensions of 5 mm × 11 mm × 55 mm. Specimens were cut from the central part of the rod in the axial direction. Before growing the fatigue crack on the specimen, a V-shaped concentrator with a depth of 1.5 mm was applied, from the top of it a fatigue crack with a length of 1.5 mm was further grown. The number of cycles for growing the fatigue crack was more 3000. Cooling of specimens with liquid nitrogen was performed in the cryogenic chamber in the ethanol  (As a result of CRF with the degree of  effect) b,c. The e = 0.56 leads to an increase in the twinning intensity inside austenitic grains. In other words, the proportion of microvolumes in which twinning develops in two systems increases, shear bands additionally appear  effect  effect e,f. In t) effect a. These ) effect b. The em) effect c.e = 1.00 and e = 1.71, the structure is transformed from predominantly lamellar to block trapezoidal and further to equiaxed grain, as evidenced by the presence of three types of areas in the structure  crystal lattice are also observed in the structure, however, according to the dark-field analysis, a small amount of ε-martensite with a hexagonal close-packed (HCP) crystal lattice is present in the package structure b. e = 2.14 leads to the formation of an equiaxed grain structure in the cross section of the rod  and yield strength (σ0.2) to the degree of the true strain are given in e = 2.14, the ultimate tensile strength made 1410 MPa with the yield strength of 1405 MPa. The value of the elongation to fracture (δ) for the steel under study decreases from 67% to 12% in the result of the deformation with the degree up to e = 0.56 and is practically maintained at this level with the increase in the deformation degree up to e = 2.14  at room temperature continuously decreases as the deformation degree increases from e = 2.14 a. Howeve dimples . However dimples a. In the dimples b,c, whicfinement  by increfinement d–f. It s2 at room temperature up to 1.2 MJ/m2 at the temperature of −190 °C  specimens made of steel under study in the initial state has an expressed thermal sensitivity within the temperatures range under study and decreases from 2.2 MJ/m −190 °C b. The ov2, the deformation develops mostly by the gliding of dislocations; if SFE is within the range of 18 to 35 mJ/m2, then the deformation is accompanied by twinning; if SFE does not exceed 18 mJ/m2, then the formation of strain-induced martensite is mainly observed. However, when SFE is below 13 mJ/m2, then ε-martensiteis formed instead of α′-martensite [2 [To identify the patterns of evolution in the structure of the 321 MASS, research was completed into the structural state in the longitudinal and cross section of a rod after varying degrees of deformation. According to the data given in reference , if the nsite [2 ,27,28, ie = 0.56) according to the twinning mechanism—TWIP effect, that forms the barrier for the glide of dislocations with the formation of dislocation cells , the process of strain-induced twinning on secondary systems is intensified, which leads to the transformation of the lamellar structure obtained at the first stage into a block trapezoidal structure in the cross section. However, it provides the ground for strain-induced martensite transformation (SIMT) in the result of the embryos formation and growth with further generation of martensite packages [At the second stage of structure formation dc=2αual to 1 ; γ—stack.9 mJ/m2 ,27,28.e = 2.14 when the structure is formed with the characteristic element close to cd—240 ± 10 nm, where almost no twining is observed. However, there are works [For the case here, the solution of this equation states that in theory the process of twinning is possible only for the states with a grain size exceeding 190 nm. This is satisfactorily consistent with the experimental results for the case after the deformation with the degree of Another possible reason for the almost complete absence of twins in the 321 MASS deformed structure is rod heating in the process of cold radial forging because of multiple dynamic impact of striking edges on the treated steel during CRF. According to some estimates, in the case of the dynamic deformation of metastable austenitic steel, the MASS heating at the deformation can reach 200 °C . HoweverThe literature contains various data on the mechanism of strain-induced α′-martensite formation in 304-type or 321-type MASS. Thus,  refers te = 0.56) the embryos of strain-induced α′-martensite  in the current moment; sf—maximum volume fraction of α′-martensite formed during the restriction of transformation under such deformation conditions; me—degree of true strain at 50% of sf; β—parameter defining the speed of transformation.Kinetics of strain-induced α′-martensite formation during deformation has an expressed sigmoidal nature b. In orde = 3.5 with the transformation volume fraction of 0.608. Maximum speed of the transformation is observed during deformation e = 1.5, which indicates the highest SIMT speed at the second stage of structure formation and its restriction at the third stage. This is due to the increase in the number of areas of formation and growth of α′-martensite embryos during the deformation with their subsequent exhaustion in the process of transformation [In the results determining the parameters of the equation (1), it was found that saturation of SIMT happens after the deformation at the level of ormation .This section discusses the change of mechanical properties depending on the peculiarities of the implemented structure formation process during CRF.At all stages of structure formation with CRF, the hardness and strength characteristics gradually increase as the deformation degree increases b, which The distribution of hardness over the cross section of the rod after CRF is non-uniform: thus, with the view to the increase in the general level of hardness, the maximum hardness is formed in the center of the rod, while the minimum hardness occurs at the half of the radius. This allows radial forging to be considered as a method of obtaining the non-uniform rod in its cross-section , along with well known methods of formation of non-heterogeneous gradient materials such as torsional deformation  and surfe = 0.56  specimen shows the relative work of the nucleation and growing of cracks. Thus, the increase in the degree of deformation is accompanied by the decrease in the origination of cracks, as the difference between the KCV and KCT. Within the interval of deformation from e = 0 to e = 0.56, the relative work of crack growth drastically reduces, which is caused by accumulation of the number of deformation twins [e = 0.56 to e = 2.14, the relative work of crack growth slightly increases and the relative work for crack initiation is sensibly reduced (see Impact strength of pre-cracked specimens (KCT) at the first stage of the structure formation and the elongation sharply decrease from the level of 1.58 MJ/mon twins ; while duced see a. This ie = 0.56, while impact strength drops almost by half in the temperature range from room temperature to −190 °C. In case of impact strength testing steel, which is deformed with a degree of e = 2.14, the impact strength decreases only by 25% in the investigated temperature range.The temperature dependence of the impact strength of the KCV V-notch specimens most clearly appears in the initial state and after deformation with the degree of When compared with the results of mechanical properties tests on 321 MASS steel obtained by other authors after treatment using other methods of structure formation, such as cryogenic and repetitive cold rolling, equal-channel angular pressing, high-pressure torsion, fast neutron irradiation, as well as post-deformation annealing in some cases, we have shown that patterns of change in the yield strength and elongation to fracture coincide with the data presented by other authors . HoweverThe research can be extended further to find the possibilities to increase the plasticity and strength without substantial reduction of strength properties through the process of recovery, recrystallization and reverse martensite transformation during post-deformation thermal treatment of the 321 MASS.e = 2.14 occurs in three stages in the rod cross section: in the first stage (up to e = 0.56), the lamellar structure of austenite is formed as a result of the formation of shear bands and deformation twins; in the second stage (e = 1.00–1.71), the lamellar structure is transformed into a trapezoidal one; in the third stage (e = 2.14), the equiaxed grain structure is formed. During CRF, the band austenitic-martensitic structure is formed in the longitudinal section. The kinetics of SIMT show that the onset is observed at the first stage when the martensite embryos formation occurs; the maximum speed of the transformation is registered at the second stage when α-martensite packages and a few areas of ε-martensite are formed; SIMT slows down at the third stage because of the exhausted areas of α′-martensite embryos formation and the stabilization of the remaining austenite due to the total structure refinement.The results of the research of the 321 MASS microstructure evolution during CRF have shown that the structure formation during deformation with degrees up to e = 2.14 does not cause a change in the level of elongation. Reduction of area smoothly decreases when the cumulative deformation degree increases. The impact strength of precracked specimens (KCT) as well as elongation decreases sharply at the first stage of structure formation, and there is a smooth increase at the second and third stages. However, the impact strength of V-notch specimens (KCV) continuously decreases when the deformation degree increases. The temperature dependence of the impact strength of the V-notch specimens (KCV) most clearly appears in the initial state and after deformation with a degree of e = 0.56, while the impact strength drops almost by half in the temperature range from room temperature to −190 °C. In case 321, MASS is deformed with a degree of e = 2.14, while the impact strength decreases only by 25% in the specified temperature range.At all identified stages of structure formation during CRF, the hardness and strength characteristics almost uniformly increase with the overall structure dispersion. The distribution of hardness over the cross section of the rod after CRF is non-uniform. A significant drop of elongation to fracture is observed at the first stage of structure formation. It is caused by the fragmentation of austenite by deformation twins and shear bands. An increase in the deformation degree up to


Hepatitis E viral infection is now emerging as a global health concern, which needs to be addressed. Mechanism of viral replicationand release is attributed by the different genomic component of HEV. However, few proteins/domain like X and Y domain remainunexplored, so we aim to explore the physiochemical, structural and functional features of HEV ORF-1 X domain. Molecular modelingof the unknown X domain was carried out using Phyre2 and Swiss Model. Active ligand binding sites were predicted using Phyre2.The X-domain protein found to be stable and acidic in nature with high thermostability and better hydrophilic property. Twelvebinding sites were predicted along with putative transferase and catalytic functional activity. Homology modeling showed 10 bindingsites along with Mg2+ and Zn2+ as metallic heterogen ligands binding to predicted ligand-binding sites. This study may help todecipher the role of this unexplored X-domain of HEV, thereby improving our understanding of the pathogenesis of HEV infection. Hepatitis E virus (HEV) is recently evolving as a global emergingdisease with neurological, haematological manifestations inaddition to acute and chronic liver infection , 2. WideHEV, a small (~32nm), non-enveloped, single stranded (+) senseRNA virus is the main aetiological agent of Hepatitis E infection. On the Recently, molecular study by Parvez MK, 2017  suggesteSo far, HEV ORF1X domain is known to interact with cellularADP-ribose protein (involved in host pathogenesis), as a putativeIFN antagonist in HEV replicating hepatoma cell , 18. AlsHowever, structure of this X domain is not reported yet. Also thedetailed physiochemical characterization and putative structurewith ligand binding active sites is not elucidated, so we proposedan in-silico 3-D structure prediction of HEV X domain usinghomology modelling.The amino acid sequence of X-Domain (HEV ORF 1) protein wasobtained from NCBI sequence database(http://www.ncbi.nlm.nih.gov/polyprotein/NP_056779.1). Themain source of the sequence with gene ID-1494415 of HEVgp01ORF-1; Seq: NC_001434.1 (4 … 5085). Polyprotein NP_056779.1;protein: protein structural polyprotein pORF1; Gene- ORF1;Organism: Hep E virus genotype 1 (IsolateHuman/China/HeBei/1987(HEV); UniProtKB:>sp/Q81862/785-942;Pfam (X-Protein/Domain: 785-942). Due tounavailability of 3-D structure in PDB, modelling of thisunexplored domain was undertaken utilizing 158 a. a. longsequence of X domain.Physiochemical properties of the retrieved sequence weredetermined using two web-based servers. ProtParam tool(Expasy) (http://us.expasy.org/tools/protparam.html)employed for the prediction of amino acid composition,instability and aliphatic indice, extinction coefficients and grandaverage of hydropathicity (GRAVY) . TheoretThe self-optimized prediction method with alignment (SOPMA)software  and PSIPProtein-protein binding sites were predicted by profISIS  byidentThere is no experimentally deduced 3D structure available for Xdomain protein in protein data bank (PDB), therefore homologymodelling of the protein (X domain) was done using twoprogram Swiss Model and Phyre2(http://www.sbg.bio.ic.ac.uk/phyre2) , 28. SecThe amino acid sequence of HEV X domain was retrieved inFASTA format andused as query sequence for determination ofphysiochemical parameters. The instability index of 37.18 (<40)indicated the stable nature of X domain protein . The proThe default parameters  were considered by SOPMA for the secondary structureprediction with >70% prediction accuracy. Utilising 511 proteins(sub-database) and 15 aligned proteins, SOPMA predicted 40.51%of residues as random coils in comparison to Alpha helix(34.81%), extended strand (20.25%) and Beta turn (4.43%) asshown in 42.41% helix confirmation , 44.30% loop (L)followed by 13.29% beta strand (E=extended strand in beta sheetconformation) was predicted in X domain. Intrinsic disorderprofile was computed using DISOPRED and >90% of the aminoacid are below the confidence score of 0.5 for disorderedcondition, suggested the lowest possibility of distortion andconferred the high stability to the predicted protein.Binding sites were predicted using predict protein software(profISIS), where 12 different protein binding sites wereidentified at positions viz.: 28-30; 46-47; 49; 59-60; 73-78; 88-89; 93;108; 128; 131-133; 135; 141 (data not shown). Gene ontologypredicted and categories the functional aspects as cellular,molecular and biological, where this X domain protein found tobe extracellular or the part of host cell or membrane; metabolicprocesses such as primary and cellular metabolic processesincluding cyclic, heterocyclic and aromatic compoundmetabolism processes (data not shown).Molecular function including binding (Score: 49) involvesheterocyclic (Score: 49), organic (49), cyclic compound binding;small molecule (Score: 32) and nucleic acid binding activity whereas and catalytic activity (Score: 26) includetransferase activity (Score: 19) with nucleotide transferase activity(Score: 40).X domain target sequence was inserted as input (fasta format) inSwiss-Model workspace. The Swiss-MODEL template library(SMTL) was searched with HHBlits  resultedStereochemical quality of the Swiss model predicted X-domainstructure was evaluated by plotting Ramachandran map (PROCHECK). 85.1% of the total residues (137) were found in thecore  whereas 13.2% of residues were in the allowed  regions. Disallowed region constitute of 1.8% of the residues.Good quality model of X domain was predicted by analyzing 118structures of good resolution (2.0 Å) and R-factor (<20%).PROCHECK analysis showed max deviation of 21.0 (residueproperties), with bond length/angle of 5.8 and 77.8% planargroups within the limits.Similarly, the homology modelling of X domain was performedby Phyre2.Based on the 6 templates , protein model was generated with87% of the residues modelled at >90% confidence  withcoordinates (A): X: 51.738, Y: 33.604, Z: 42.515 . Secondary structure prediction by Phyre2was described as Disordered (13%), Alpha helix (36%), and betastrand (22%) (data not shown).Phyre2 predicted structural model was evaluated for the stereochemicalquality using Ramachandran map (PROCHECK). The84.1% of the residues were found in the core  whereas12.1% of residues were in the allowed  regions. However2.3% residues were aligned in generously allowed region , whereas disallowed region constituted 1.5% of the residues.Among residual properties max deviation was 4.1, bondlength/angle 10.5 with 2 cis-peptides with 98.3% planar groupswithin limits.The model was structured based on multi-template/ab initiowith confidence score of 87. The 3D ligand site prediction in thefinal selected model was based on the cluster 1, showing 23ligands and 18 structures. Total of 10 binding sites werepredicted, at residue no. 21 (Asn: 12 contacts), 22 (Ala: 11contacts), no. 31-37 , no.131 (Pro: 10 contacts). Mg (1) and Zn (6) waspredicted as metallic heterogen ligands, binding to the predictedligand binding site of the modelled X domain .We report the structural model of HEV X domain with predictedactive site for ligand binding. This provides insights into thefunctional role of X-domain in viral pathogenesis.VT is the recipient of ICMR JRF/SRF fellowship provided byIndian Council of Medical Education and Research (ICMR), NewDelhi.None


Clitoria ternatea L. flower extract (CTE), on the inhibition of pancreatic α-amylase, in vitro starch hydrolysis, and predicted the glycemic index of different type of flours including potato, cassava, rice, corn, wheat, and glutinous rice flour. The application in a bakery product prepared from flour and CTE was also determined. The results demonstrated that the 1% and 2% (w/v) CTE inhibited the pancreatic α-amylase activity by using all flours as a substrate. Moreover, 0.5%, 1%, and 2% (w/v) CTE showed a significant reduction in the glucose release, hydrolysis index (HI), and predicted glycemic index (pGI) of flour. In glutinous rice flour, 1% and 2% (w/v) CTE had a significantly lower level of rapidly digestible starch (RDS) and slowly digestible starch (SDS) with a concomitant higher level of undigested starch. The statistical analysis demonstrated strong positive significant correlations between the percentage of CTE and the undigested starch of wheat and cassava. The addition of 5%, 10%, and 20% (w/w) CTE significantly reduced the rate of starch digestion of the wheat bread. The pGI of bread incorporated with 5% CTE (w/w) was significantly lower than that of the control bread. Our findings suggest that CTE could reduce the starch digestibility, the HI, and pGI of flour through the inhibition of carbohydrate digestive enzymes. Taken together, CTE may be a potent ingredient for the reduced glycemic index of flours.This study aimed to assess the effect of the  Carbohydrates are one of three basic macronutrients that produce energy for our body. Nowadays, edible flour enriched with carbohydrates has been made from several parts of plants, including roots, seeds, and germs . Flour iThe predicted glycemic index (pGI) is a common technique used to measure the rate of carbohydrate hydrolysis in foods . It has Clitoria ternatea L., commonly known as Butterfly pea, is a plant species belonging to the Fabaceae family. This plant is widely distributed in tropical zones such as Asia, the Caribbean, and Central and South America. In traditional Ayurvedic medicine, Clitoria ternatea L. has been used for treating stress and depression and enhancing memory [Clitoria ternatea has been reported to contain rutin, kaempferol, delphinidin, and related glycosides [Clitoria ternatea has been used as a colorant in various foods, beverages, and desserts in Asia. This colorant flower is regularly mixed with rice, bread, cookies, flours, and other traditional foods and desserts with a variety of ratios. For example, some traditional Thai desserts are made using cassava flour or glutinous rice flour mixed with butterfly pea juice at various concentrations to color and are then steamed until cooked. Moreover, sticky rice noodles are made by the mixture of rice flour with CTE juice. Although the pancreatic α-amylase and α-glucosidase inhibitory activity of CTE is well-documented, studies regarding its effect of pancreatic α-amylase action and in vitro starch digestibility using various types of flour have not been taken. Particularly, the potential food application of CTE in flour-based products remains unknown. Therefore, the aim of the present study was to investigate the effect of Clitoria ternatea L. flower extract on the activity of pancreatic α-amylase, in vitro starch hydrolysis, and predicted glycemic index of potato, cassava, rice, corn, wheat, and glutinous rice flour. The application in bread prepared from wheat flour and CTE was also determined.g memory . There hg memory , antipyrg memory , anti-ing memory , and antg memory . Clitoriycosides . Our recycosides . In addiCommercial flours including potato, rice, glutinous rice, wheat, corn, and cassava flours were purchased from a supermarket. Porcine pancreatic α-amylase Type VI-B  and 3,5-dinitrosalicylic acid were purchased from the Sigma-Aldrich Chemical Co., Ltd. . Amyloglucosidase was obtained from Roche Diagnostics . The glucose oxidase-peroxidase (GOPOD) kit was purchased from HUMAN GmbH .Clitoria ternatea L. was purchased from a local herbal drug store, Bangkok, Thailand. The extraction was performed according to a previous study [The dried flower of us study . The conFlour (0.25 g) was dissolved with 50 mL of boiled water at 100 °C and stirred for 10 min. The flour solution was allowed to cool at room temperature for 10 min. Furthermore, the CTE powder was dissolved in 0.1 M phosphate buffer saline (PBS) or 0.2 M sodium acetate buffer. The CTE solution was vortexed for 10 min. The CTE solution was added into the flour solution  and subjected to in vitro digestion.2SO3, and 1% NaOH in distilled water) and heated at 100 °C for 10 min. Then, 40% potassium sodium tartrate (250 µL) was added to stabilize the color. After cooling at room temperature, the absorbance was measured at 540 nm. The pancreatic α-amylase inhibitory activity was calculated as the percentage inhibition. A control was prepared using the same procedure, replacing the CTE solution with 0.1 M PBS.The activity of pancreatic α-amylase was carried out using a modified procedure of Adisakwattana et al. . Fifty mThe in vitro digestion of flour and CTE was performed according to a previous method with some modifications ,22. FiftThe starch fraction was calculated based on the in vitro starch digestibility of samples. Rapidly digestible starch (RDS) was calculated as the amount of glucose present in the sample at 20 min of the in vitro digestion, whereas slowly digestible starch (SDS) was calculated as the difference between the amount of glucose measured at 120 min and 20 min ,23. The Wheat flour and other dry ingredients as a % on the weight of flour basis, including sugar (5%), salt (1.8%), yeast (3%), Benecel methylcellulose, and hydroxypropylmethylcellulose (2%) , were mixed using a Kitchen-Aid bowl mixer at speed 53 rpm for 1 min. The levels of CTE incorporated into this formulation were 5%, 10%, and 20% (w/w) of the wheat flour basis, by adding to mix with other dry ingredients. Based on the wheat flour, vegetable oil (6%), white egg (40%), and milk (70%) were added and then mixed together at speed of 160 rpm for 10 min. After that, the batter was poured into a mold, placed at 30 °C and 90% relative humidity for 50 min, and baked at 150 °C for 40 min. The loaf was removed from the mold and cooled at room temperature. The bread sample was packed in a sealed polyethylene bag until analysis. After baking, the in vitro starch hydrolysis of the bread with or without CTE was determined to indicate the in vitro starch digestibility and pGI. The bread samples were weighed (1 g) and mixed with distilled water (10 mL) while stirring for 10 min. The in vitro starch digestion of the bread was performed according to the above-mentioned method. A control bread was prepared using the same procedure, without the CTE.p < 0.05 was considered statistically significant.The data were expressed as mean ± standard error of the mean (SEM). The statistical significance of the results was evaluated by one-way ANOVA using Duncan multiple comparisons, using SPSS version 22.0 . The Pearson’s correlation coefficients (r) were calculated between the CTE concentration and undigested starch. p < 0.05). As shown in p < 0.05). At 2% (w/v) CTE, the potato flour had the highest percentage of pancreatic α-amylase inhibitory activity, followed by glutinous rice, rice, wheat, corn, and cassava, respectively.The percentage of pancreatic α-amylase inhibitory activity after mixing the CTE into flour is shown in The results of in vitro starch digestibility of flour at different concentration of CTE are shown in p < 0.05). Interestingly, the addition of 2% (w/v) CTE caused the reduction of pGI of the glutinous rice, wheat, and cassava flour from the high value to the medium value (GI < 70).p < 0.05). The addition of CTE at 0.5%, 1%, and 2% (w/v) caused a reduction in the SDS content of glutinous rice, corn, and cassava flour (p < 0.05). However, the CTE did not alter the SDS content of potato flour. The observed results also found that only the glutinous rice flour significantly increased the undigested starch with the addition of CTE (p < 0.05). r = 0.650 and 0.758, respectively; p < 0.05). However, no significant correlation was observed between the concentration of CTE and other flours, including potato flour, rice flour, glutinous rice flour, and corn flour.The RDS, SDS, and undigested starch of flours are presented in p <0.05). As shown in Cross sections of bread made from wheat flour and CTE are shown in O-(2-rhamnosyl) rutinoside, and rutin [Starchy foods or ingredients are digested by amylolytic α-amylases and α-glucosidase enzymes, including maltase-glucoamylase and sucrase-isomaltase, at the brush border of the small intestine . Thereafnd rutin . In partnd rutin ,28. Morend rutin ,30. A stnd rutin  observednd rutin  also expnd rutin . The intnd rutin . FurtherFor nutritional purposes, the starch in food is generally classified into three categories, including rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) . In termSeveral studies have reported that the plant-based diets containing polyphenols alter the glycemic index of various foods. The current study found that the addition of CTE caused the rate of carbohydrate digestion and pGI of wheat bread to slow down. Our results are in agreement with Reshmi et al. , who repThe present study demonstrated that CTE could inhibit pancreatic α-amylase activity, leading to a reduction of maltose release from flour. The addition of CTE to flour alters in vitro starch digestibility, resulting in a reduction in the amount of glucose released from the various types of flour and a subsequent reduction of HI and pGI. Moreover, the addition of the CTE reduced the starch digestibility rate and pGI of wheat bread. The findings indicate that the addition of CTE into flour can inhibit the starch digestibility of flour through the inhibition of carbohydrate digestive enzymes, including pancreatic α-amylase and intestinal α-glucosidase. We suggest that CTE may be a potent ingredient for the reduced glycemic index of flours.


SHANK genes are associated with autism. Deletions and truncating mutations suggest haploinsufficiency for Shank3 as a major cause of disease which may be analyzed in appropriate Shank deficient mouse models. Here we will focus on the functional analysis of missense mutations found in SHANK genes. The relevance of most of these mutations for Shank function, and their role in autism pathogenesis is unclear. This is partly due to the fact that mutations spare the most well studied functional domains of Shank3, such as the PDZ and SAM domains, or the short proline-rich motifs which are required for interactions with postsynaptic partners Homer, Cortactin, dynamin, IRSp53 and Abi-1. One set of mutations affects the N-terminal part, including the highly conserved SPN domain and ankyrin repeats. Functional analysis from several groups has indicated that these mutations  interfere with the critical role of Shank3 for synapse formation. More recently the structural analysis of the SPN-ARR module has begun to shed light on the molecular consequences of mutations in the SPN of Shank3. The SPN was identified as a Ras association domain, with high affinities for GTP-bound, active forms of Ras and Rap. The two autism related mutations in this part of the protein, R12C and L68P, both abolish Ras binding. Further work is directed at identifying the consequences of Ras binding to Shank proteins at postsynaptic sites.Genetic defects in  Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by delayed acquisition of speech, deficits in social interactions and stereotypic behaviors. In recent years several molecular genetic studies in large cohorts of patients have shown that the pathogenesis of ASDs involves a strong genetic component  are major scaffold proteins of the postsynaptic density (PSD); via multiple interactions they connect different types of glutamate receptor complexes with signaling molecules and the actin cytoskeleton of the dendritic spine. The ability of Shank3 to multimerize via its C-terminal SAM domain has led to the suggestion that formation of Shank clusters is a key event in PSD assembly , ruling out a dominant effect on disease. Whereas, all other mutations are extremely rare, R300C is found 15 times in a database of exomes of 64.000, mostly healthy individuals (Exac database), suggesting that it could be a polymorphism rather than a pathogenic mutation. In contrast, P141A and Q312R variants occur de novo in the affected children  show a much milder behavioral phenotype than mice lacking most variants of the protein [Shank3B−/− mice , and in the long proline rich segment between PDZ and SAM domains which has been characterized as an “intrinsically disordered region.” As a consequence, it has been difficult to design functional assays where one could actually show that these mutations interfere with specific functions of the Shank3 protein. Intriguingly, mice lacking only the Ank repeat containing forms of Shank3 . In addition, the Ank domains are preceded by a conserved domain which was initially overlooked when Shank domains were assigned. We termed this the Shank/ProSAP N-terminal (SPN) domain. This about 90 amino acid domain is most similar to the so-called FUsing both unbiased approaches such as yeast two hybrid screening, as well as educated guesses based on functional data, several interaction partners for the Ank domain have been identified: (a) Sharpin, a cytosolic signaling protein  is similar to that observed in Ras association domains. Indeed we observed that the SPN binds to active (GTP bound) Ras family members  with high affinity, whereas GDP-bound Ras proteins do not bind.Initially we observed that the SPN domain is locked in an intramolecular interaction with the Ank domain, blocking access of Ank domain ligands α-fodrin and Sharpin  was fully restored to control levels by transfection with a shShank3-resistant form of WT Shank3 and the L68P mutant, suggesting that the L68P mutant supports normal excitatory synaptic transmission and does in this respect not result in a loss of function , the mGluRI signaling pathway leading to phosphorylation of ERK1/2 and CREB was impaired. Overexpression of shShank3-resistant Shank3 WT and also full-length mGluR5 were able to rescue DHPG-induced ERK1/2 activation, while the Shank3 R12C mutant was not able to do so  could be downstream effectors of Ras signaling, in a so far unidentified signaling pathway. Alternatively, Shank3 could function to sequester active G-proteins, thus limiting their availability for other pathways. Indeed, such a scenario has been observed as both Shank1 and Shank3 were reported to negatively regulate integrin activation. This occurs due to sequestration of active Rap1 proteins via the Shank SPN domain. Both SPN domain mutations (L68P and R12C) that disrupt the binding of Shank to G-proteins also impair the inhibitory effect of Shank3 on integrin activation. Overexpression of Shank3 constructs in the rat hippocampal neurons showed that WT Shank3, but not the L68P mutant suppress formation of filopodia by inhibiting integrin activity. Also, overexpression of Shank3 WT in Shank3 KO cortical neurons plated on laminin inhibited β1-integrin activity in neuronal growth cones, while overexpressed L68P mutant failed to do so  actually bind to the Ank repeats. There is no single motif in either interaction partner which has been mapped with some precision, and there is also no consensus between these three possible partners. Further analysis, accompanied by structural analysis of a possible Ank/ligand complex might help to elucidate the relevance of ASD-associated variants here.A second open question is: how do the deficits in molecular interactions which have been identified for several variants, actually correlate with the cellular phenotype observed in neuronal cells? This is currently most unclear for the R12C mutant, which alters F-actin content as well as the form of dendritic spines, which interferes with mGluR5 signaling and which disrupts the transsynaptic strengthening of synaptic connections (see Table All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.