Furthermore, ORF3 and methyltransferase competed with each other for associating with the X-domain. These findings provide molecular understanding of the interaction between the HEV macro domain, methyltransferase and ORF3, suggesting an important role of the macro domain in the life cycle of HEV.Hepatitis E virus (HEV) is a major cause of hepatitis in normal and organ transplant individuals. HEV open reading frame-1 encodes a polypeptide comprising of the viral nonstructural proteins as well as domains of unknown function such as the macro domain (X-domain), V, DUF3729 and Y. The macro domain proteins are ubiquitously present from prokaryotes to human and in many positive-strand RNA viruses, playing important roles in multiple cellular processes. Towards understanding the function of the HEV macro domain, we characterized its interaction partners among other HEV encoded proteins. Here, we report that the HEV X-domain directly interacts with the viral methyltransferase and the ORF3 proteins. ORF3 association with the X-domain was mediated through two independent motifs, located within its N-terminal 35aa (amino acids) and C-terminal 63-123aa. Methyltransferase interaction domain was mapped to N-terminal 30-90aa. The X-domain interacted with both ORF3 and methyltransferase through its C-terminal region, involving 66 Hepatitis E virus (HEV) causes acute viral hepatitis, which is a major public health concern in developing and resource poor countries2Hepevirus in the family of Hepeviridae. It is a positive-sense, single strand, nonenveloped RNA virus with a 7.2 kb genome. It consists of a 5′-noncoding region (NCR) of 27 to 35 nucleotides, followed by three known open reading frames: ORF1, ORF2 and ORF3 and a 3′-NCR of 65 to 74 nucleotides, ending with a poly (A) tail of variable length810121720HEV is the only member classified as a Among the domains of unknown function, the X-domain is designated as a macro domain, owing to its homology with the macro domain of the histone H2A225in vitro. However, they have relatively poor ADP-ribose 1″-phosphohydrolase activity22The macro domains are also present in many positive strand RNA viruses such as the SARS CoV (Severe Acute Respiratory Syndrome CoV), the Sindbis virus and the Rubella virus, where it is referred as the X-domainThis study was initiated to understand the role of the X-domain during the life cycle of HEV. We have identified the interaction partners of the X-domain among the HEV encoded proteins and demonstrate that the X-domain directly and selectively associates with the viral methyltransferase and the ORF3 proteins. Further investigations were carried out to map the interaction domain, evaluate the specificity and establish whether the interaction partners competed or cooperated with each other to form a multi protein complex. Significance of these interactions during the life cycle of HEV is discussed.Y2H gold and Mav203) were used to perform the assays. Y2H gold contains 4 independent reporters: AUR1-C [confers resistance to Aureobasidin A (Ar+)], HIS3 [permits growth on histidine deficient (H−) medium], ADE2 [permits growth on Adenine deficient (A−) medium] and MEL1 . These reporters are expressed upon successful interaction between the BD and the AD fusion proteins. Two different methods were followed to evaluate the interaction between the bait and the prey proteins in Y2H Gold: (a) replica plating of the cotransformants into histidine deficient growth medium containing increasing quantities of 3-AT , a competitive inhibitor of the enzyme imidazoleglycerol-phosphate dehydratase (encoded by the HIS3 reporter gene), which catalyzes the sixth step in histidine biosynthesis pathway. If two proteins interact with high affinity, more imidazoleglycerol-phosphate dehydratase is produced, which requires higher quantity of 3-AT to be inhibited. Therefore, ability of the cotransformants to grow on increasing amount of 3-AT reflects the strength of interaction of the test proteins. Growth of the colonies were monitored after 3 days of replica plating and relative growth was scored in a scale of “+” to “+++” where +, ++ and +++ indicates poor, average and strong growth, respectively. (b) Quantitative estimation of α-galactosidase activity (encoded by the MEL1 reporter gene) by performing a liquid α-galactosidase assay in vitro in the presence of its substrate p-nitrophenyl-α-D-galactoside. Mav203 contains HIS3 [permits growth on H− medium], URA3 [permits growth on Uracil deficient (U−) medium] and lacZ  reporters to monitor the protein-protein interactions. Strength of the interaction between the bait and the prey proteins in Mav203 was evaluated by scoring the colony growth on increasing concentrations of 3-AT, as mentioned above.In order to identify the direct interaction partners of the HEV macro domain, a GAL4 transcription factor based Yeast Two Hybrid (Y2H) assay was designed. Two different host strains  and expression of fusion proteins were confirmed by western blot of the whole cell extract of the transformed colonies using an antibody against the myc-tag, located between the GAL4 BD and our protein of interest. All the constructs produced expected size of the fusion protein  to monitor self-activation of the reporter genes. No background reporter activity was observed in the AD X+BD cotransformed colonies , genotype-3 ORF3 fused to BD (BD g3ORF3) and genotype-3 methyltransferase fused to BD (BD g3Met) produced similar results as in genotype-1  and S2. A coimmunoprecipitation (CoIP) assay was performed to confirm the above observations. Huh7 human hepatoma cells were transiently transfected with expression plasmids coding for the C-terminal HA-tagged X (pUNO X-HA) and N-terminal myc-tagged ORF3 (pVITRO ORF3-myc) or empty vector (pUNO). 48 hours post-transfection, whole cell extract was immunoprecipitated (IP) with either rabbit pre immune serum (RS) or HA and myc antibodies. As expected, both HA and myc antibodies could pull down ORF3 and X-domain, respectively, confirming their association . SimilarA series of deletion mutants of the ORF3, methyltransferase and X-domain were generated to map the minimal region of interaction. Y2H analysis of the interaction potential between the full length X-domain (AD X) and 7 overlapping deletion fragments of the ORF3  and 7 overlapping deletions of the methyltransferase (BD D1-D7 met) revealed that removal of 210aa from the C-terminus significantly increased the strength of the interaction,  to glutamic acid (E) R93E or 94 leucine (L) to arginine (R) L94R lead to partial destruction of the β-sheet present between 90th-111thaa, whereas altering 101 and 102 leucines (L) to glutamic acid (E) LL101,102EE lead to almost complete disappearance of the same β-sheet. Except for the 94th L, which was conserved in 60% sequences, all other altered amino acids  were fully conserved across all HEV isolates analyzed  of the X-domain was sufficient for interacting with the ORF3 . Howeveranalyzed . SecondaAbove mentioned point mutations were introduced into the full length X-domain, followed by Y2H analyses. R93E or L94R substitutions were ineffective in preventing the interaction between the X and the ORF3 . HoweverCoIP assays were performed to confirm the above observation. The LL101,102EE mutations were introduced into the pUNO X-HA plasmid (named pUNO D11 X-HA). Huh7 cells were transfected with the empty vector (pVITRO) or pVITRO ORF3-myc along with the wildtype X (pUNOX-HA) or LL-EE mutant X (pUNO D11 X-HA). Immunoprecipitation of the whole cell extract with HA antibody followed by immunoblotting with myc antibody demonstrated coimmunoprecipitation of ORF3 only from cells expressing the wild type X-domain, but not from cells expressing the LL101,102EE mutant X .−, LTH−Ar+ and LTH−+5 mM 3-AT media  pull down assay using partially purified recombinant proteins. GST fused X or LL101,102EE mutant X (D11 X) and His-tagged ORF3 or methyltransferase were affinity purified from ectively . Both ORectively . Specifiectively .Both ORF3 and methyltransferase lost the ability to interact with the LL101,102EE mutant X-domain, raising the possibility that they might compete with each other for binding to the X-domain. A pull down assay was performed to evaluate whether the ORF3 and methyltransferase competed with each other for binding to the X-domain. Approximately same molar equivalents (500 fmol) of the ORF3 and the methyltransferase proteins were allowed to bind with 500 fmol of the GST-X protein. Bound fractions were compared to that obtained by incubating 500 fmol ORF3 with GST-X or 500 fmol methyltransferase with GST-X. With reference to the input amount  of pBRIDGE vector and methyltransferase was cloned in the MCS2, under control of the MET 25 promoter (active only in the absence of methionine). The BD ORF3-Met and AD-X cotransformants revealed that ORF3 interacted with the X-domain in the presence of methionine (1mM methionine was supplemented in the medium to suppress MET25 promoter activity). Upon replica plating of the same colonies onto various selection media lacking methionine, growth was observed only on the LTM− plates  assay− plates . No growal plate  indicatial plate . As a coal plate . Similaral plate . We alsoal plate  thus rulTaken together, both the pull down and Y3H assays demonstrate that ORF3 and methyltransferase compete with each other for binding to the X-domain and they do not form a trimeric complex. Further, as there is no direct interaction between ORF3 and methyltransferase, relative abundance and affinity of these proteins dictate their ability to associate with the X-domain.Macro domains and RNA methyltransferase are present in a wide range of living organisms, including human, which is a host for the HEV. Since the HEV macro domain and RNA methyltransferase interacted with each other, we wondered whether such interactions are also prevalent in human and whether the virus and the host counterparts cross-talk with each-other.Y2H analysis of the HEV X-domain interaction with 4 different human RNA methyltransferases did not provide any positive result . Y2H anaNext, Y2H analysis was conducted to identify possible interactions between human macro domain proteins and RNA methyltransferases. Out of the 4 human RNA methyltransferases and 7 macro domain proteins tested, only human CMTR2 was able to associate with C20orf133 . These ein vivo set-up. Use of two different yeast strains and domain swapping ruled out the possibility of experimental artifacts; (b) CoIP assays established that the interactions obtained in Y2H assay were strong enough to be reproduced in human hepatoma cells, in the presence of many other potential host interaction partners of the X, methyltransferase and the ORF3; (c) an in vitro pull down assay using partially purified recombinant proteins confirmed that the two interaction partners possess the required motif/signature sequence to associate with each other. Only those viral factors, which could interact with the X-domain in the above three assays were considered as true interaction partners. Therefore, according to our data, only viral methyltransferase and ORF3 happen to be the direct intra-viral interaction partners of the X-domain. Viral helicase activated only two of the four reporter genes in the Y2H gold based assay. Neither did it show positive interaction in the Mav203 based Y2H assay nor associated with the X-domain in the CoIP assay. Therefore, it is not a direct interaction partner of the X-domain. This finding is in contrast to a recent report published by Osterman and coworkersIn this study, we have identified and characterized the intra-viral interaction partners of the X-domain, which is a macro domain protein encoded by the HEV. Three independent assays were conducted to validate the true interaction partners: (a) Y2H assay identified direct interaction partners of the X-domain in a heterologous and Our efforts at mapping the region within the ORF3 protein that interacts with the X-domain identified two distinct regions: an N-terminal hydrophobic domain I and a C-terminal proline rich region3233Viral methyltransferase interacted with the X-domain through its N-terminal region, the N-terminal 30-60aa being the minimum sequence required for the association. N-terminal 1-60 and 1-90aa of the methyltransferase could grow better than the full length protein and displayed more α galactosidase activity, indicating their stronger binding with the X-domain than the full length methyltransferase. These data also suggests that the C-terminal region might be imposing structural constraints by masking the motif(s) required for associating with the X-domain or some other host protein may bind to the C-terminal region in such a manner that the N-terminal motif(s) remain inaccessible. Since HEV methyltransferase is known to be involved in capping of the 5′-end of the viral genomic RNA6Our study also reveals that the interaction between RNA methyltransferase and macro domain proteins is not restricted to the HEV. In our limited analyses, we observed that one of the human RNA methyltransferases, CMTR2, interacts with the human macro domain protein C20orf133. CMTR2 is a cap specific RNA methyltransferase having mRNA (nucleotide-2′-O ribose) methyltransferase activity. It is involved in 7-methylguanosine mRNA capping, like the HEV methyltransferase and is also conserved in a broad range of organismsDeletion and subsequent point mutation studies identified that two consecutive leucine residues  located towards C-terminus of the X-domain are essential for its binding with both ORF3 and methyltransferase. Comparison with known protein binding motifs did not indicate any homology of the dileucine-containing region to known motifs. Thus, it is unlikely that the two leucine residues are essential amino acids of a linear motif. It could be possible that they maintain a favourable structure or permissive electrostatic environment for binding of the X-domain with the ORF3 or methyltransferase. Changing leucine to glutamic acid might be collapsing the structure or altering the electrostatic environment . Similar mechanism may hold true for the observed effect of the II-EE mutation.Our data also suggests that the methyltransferase differs from the ORF3 in binding to the X-domain because the II66,67EE mutation, which partly reduced the binding of the ORF3 to the X-domain, had no effect or mild stimulatory effect on the methyltransferase binding to the X-domain. Thus, the methyltransferase and the ORF3 might be associating with different epitopes/conformations/structurally distinct regions of the X-domain. Structural studies are essential to delineate the actual mechanism. Nevertheless, genetic mutants identified in this study will be instrumental in investigating the functional significance of the interaction between X and methyltransferase or ORF3 during the life cycle of HEV. The II66,67EE and LL101,102EE mutations can be introduced in the HEV genome to investigate the functional significance of the interaction between the ORF3, methyltransferase and the X-domain. It is worth mentioning that the C-terminal region of the HEV macro domain is highly conserved: the 66, 67 isoleucine and the 101, 102 leucines are absolutely conserved across all genotypes and isolates of the HEV. Moreover, 101, 102 leucines are also conserved in the macro domains of other positive strand RNA viruses such as Rubella, Sindbis, SARS and Semliki forest Virus. It would be interesting to investigate whether the macro domains of these viruses interact with their methyltransferases.Through Y3H and GST-pull down assay, we have demonstrated that the ORF3 protein displays a higher affinity for binding with the X-domain than the methyltransferase. ORF3 is a viral late protein, synthesized from the subgenomic RNA, which is generated after replication of the genomic RNA4142Y2H gold strain, Aureobasidin A (630466) and X-α gal (630463) were from Clontech . Mav203 strain was from Life Technologies . Amino acid supplements, 3-Amino-1, 2, 4-triazole , IPTG (I6758) were from Sigma-Aldrich . Anti-His (SC-57598), anti-myc (SC-789) and anti-Flag (SC-807) antibodies were from Santa Cruz Biotechnology . Lysozyme (10153516103) was from Roche . Ni NTA agarose (25215) and Glutathione agarose (16100) were from Thermo Scientific . Peptide affinity purified rabbit polyclonal antibody against the macro domain (X) of HEV was generated at Genscript  using KLH (Keyhole Limpet hemocyanin) conjugated peptide antigens. Peptide sequence is: CPDYRLEHNPKRLEA. Pre immune serum was collected from the animals prior to immunization. ELISA using peptides as antigen and pre immune serum as negative controls confirmed their titres to be >1:512,000. In competition assays, corresponding peptides could block the binding of cognate antibodies. Functionality of the antibody in western was validated by testing its ability to detect the purified X-domain (from bacteria) and the X-domain expressed in Huh 7 cells.Yeast vectors, Desired fragments were either PCR amplified using specific primers and template or isolated by restriction digestion and ligated into the target vector following standard protocolsY2H Gold and Mav203 strains were transformed using Lithium Acetate, following manufacturer’s instruction. Transformation mixture was plated on LT− Synthetic Dropout media. Three days post-transfection, colonies were used for replica plating, whole cell extract preparation or quantitative liquid α-galactosidase assay. For whole cell extract preparation, spheroplasts were prepared by treatment with Zymolyase (5units/106 cells), followed by lysis in Laemmli buffer .Y2H Gold or Mav203 cells were grown on LT− plates, followed by replica plating into required selection medium. For Y2H Gold, LTH−, LTHA−, LTH−Ar+, LT− +X-α gal, LTH−+5 mM 3-AT, LTH−+10 mM 3-AT and LTH−+20 mM 3-AT were used. Plates were grown for 3 days at 30 °C in a humidified incubator, followed by visual scoring of colony growth in a scale of “+” to “+++” where +, ++ and+++ indicates poor, average and strong growth, respectively.Lithium Acetate transformed Y2H gold along with the AD X, in the required combination and plated on LT− plates supplemented with 1 mM methionine (compared to 0.13 mM methionine in regular Y2H selection plates). Eight random colonies from each plate were replica plated into LTHA−, LTH− Ar+, LT−+X-α-gal, LTH−+5 mM 3-AT and LTH−+10 mM 3-AT plates, supplemented with 1 mM methionine. A second set of replica plating was done into LTM− plates to induce MET 25 promoter dependent protein expression from MCS2. Four days post incubation at 30 °C in a humidified chamber, colonies from LT− or LTM− plates were again replica plated into various selection media containing 1 mM methionine or no methionine. Colony growth was scored on day four-post incubation. Data are representative of three independent experiments.pBRIDGE vector containing GAL4-BD fused ORF3 cloned under the control of constitutive ADH1 promoter (in MCS1) and methyltransferase in a second expression cassette (in MCS2), under the control of methionine deprivation induced MET25 promoter, was used to perform the Yeast Three Hybrid assayY2H Gold, following manufacturer’s instruction . Briefly, Y2H Gold cotransformants expressing the test protein pairs to be analyzed were inoculated in 5 ml of LT− or LTM− medium in triplicates and grown till the A600 reached ~1.0. A600 of each culture was noted for downstream calculations. Cells were collected by centrifugation at 3000 g, resuspended in 100 μl of Z buffer , lysed by three times freeze-thaw in liquid nitrogen, followed by centrifugation at 10,000 g for 10 minutes to clarify the extract. 16 μl of the extract was assayed in triplicate for each sample in a 96 well plate. 43 μl of assay buffer  was added to each well and incubated at 30 °C for 60 minutes. Reaction was terminated using 136 μl of stop solution (1 M Na2CO3 in deionized H2O). Absorbance was measured at 410 nm. α-galactosidase units were calculated using the following formula after subtracting the obtained readings from that of the blank sample. [milliunits/(ml × cell)] = A410 × Vf × 1,000/[(ε × b) × t × Vi × A600], t = elapsed time (in min) of incubation, Vf = final volume of assay, Vi = volume of culture medium supernatant added, A600 = Absorbance of overnight culture, ε × b = p-nitrophenol molar absorptivity at 410 nm × the light path (cm) = 10.5 (ml/μmol) for 200-μl. α-galactosidase units are represented as mean ± SEM, analyzed using “GraphPad Prism 5.0” by the student t- test (two-tailed). P < 0.05 was considered significant.A liquid α-galactosidase assay was performed to quantitate the α-galactosidase activity in 2. Cells were transfected using Lipofectamine 2000, following manufacturer’s protocol .Huh7 human hepatoma cells were maintained in Dulbecco’s modified Eagle medium (DMEM) containing 10% Fetal Calf Serum (FCS), 50 I.U./mL Penicillin and Streptomycin, in 5% CO43−, 137 mM NaCl, 2.7 mM KCl) and lysed in 500 μl of Co-immunoprecipitation (CoIP) buffer  supplemented with protease inhibitor cocktail . The lysates were clarified by centrifugation for 10′ at 13,000 g. For co-immunoprecipitation, an equal amount of protein was incubated with 1 μg of corresponding antibody overnight at 4 °C, followed by incubation with 100 μl of 10% protein A-sepharose beads for 1 hour. The beads were washed three times in the CoIP buffer, incubated at 95 °C in Laemmli buffer  for 5′ and resolved by SDS-PAGE, followed by western blotting using required primary antibodies and corresponding horseradish peroxidase-conjugated secondary antibodies. Protein signals were detected by Enhanced Chemiluminiscent using a commercially available kit .48 hours post transfection; cells were washed once with phosphate-buffered saline  and treated with 1 μg/ml lysozyme followed by sonication. Soluble proteins were incubated for 30 minutes with Glutathione agarose beads, followed by washing thrice in PBS. The fusion protein was eluted by incubation in 20 mM Glutathione in 50 mM Tris (pH- 8.0). N-terminal His-tagged methyltransferase cloned in pET28a vector was transformed into Rosetta pLysS cells. Cells were grown to A600 ~0.5 and induced with 0.5 mM IPTG for 16 hours at 18 °C. Pellet was resuspended in Buffer B  and treated with 1 ug/ml of lysozyme followed by sonication. Soluble methyltransferase protein was precipitated in 30% Ammonium Sulphate, resuspended in buffer C (Buffer B + 20 mM imidazole) and incubated with Ni-NTA superflow agarose for 2 hours at 4 °C, followed by washing thrice in Buffer C. Bound protein was eluted in 500 mM imidazole. His-tagged ORF3 expression vector (pRSET ORF3) has been described600 ~0.5, induced with 0.5 mM IPTG for 16 hours at 37 °C, resuspended in Buffer D  and sonicated. Inclusion bodies were recovered, solubilized in Buffer E  and incubated at room temperature for 1 hour, followed by centrifugation at 15,000 g for 20′ at 10 °C. Supernatant was diluted in Buffer F  and loaded on HisTrap FF column . Bound protein was eluted using Buffer G (Buffer F + 250 mM imidazole). Imidazole was removed by dialysis. In all cases, purification steps were monitored by SDS-PAGE followed by Coomassie blue staining and western blotting using anti-X (for GST-X), anti-His (for methyltransferase and ORF3) and anti-ORF3 (for ORF3) antibodies. Approximate concentrations of purified proteins were determined by running them along with known quantities of purified Bovine Serum Albumin and quantifying the band intensities.Wild type and LL-EE mutant X was cloned as GST fusion into bacterial expression vector pGEX4T-1 and transformed into BL-21pLysS cells. Cells were grown to AA pull down assay was conducted using the purified wild type X (GST-X) or LL-EE mutant (GST-D11) X proteins as bait. 25 ng of the GST-tagged protein was mixed with the same quantity of purified ORF3-His or Met-His in PBS containing protease inhibitor cocktail, 25 μl of Glutathione agarose beads and incubated on a flip flop shaker at 4 °C for 1 hour. Samples were centrifuged at 500 g, 1′, 4 °C to settle the beads containing the bound proteins. Beads were washed thrice in PBS + 0.1% Triton X 100. Bound proteins were eluted by incubation at 95 °C for 5′ in Laemmli buffer. Aliquots of the eluted proteins were resolved by SDS-PAGE and western blotted using anti-X and anti-his antibodies. 40% (10 ng) of proteins used for the pull down assay were resolved as input.For monitoring competition between the methyltransferase and the ORF3 proteins for binding to the X-domain, a pull down assay was conducted by using approximately same molar equivalents of each protein (~500 fmol) in the binding mixture. Based on the size of the fusion protein and quantitation obtained from comparison with BSA standard in coomassie stained gel, 1 pmol of GST-X, Met-His and ORF3-His corresponded to 50 ng, 40 ng and 16 ng proteins, respectively. For comparing the affinity of interaction, increasing quantity of the Met-His or ORF3-His (1 and 2 pmols) was added without altering rest of the parameters.How to cite this article: Anang, S. et al. Identification of critical residues in Hepatitis E virus macro domain involved in its interaction with viral methyltransferase and ORF3 proteins. Sci. Rep.6, 25133; doi: 10.1038/srep25133 (2016).


The surface properties and dynamics of PKD-like domains from ColG and ColH differ. Clostridium histolyticum collagenases ColG and ColH are segmental enzymes that are thought to be activated by Ca2+-triggered domain reorientation to cause extensive tissue destruction. The collagenases consist of a collagenase module (s1), a variable number of polycystic kidney disease-like (PKD-like) domains (s2a and s2b in ColH and s2 in ColG) and a variable number of collagen-binding domains (s3 in ColH and s3a and s3b in ColG). The X-ray crystal structures of Ca2+-bound holo s2b  and holo s2a , as well as of Ca2+-free apo s2a  and two new forms of N-terminally truncated apo s2 , are reported. The structurally similar PKD-like domains resemble the V-set Ig fold. In addition to a conserved β-bulge, the PKD-like domains feature a second bulge that also changes the allegiance of the subsequent β-strand. This β-bulge and the genesis of a Ca2+ pocket in the archaeal PKD-like domain suggest a close kinship between bacterial and archaeal PKD-like domains. Different surface properties and indications of different dynamics suggest unique roles for the PKD-like domains in ColG and in ColH. Surface aromatic residues found on ColH s2a-s2b, but not on ColG s2, may provide the weak interaction in the biphasic collagen-binding mode previously found in s2b-s3. B-factor analyses suggest that in the presence of Ca2+ the midsection of s2 becomes more flexible but the midsections of s2a and s2b stay rigid. The different surface properties and dynamics of the domains suggest that the PKD-like domains of M9B bacterial collagenase can be grouped into either a ColG subset or a ColH subset. The conserved properties of PKD-like domains in ColG and in ColH include Ca2+ binding. Conserved residues not only interact with Ca2+, but also position the Ca2+-interacting water molecule. Ca2+ aligns the N-terminal linker approximately parallel to the major axis of the domain. Ca2+ binding also increases stability against heat and guanidine hydrochloride, and may improve the longevity in the extracellular matrix. The results of this study will further assist in developing collagen-targeting vehicles for various signal molecules. The two classes of collagenase, ColG and ColH, differ in domain structures  leads to higher affinity (Kd = 3.39 × 10−7 M)  and bacterial collagenases -fusion protein was achieved using previously described methods . Using the homology-modeled s2  was crystallized from 3 M ammonium sulfate, 0.1 M MES pH 4.5, 15%(w/v) PEG 4000 at 290 K, whereas holo s2a (at a concentration of 4.8 mg ml−1) was crystallized from 30%(w/v) PEG 4000, 0.2 M MgCl2, 0.1 M Tris–HCl pH 8.5 at 17°C. These crystals were subsequently soaked in 15%(w/v) PEG 4000, 0.2 M MgCl2, 50 mM CaCl2 , 0.1 M Tris–HCl pH 8.5 before initial unit-cell characterization and data collection. Meanwhile, holo s2b (at a concentration of 13.7 mg ml−1) was crystallized from 35%(w/v) PEG 5000, 0.2 M ammonium sulfate, 0.1 M MES pH 6.5 at 277 K. Both crystal forms of s2 (12.0 mg ml−1) were grown from 41–49% 2-methyl-2,4-pentanediol (MPD), 100 mM bis-tris pH 5.5, 0.1–0.3 M ammonium acetate at 277 K. The in-house X-ray diffraction facility  was used for initial characterization of each PKD-like domain crystal, and in the case of the s2a crystals was also used for data collection at 113 K. s2b and s2 crystals were cryocooled and subsequently stored in liquid nitrogen until data collection. Diffraction data were collected at 109 K on the 19-ID beamline of the Advanced Photon Source at Argonne National Laboratory, USA.Initial conditions suitable to grow crystals of apo s2a and holo s2b and two crystal forms of apo s2 were identified by the sitting-drop method using a high-throughput screen . Subsequent crystallization trials using the initial conditions were carried out using the hanging-drop method. Apo s2a  was used as the search model during the structure determination of s2. s2 form I was subsequently used as the search model during structure determination of s2a and s2b. Four molecules were found in the asymmetric unit of the apo s2a crystals, while eight molecules were found in the asymmetric unit of the holo s2a crystals. Meanwhile, two molecules were found in the asymmetric unit of the holo s2b crystals and each form of the apo s2 crystals.Each s2a data set was indexed and scaled using MIFit . In the thermal denaturation trials for s2b and s2, the protein concentration was 3 µM. In the chemical denaturation trials, the protein concentration was 1.5 µM. In the case of s2a, the protein concentration for both thermal and chemical denaturation was 5 µM. Each holo PKD-like domain was supplemented with 2 mM CaCl2, while each apo PKD-like domain was supplemented with 2 mM EDTA. In all cases, the protein was diluted in 10 mM Tris–HCl pH 7.5, 100 mM NaCl. When heat was used as the denaturant, each domain was exposed to temperatures that linearly increased from 280.5 to 363 K in 2.5 K increments. When guanidine hydrochloride (Gu–HCl) was used as the denaturant, each domain was exposed to concentrations of denaturant that increased linearly from 0.0 to 5.8 M in 0.2 M increments. ΔGHOH, CM and m values were calculated as described previously , Ca2+-free apo s2a (space group P61), Ca2+-bound holo s2b (space group P21) and two new forms of N-terminally truncated wild-type apo s2 (space groups P21 and P212121) are reported for the first time. Of the novel s2a and s2b structures, that of s2b was solved at higher resolution and therefore is described in the most detail. New insights into s2 are subsequently reported.The X-ray crystal structures of Ca3.1.a). The eight holo s2a molecules are virtually identical (average r.m.s.d. of 0.2 ± 0.1 Å). Here, the molecules spiral along the crystallographic  axis. Along this axis, molecule pairs A and G, B and E, C and F, and D and H are related by NCS translation that results in an off-origin peak that is 63.9% of the origin peak in the Patterson map.In the following discussion, holo s2a will be described first Fig. 2a. The eC and D being the most similar (r.m.s.d. = 0.2 Å) and molecules A and D being the most divergent (r.m.s.d. = 0.8 Å). Molecules A and B, as well as molecules C and D, are related by a noncrystallographic twofold. Temperature factors for each structure are relatively high , with molecules h Table 1, possibl2+ reorients the interacting residues Asn685 and Ser686. While neither structure could be refined using anisotropic B factors, comparison of B factors revealed that with the exception of the N-terminal residues, no significant change in B factors occurred upon Ca2+ binding.The holo and apo structures resemble each other (r.m.s.d. = 0.6 ± 0.2 Å). As expected, the most notable difference between the structures occurs near the N-terminus, where CaA through an approximately 126° rotation of the ψ bond of Ile692. To accommodate the change, Tyr696 packs with Phe706 and is involved in a Δ4 Tyr corner, in which the side-chain hydroxyl group hydrogen-bonds to the backbone four residues prior to it . A second bulge between β-strands B and B′ is stabilized by a hydrogen-bonding network that features a monodentate interaction between Asn735 and Ser708.Unlike s2b or s2, s2a truncates β-strand 3.2.2+ was found near the N-terminal linker . Each holo s2b structure begins at residue 766 and the last residue is 860. The PKD-like domain resembles a V-set Ig fold that lacks two strands (Omit β-strand C′ of the Ig fold corresponds to β-strand D of the PKD-like domain fold). β-strands B, D, and E in the PKD-like domain structures are shorter than the corresponding β-strands of a prototypical V-set Ig fold . In the structures, one Caer Fig. 2c. Each A and B and help to form a ridge along the ABE face . The second bulge helps to form a prominent ridge. Surface-exposed aromatic residues are found on both sides of the ridge and are discussed later.Two prominent features are the conserved bulges in the domain that interrupt β-strands ce Fig. 2. The fir98 Fig. 2c. The sB factor for molecule A is 11.7 Å2 and that for molecule B is 9.9 Å2). Anisotropic B-factor analyses using the Anisotropic Network Model (ANM) web server .Despite being solved in two crystal forms, the crystal structures of forms I and II of apo s2 from ColG are similar. Each asymmetric unit contains two noncrystallographic twofold symmetry-related molecules. All four molecules are virtually identical to each other (r.m.s.d. of <0.5 Å). Each structure begins at residue 685, although the first two residues (Gly-Ser) are remnants from GST-tag cleavage. The last residue is either 770 or 771. The NCS-related molecules are stabilized by antiparallel-type intermolecular interactions between β-strands 09 Fig. 2d.3.4.2+ chelation. The residues involved in Ca2+ chelation will be discussed in the next section. Peptidase M9 family members are all thought to be collagenases and consist of subfamilies A (Vibrio) and B (Bacillus and Clostridium). M9A enzymes lack CBDs, and consequently may utilize different approaches to collagenolysis. Therefore, this section will discuss M9B-derived PKD-like domains exclusively, and will utilize s2b numbering. The PKD-like domains found in M9B enzymes share two conserved clusters of residues . This stretch is conserved for its Ca2+-binding and architectural importance. The first two Asp residues chelate Ca2+. The invariant Tyr810 is accommodated by the second β-bulge. Asp813 is sometimes replaced by an acidic Glu residue. The side chain of Glu may easily fulfill the role of the side chain of Asp813, which terminates β-strand C and stabilizes the subsequent sharp turn by hydrogen-bonding to the amide of Gly815. Phe814 stacks against the conserved His828 so that the imidazole ring can also form a salt bridge with the conserved Asp816. Gly817 allows Asp816 to also stabilize the turn by hydrogen-bonding to the amide of Ser818. Although a sharp turn follows β-strand C in all PKD-like domains, the type of turn is different. In s2b, the insertion of Asp819 results in the region adopting an ω-loop (i→i + 10). Both s2a and s2 lack Asp819 in this stretch, and subsequently each is involved in an α-turn (i→i + 4) that forms a β-hairpin. The second conserved stretch, 825NPxxYHxxxxYxVxLxVxDGxxG847, forms β-strands E and F. Tyr830 and Tyr836 stack against each other to stabilize the interactions between the sheets. Tyr836 further stabilizes the sheets by forming a Δ4 Tyr corner. Asp844 is responsible for one of the axial interactions with Ca2+. The β-strands also wrap around the conserved turn and β-strand C to form the most unique feature of the PKD-like domain. Gly847 allows β-strands F and G to be separated by a β-turn (i→i + 3).Sequence comparison of divergent PKD-like domains revealed conserved residues that are essential for the overall fold and for Caes Fig. 3. The fir3.5.2+ coordinations in s2a, s2b and s2 are virtually identical to each other. Since holo s2 has been described, this section describes the binding sites in s2a and s2b in detail. One calcium-binding site with a pentagonal bipyramidal geometry was identified near the N-terminus in each domain . The calcium coordination in s2 . Based on the sequence alignment : s2b utilizes OG from both Ser845 and Ser846 in lieu of Asp755 in s2a.The Cain Fig. 4. The penin Fig. 4 are simint Fig. 3 of PKD-lle Fig. 4b: s2b u3.6.2+ chelation appears to align the N-terminal linker approximately parallel to the major axis of the domain . In s2b, Ca2+ chelation by Asn774 and Lys775 could stabilize a 310-helix (residues 770–774) that aligns with the cylinder axis. In s2 and s2a, the N-terminal residues are positioned so that the N-terminal linker could also be positioned parallel to the major axis of the domain. The residues prior to Asn685 cannot be observed in the electron density, and consequently the secondary structure of the region remains ambiguous. Structural comparison of the Ca2+-binding site between the apo and holo PKD-like domains revealed that Ca2+ has a varied influence on the loop between β-strands B′ and C. The proline positioned between the aspartates equivalent to Asp802 and Asp804 restricts the loop flexibility so that minimal change occurs between the apo and holo states. However, in s2, which does not have a proline positioned between Asp713 and Asp715, the Ca2+ interaction moves Asp715 2 Å from the binding site .CaB factors of s2b and s2 are low and the average B factors of apo and holo s2a are similar  are very similar to each other . Holo s2, however, does not follow this trend . Comparison of holo and apo structures revealed a stark contrast between s2a and s2 in the influence of Ca2+ . Decreases in the CαB factor in holo s2 are found in three stretches  that are immediately preceded by stretches in which the B factor is higher . In the holo s2 structure the mid-section became more flexible, although both terminal regions became more rigid. Differences in crystal packing could account for the reversal in dynamics, although it is possible that the crystal packing is a consequence of dynamic changes. Both termini of holo s2 are pinned down by symmetry-related molecules that could suppress their dynamics, while the mid-section of the barrel lacks the intermolecular interactions observed in the apo state. In apo s2, intermolecular antiparallel β-sheet interactions involving β-strand A could suppress dynamics of the region.Overall, the average r Table 1. Despite2+ did not increase the B factor of the mid-section . In the s2a structures the termini are the most flexible. As mentioned, the B-factor trends in all holo and apo s2a structures are similar despite the difference in crystal packing. CαB factors for the mid-section of holo s2b are low  and suggest that s2b behaves similarly to s2a. Overall, the starkly contrasting dynamics between ColG-derived and ColH-derived PKD-like domains suggest diverging roles during collagenolysis.In contrast, comparison of the holo s2a and apo s2a structures revealed that Caon Fig. 5a. In thow Fig. 5b and su3.7.Tm of ∼373 K), but they gain further stability in the presence of Ca2+. The fluorescence of Trp812 in s2b was monitored, while in s2a and s2 the fluorescence of Trp723 was monitored. During fluorescence-monitored thermal denaturation, none of the PKD-like domains fully unfolded in the holo state . Such hyperthermostability has also been observed for the holo states of s3 . In contrast to heat, Gu–HCl is thought to denature proteins predominately by disrupting hydrophobic interactions  is approximately the same for all PKD-like domains. In addition to reorienting the N-terminal linker, the proposed Ca2+-induced helical base of the N-terminal linker may have a partial capping effect that shields the core against Gu–HCl. It is also well documented that metalloproteins are more stable in the presence of their metal ligand . Interestingly, these residues are also located along the previously mentioned ridge. These residues could be involved in collagen binding given that aromatic residues are found at the hotspot of the collagen-binding pocket in CBD. In s3b, mutations of Tyr970, Ty994 and Tyr996 to Ala greatly reduced binding to collagenous peptide as monitored by surface plasmon resonance . In this model, the surface aromatic residues on s2a-s2b may either span across multiple tropocollagen molecules on the surface of the fibril or bind along one tropocollagen molecule when the binding surface of s3 is docked onto the collagen fibril surface. The interactions may serve to prevent the collagen-binding segment from diffusing away after the s3–collagen interaction is transiently broken. Likewise, the domains may provide loose contacts with the collagen fibril that allow the enzyme to scan the fibril surface for optimal regions for tight CBD interaction. In these roles, the PKD-like domain strengthens collagen avidity so that only one CBD is required for collagen binding. The zinc ion involved in activation of a water molecule is approximately 115 Å away from Tyr962 found in the collagen-binding pocket of s3. In this model, the PKD-like domains may also be critical in positioning the catalytic domain with respect to CBD.A putative structure of holo ColH can be built from the homology-modeled activator domain of s1 and helical linker  β-Strand C contains the conserved tryptophan, and along with β-strand C′ (β-strand D of the PKD-like domain fold) forms a β-hairpin connected by an approximately i→i + 8 ω-loop. (iv) The turn leading into β-strand F is stabilized by a Δ4 Tyr corner.Comparison of clostridial PKD-like domain structures with V-set kappa light-chain Bence–Jones protein (PDB entry 4.Comparison of crystal structures of ColG s2 with crystal structures of ColH s2a and s2b suggests that despite common tertiary folds, PKD-like domains can be grouped into two subsets. The subset containing ColH-derived domains exhibits exposed aromatic residues and is found in M9B collagenases with a single CBD. The surface aromatic residues could be involved in secondary interactions that allow weak collagen binding. In contrast, the subset containing s2 is likely to be different; the lack of surface aromatic residues on s2 suggests that the domain is less directly involved in interactions with collagen. Overall, this subset is found in M9B collagenases with multiple CBDs. The unique differences in dynamics and surface characteristics between s2a-s2b and s2 may aid in synergistic collagenolysis.2+ repositions the linker along the barrel axis. The helical structure of the linker upon Ca2+ binding may shorten the distance between s2b and s2a, and may help to account for the previously described proteinase resistance (Ohbayashi et al., 20132+. Structural and stability data are critical for the development of PKD-like domains as part of the site-directed delivery of signal molecules such as growth factors and cytokines.Meanwhile, the N-terminal linker structure of a PKD-like domain is described for the first time in the holo s2b structure and suggests that CaColG PKD domain 2, 4jrwPDB reference: 4tn9PDB reference: ColH PKD domain 2a, apo, 4u6tPDB reference: in the presence of calcium, 4u7kPDB reference: ColH PKD domain 2b, in the presence of calcium, 4jguPDB reference: 10.1107/S1399004714027722/mn5081sup1.pdfSupporting Information.. DOI:


Music and voice bear many similarities and share neural resources to some extent. Experience dependent plasticity provides a window into the neural overlap between these two domains. Here, we suggest that research on auditory deprived individuals whose hearing has been bionically restored offers a unique insight into the functional and structural overlap between music and voice. Studying how basic emotions  are perceived in auditory stimuli constitutes a favorable terrain for such an endeavor. We outline a possible neuro-behavioral approach to study the effect of plasticity on cross-domain processing of musical and vocal emotions, using cochlear implant users as a model of reversible sensory deprivation and comparing them to normal-hearing individuals. We discuss the implications of such developments on the current understanding of cross-domain neural overlap.Our musical and vocal perception abilities have such a close relationship that some authors suggested that the former originated from the latter or vice-versa  are bionic devices that can restore the sense of hearing in profoundly deaf individuals. We argue that cochlear implant users offer a promising model to study the mechanisms of cross-domain plasticity because they undergo different trajectories of auditory development: deafness of various origins results in a variable period of auditory deprivation followed by surgical restoration of auditory input and an intense rehabilitation period, yielding variable individual auditory outcomes.Signal transmitted from the implant to the auditory nerve is impoverished compared to natural hearing. Critically, the access to pitch cues is impaired, reduced to a small number of frequency bands. As a result, cochlear implant users can potentially perceive speech relatively well in a quiet setting, but understanding it in noise, or accurately perceiving music is very challenging since both tasks rely on pitch information , Limb et al. , reportead-hoc stimuli are required to accurately compare the two domains. This could explain why no study has yet directly compared emotion processing in CI users across the domains of music and voice. To date, there is very little neuro-imaging evidence building up on the aforementioned behavioral findings. Only one study evaluated the impact of two implant processing strategies on the perception of prosody  in each domain: non-speech vocalizations and brief mono-instrumental musical excerpts.In the vocal domain, non-speech vocalizations  depicting basic emotions that are minimally conventionalized, relatively universal and fundamental to spontaneous human communication Scherer, , could bThese highly similar vocal and musical stimuli seem well-suited to study cross-domain overlap in any population and their primitive quality could be extremely useful to study plasticity in CI users.A second step would be to pair a well-controlled behavioral paradigm using those stimuli  with a neuro-imaging modality that is acceptable for use with cochlear implants. Except for a few recent exceptions, implants are not MR-compatible. Hi-density EEG  make use of overlapping structures in an optimal fashion compared to poor performers, can we boost residual neural processes in the latter group? It has been suggested that musical training can improve speech outcomes in this population Patel, , but whaCross-domain research on cochlear implant users not only offers a unique insight into auditory neural plasticity, but also has practical implications for patients' rehabilitation, implant design, and programming. We believe that highly comparable stimuli are needed to carry out such studies, together with an optimal imaging technique within a paradigm fine enough to reveal subtle behavioral and neural differences. Such scientific undertaking can further our understanding of how our brain processes vocal and musical emotions and how such cross-domain processing is affected by plasticity. Furthermore, such studies could provide objective measures to support the use of music in the rehabilitation of various disorders.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.


UBE3A gene cause Angelman syndrome (AS), a severe neurodevelopmental disorder. The paternal UBE3A/Ube3a allele becomes epigenetically silenced in most neurons during postnatal development in humans and mice; hence, loss of the maternal allele largely eliminates neuronal expression of UBE3A protein. However, recent studies suggest that paternal Ube3a may escape silencing in certain neuron populations, allowing for persistent expression of paternal UBE3A protein. Here we extend evidence in AS model mice (Ube3am–/p+) of paternal UBE3A expression within the suprachiasmatic nucleus (SCN), the master circadian pacemaker. Paternal UBE3A-positive cells in the SCN show partial colocalization with the neuropeptide arginine vasopressin (AVP) and clock proteins (PER2 and BMAL1), supporting that paternal UBE3A expression in the SCN is often of neuronal origin. Paternal UBE3A also partially colocalizes with a marker of neural progenitors, SOX2, implying that relaxed or incomplete imprinting of paternal Ube3a reflects an overall immature molecular phenotype. Our findings highlight the complexity of Ube3a imprinting in the brain and illuminate a subpopulation of SCN neurons as a focal point for future studies aimed at understanding the mechanisms of Ube3a imprinting.Mutations or deletions of the maternal allele of the  UBE3A cause Angelman syndrome (AS), a rare neurodevelopmental disorder characterized by developmental delay, lack of speech, seizures, motor abnormalities, happy affect, and sleep disturbancesUBE3A gene exhibits preferential expression from the maternal allele in a majority of neuronal subclasses, while its expression appears to be biallelic in other cell types2UBE3A is achieved through paternal expression of an antisense transcript, UBE3A-ATS, which extends across the UBE3A gene and suppresses its expression in cis4Ube3a is nearly exclusively maternal in neurons within the CA3 region of the hippocampus and in cerebellar Purkinje cells, with moderate maternal bias in the cerebral cortexUbe3a expression is preferentially maternal in neurons throughout the cortex, hippocampus, cerebellum, and thalamusUbe3a has been observed in early postnatal mouse visual cortexUbe3a allele is silenced in neurons in the mouse neocortex between birth and postnatal day 7 (P7), and at P7 granule cells in the dentate gyrus and cerebellum still exhibit paternal Ube3a expressionUbe3a expression bias in many forebrain structures has begun to emerge, detailed knowledge of allelic contributions to Ube3a expression in the basal telencephalon, particularly the hypothalamus, is lacking.Mutations or deletions of the maternal allele of Ube3am–/p+), in which a null allele of Ube3a is maternally inherited, have also been consistently observed1417Ube3a in the adult brain. Here we examine the expression patterns of UBE3A in the SCN of AS model mice and provide evidence for paternal expression of Ube3a in a subset of neurons in this circadian regulatory region. The persistence of paternal UBE3A may be important for SCN function and, together with other markers of young neurons, suggests a degree of neoteny in the molecular profile of adult SCN neurons.Sleep disturbances in individuals with AS persist throughout childhood and manifest as reduced need for sleep, difficulties falling asleep, and sleep fragmentation1112Ube3am+/p+), AS model (Ube3am–/p+), and homozygous null (Ube3am–/p–) mice. For these experiments we used a previously characterized antibody10We previously made the surprising finding that UBE3A protein is expressed in the SCN of adult AS model miceUbe3a mutant allele does not produce a detectable transcript or proteinUbe3a expression. This pattern was specific to the SCN, as regions immediately rostral or caudal to the SCN did not exhibit UBE3A that was readily apparent at this magnification  is fused to the carboxyl-terminus of UBE3AUbe3amYFP/p+) yielded UBE3A-YFP expression in virtually all neurons in the SCN and surrounding hypothalamus, including the PVN   . Notably the SCN . These dUbe3a expression is a characteristic of glial cells, young or newly differentiated neurons, and postnatal neural stem cells202223Ube3a expression and NEUN expression during postnatal neuronal development in the neocortexPaternal SOX2 (sex-determining region Y [SRY]-related HMG box 2), is a member of the SOXB1 family of transcription factors and plays important roles in maintaining neural progenitors during development and in adult neural stem cell niches25Sox2 expression has been observed in neurons of the adult rodent SCN but not in other hypothalamic regionsSox2’s role in maintaining neural stem cell populations. We hypothesized that SCN neurons expressing paternal UBE3A might also express SOX2, as both markers are strongly associated with neuronal immaturity. In contrast to NEUN, SOX2 expression was widespread across the SCN in all genotypes, similar to the pattern previously describedWe also employed other markers to determine the neuronal identity of paternal UBE3A-expressing cells. One such marker, zeitgeber time (ZT) 8 on a 12:12 light/dark (LD) cycle   cycle . Average, n = 3) , illustr, n = 3) . While o, n = 3) . These dt test, n = 3 mice)  is a neuropeptide that is critically important in the SCN for intercellular communication from light-responsive “core” neurons to shell neurons, which receive minimal retinal input 3 mice) . While s 3 mice) . Further 3 mice) .Ube3a does not disrupt expression levels of either AVP or VIP across the SCN. Furthermore, our results reveal that UBE3A-positive neurons in AS model mice are more likely to express AVP than VIP in the SCN, but paternal Ube3a expression was not restricted to either of these SCN populations.Taken together, these findings show that loss of maternal PER2) is a key component of the transcriptional-translational feedback loops that drive circadian rhythms in the SCNmPer2::mPER2-LUC reporterUbe3a expression occurs in SCN neurons that participate in circadian timing.Period homolog 2  = 0.4777, p = 0.6337, n = 4–5 mice) (Ube3am–/p+ miceUbe3a does not detectably perturb BMAL1 levels in the SCN.–5 mice) . Further–5 mice) . HoweverUbe3a expression might be a more general feature of neurons that undergo rhythmic changes in gene expression in the adult brain. We examined paternal Ube3a expression in the BNST and CeA of adult UBE3A-YFP-expressing and AS model mice del mice . UBE3A-Yexpected . SurprisYFP mice . PaternaYFP mice , confirmYFP mice ; howeverYFP mice . In a siYFP mice , whereasYFP mice , presentYFP mice . Thus, rUbe3a expression occurs in neurons throughout the SCN in adult mice. Neurons expressing paternal Ube3a were also likely to express SOX2, a marker of newly differentiated neurons, and were less likely to express the mature neuronal marker NEUN. Despite having molecular characteristics of immature neurons, paternal UBE3A-expressing neurons showed a time-of-day dependent change in PER2 levels, suggesting that they were functionally integrated into SCN circuitry. The BNST and CeA, regions of the extended amygdala that exhibit strong circadian rhythms of clock gene expression and control motivational behaviors, also exhibited paternal Ube3a expression in the adult. Our findings provide additional evidence for the persistence of markers of neuronal immaturity in the adult SCN, and highlight discrete groups of basal forebrain neurons in which paternal Ube3a appears to “escape” imprinting during postnatal development – knowledge that could be exploited to further elucidate molecular mechanisms of paternal Ube3a silencing.In this study, we have established that paternal We have provided evidence for the neuronal identity of paternal UBE3A-positive cells in the SCN. Paternal UBE3A expression exhibited substantial overlap with AVP, PER2, and BMAL1, proteins that are abundantly expressed in SCN neurons and are essential for SCN function and circadian rhythmicity. Due to its incomplete expression pattern in the SCN, the canonical neuronal marker NEUN was largely uninformative in defining the neuronal identity for paternal UBE3A-positive cells. Indeed, NEUN was expressed in SCN cells to the exclusion of paternal UBE3A and vice versa. Because previous observations suggest an inverse relationship between paternal UBE3A and NEUN expression in maturing neocortical neuronsUbe3a10It is possible that paternal UBE3A immunofluorescent signal observed in the SCN of AS model mice could be partially mediated by paternal UBE3A expression in glia, which biallelically express Ube3a expression, previously ascribed to immature neurons, constitutes further evidence of an immature molecular phenotype in adult SCN neurons. In addition to minimal NEUN expression41The persistence of paternal Paternal UBE3A-positive neurons in the adult SCN bear a molecular resemblance to newly generated neurons, which have yet to integrate into surrounding circuitry. Although the details of adult neurogenesis in the SCN specifically are not well understood, neurogenesis occurs at constitutive but low levels in the adult mouse hypothalamus4344Ube3a imprinting in the SCN that is not typical of most neurons. It remains to be determined whether this feature of murine Ube3a expression is conserved in the human brain. Nevertheless, the identification of novel sites in the brain in which Ube3a imprinting is differentially regulated has broad implications. The most promising therapeutic strategies for AS take advantage of unsilencing of the paternal UBE3A allele46Ube3a expression bias, such as those of the SCN, may allow inroads toward further understanding of the molecular mechanisms that regulate Ube3a imprinting, and thereby, the refinement of AS therapeutics.In this study we provide evidence that SCN neurons maintain persistent expression of paternal UBE3A protein, revealing a relaxation of Ube3am–/p+)  mice and their wildtype littermates were generated by breeding Ube3am+/p– females with wildtype males. Experimental litters that included Ube3am–/p– mice were generated by breeding Ube3am+/p– females with Ube3am+/p– males. Experimental trios of Ube3aYFP miceUbe3am+/pYFP females to Ube3am+/pYFP males, yielding Ube3am+/p+, Ube3amYFP/p+, and Ube3am+/pYFP mice. Homozygous Ube3aYFP mice were excluded from experiments.All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of North Carolina School of Medicine and were performed in accordance with the guidelines of the U.S. National Institutes of Health. AS modelUbe3aYFP reporter mice and their control littermates were collected at P30; all others were 2–5 months old at time of perfusion. Males and females were balanced in number across genotypes. Unless otherwise stated, mice were perfused between ZT4–ZT9. At the indicated ZTs, mice were deeply anesthetized with sodium pentobarbital  before being transcardially perfused with room-temperature phosphate-buffered saline (PBS) immediately followed by room-temperature phosphate-buffered paraformaldehyde (pH 7.3). Optic nerves were severed prior to removing the brains from skulls, and the brains were then placed in fixative overnight at 4 °C. Fixed brains were then cryoprotected in 10%, 20%, and 30% sucrose in PBS at 4 °C for 12 hours each. Brains were then frozen in dry ice, and coronal sections (40 μm thick) were collected using a freezing sliding microtome . Sections were stored in a cryopreservative solution  at −20 °C.Mice were housed in 12:12 LD and given ad libitum access to food and water prior to perfusion. 1 Alexa Fluor 568 (Cat. #A-21124), goat anti-mouse IgG2A Alexa Fluor 488 (Cat. #A-21131), goat anti-rabbit Alexa Fluor 568 (Cat. #A-11011), goat anti-rabbit Alexa Fluor 633 (Cat. #A-21071), and goat anti-chicken IgY Alexa Fluor 488 (Cat. #A-11039). Where specified, sections were incubated for 30 min following secondary antibodies with NeuroTrace 640/660 Deep-Red Fluorescent Nissl Stain , diluted 1:200 in PBS, prior to final washes. In all experiments, 4′,6-diamidino-2-phenylindole (DAPI) was added during the secondary antibody incubation at a concentration of 700 ng/ml for nuclear counterstaining and delineation of the boundaries of the SCN. Perfusions, tissue sectioning, and immunostaining were performed by experimenters blind to genotype. Brain sections compared within figures were stained within the same experiment under identical conditions.Tissue sections were washed several times in PBS and blocked in PBS containing 5% normal goat serum and 0.2% Triton X-100 (NGST) for 1 hour at room temperature. Blocked tissue sections were incubated with primary antibodies diluted in NGST for 48 hours at 4 °C. Sections were then washed several times in PBS containing 0.2% Triton X-100 before incubation with secondary antibodies (diluted in NGST) for 1 hour at room temperature. The following primary antibodies were used: mouse anti-UBE3A ; mouse anti-NEUN ; rabbit anti-SOX2 ; rabbit anti-VIP ; rabbit anti-AVP ; mouse anti-BMAL1 ; rabbit anti-PER2 ; rabbit Anti-GFAP ; and chicken anti-GFP . The following secondary antibodies from Invitrogen  were used at a 1:500 concentration: goat anti-mouse IgGrd ventricle and surrounding hypothalamic regions, or the BNST or CeA, were acquired by tiling 3 × 3 micrographs of 1024 × 1024 pixels that were stitched using ZEN software. Images compared within figures were acquired using identical acquisition parameters. All images to be compared underwent identical manipulations for brightness and contrast, unless otherwise stated in the figure legends.Immunostained sections containing the SCN were imaged using a Zeiss LSM 710 confocal microscope equipped with ZEN Imaging Software . Single-plane optical slices of 1.8 μm in thickness were acquired using a 20× objective (Plan-Apochromat 20 × /0.8 M27) at a pixel dwell time of 3.15 μsec with 4× averaging. Images containing the SCN, the 3http://imagej.nih.gov/ij/). We used average intensity measurements to address whether changes in UBE3A expression altered protein levels of AVP, VIP, and BMAL1, in cell bodies as well as in neuronal processes. We also used a cell-counting approach to examine the degree of overlap of UBE3A-positive and SOX2-positive, NEUN-positive, VIP-positive, or AVP-positive cells in the SCN. To quantify total SCN signal of a given fluorophore, regions bounding the left and right SCN were created manually for each image using the DAPI channel, by an experimenter blind to genotype. The regions were applied to the channel of interest, and the mean gray values within SCN regions were measured. An oval region of a standard size across all images (150 μm × 300 μm) was also placed in the adjacent lateral hypothalamus beyond the SCN boundaries, and the average intensity within this region was subtracted from the average intensity in the SCN to account for variability in background fluorescence. Intensity measurements in the lateral hypothalamus regions  were not different across genotypes or ZTs. Expression measurements of proteins other than UBE3A, whose staining pattern made the genotype evident, were performed blind to genotype. To quantify the degree of colocalization, cell bodies positive for each immunofluorescent signal within each SCN region (defined by DAPI counterstaining) were manually counted, and the percentage of cells positive for both fluorophores was calculated. Sections containing rostral, central, and caudal regions of the SCN were imaged for all conditions and included for quantification in a balanced fashion.Quantification of immunofluorescent signals was performed in ImageJt tests and one-way ANOVA were used where indicated. Data are represented as mean ± s.e.m. (error bars). P values less than 0.05 were considered statistically significant.Statistical analyses were performed using GraphPad Prism version 6.00 for Windows . Unpaired two-tailed Student’s How to cite this article: Jones, K. A. et al. Persistent neuronal Ube3a expression in the suprachiasmatic nucleus of Angelman syndrome model mice. Sci. Rep. 6, 28238; doi: 10.1038/srep28238 (2016).


C-type natriuretic peptide (CNP) is a paracrine factor that stimulates long bone growth, axonal path finding and inhibits meiosis in the oocyte . The bioGC-B is a homo-oligomer, possibly a dimer, containing a glycosylated extracellular ligand-binding domain, a single membrane-spanning region, and intracellular kinase homology domain (KHD), dimerization domain and C-terminal GC catalytic domain. Phosphorylation of the region leading into and at the beginning of the kinase homology domain is required for CNP activation of GC-B and dephosphorylation inactivates the enzyme .Homozygous inactivating mutations in GC-B result in Acromesomelic Dysplasia, Type Maroteaux (AMDM) dwarfism -6, and hMore than fifteen inactivating missense mutations in GC-B have been identified in humans. These mutations are randomly distributed from the N-terminus (P32) to the C-terminus (G959A) of the enzyme, consistent with two potential mechanisms. The first involves multiple processes like disruption of CNP or GTP binding to the extracellular or catalytic domains, respectively. The second more general mechanism involves conformational changes in secondary, tertiary or quaternary structure that preclude catalytic domain formation or activation.Previous investigators reported that 11 out of 12  missenseAMDM mutants are incompletely processed- Post-or co-translational processing of the four intracellular AMDM causing mutants  was compared against WT-GC-B and GC-B-7A, a mutant containing alanine substitutions for all known phosphorylation sites [on sites , in reduon sites -14. The The AMDM GC-B Mutants Are On the Cell Surface and Bind CNP- 125I-CNP binding assays were performed on live 293T cells transfected with the individual AMDM mutants, WT-GC-B as a positive control, and GFP as a negative control (Fig. AMDM mutants have dramatically reduced GC activity-The ability of the GC-B mutants to form active catalytic domains that can be stimulated by CNP or non-ionic detergent and manganese was tested in membranes from transfected 293 cells (Fig. We conclude that glycosylation of GC-B is required for active catalytic domain formation and that the majority of GC-B mutations that reduce stature inactivate the enzyme by decreasing receptor glycosylation not by inhibiting trafficking to the plasma membrane.


Using these maps as a constraint, we derived 120 conformations of the antibody via structural flexible docking of the crystal structure to these maps by targeted molecular dynamics simulations. Statistical analysis of the various conformations disclosed the antibody 3D conformational flexibility through the distribution of its domain distances and orientations. This blueprint approach, if extended to other flexible proteins, may serve as a useful methodology towards understanding protein dynamics and functions.Commonly used methods for determining protein structure, including X-ray crystallography and single-particle reconstruction, often provide a single and unique three-dimensional (3D) structure. However, in these methods, the protein dynamics and flexibility/fluctuation remain mostly unknown. Here, we utilized advances in electron tomography (ET) to study the antibody flexibility and fluctuation through structural determination of individual antibody particles rather than averaging multiple antibody particles together. Through individual-particle electron tomography (IPET) 3D reconstruction from negatively-stained ET images, we obtained 120  Understanding how proteins function in isolation and in their native context requires merging several molecular-level techniques that explore the interplay of protein structure and dynamicsTransmission electron microscopy (TEM) serves as a tool for individual protein imaging at atomic resolution, while electron tomography (ET) images an individual protein particle from a series of tilting angles. The first 3D reconstruction of an individual protein particle, fatty acid synthetase, was reconstructed in 1974 by Walter Hoppe and his colleagues through aligning and merging tilted images acquired from a negatively-stained sample2Even though a few reconstructions of individual molecules had been reported after Hoppe34567891010In this study, we further employed this IPET method to study the dynamics of one of the most well-known flexible proteins: the IgG1 antibody. Through particle-by-particle 3D reconstructions, we reconstructed a total of 120 density maps at an intermediate resolution from negatively-stained ET images. By flexibly docking the crystal structure onto these 3D reconstruction maps, we subsequently achieved 120 conformations of the antibody particles via targeted molecular dynamics (TMD) simulationsab domains and one Fc domain. The domain sizes and shapes were similar to those of the corresponding crystal structures  was performed by optimized negative-staining (OpNS) EM technique14ab-initio 3D structure of a single and individual particle of protein from a series of tilt images by electron tomography (ET). Compared to conventional ET 3D reconstruction methods that use large-size whole-micrographs, IPET demonstrated the adverse effects from image distortion and the measuring tilt-errors (including tilt-axis and tilt-angle errors), both of which play a major role in limiting the reconstruction resolution. The method includes a “focused electron tomography reconstruction” (FETR) algorithm to improve the resolution by decreasing the reconstructed image size so it contains only a single-instance protein. As a result, it can tolerate certain levels of image-distortion and tilt-errors, but can precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. FETR does not require an assumption that particles share a common structure or a pre-given initial model to study protein structures and dynamics. The use of IPET/FETR could potentially avoid some problems in single-particle reconstruction methods, such as initial model bias, uneven distribution of resolution, or absence of flexible domains.IPET is an EM method for determining an ab-inito 3D reconstruction with IPET  of an individual antibody particle in each tilt image (from −60° to + 60° in a step of 1.5°) were only ~0.1 to ~0.26 in the raw micrographs, the overall shape of each individual antibody particle was still visible . The imaith IPET . During ith IPET .c domain density map c domain of crystal structure was well matched to the Fc domain of the final 3D reconstruction in domain shape and size as well as its contained “hole”. The crystal structure showed the “hole” diameter in the Fc domain is ~25 × ~30 Å, which was surrounded by heavy chain CH domains (CH2 and CH3) in diameters of ~18 × ~21 × ~30 Å. A reasonable match in size and shape to the 3D reconstruction suggested our reconstruction confirmed that the resolution was within above measured range, i.e. ~20 Å to ~30 Å. Despite the different strategies in determination of the resolution, a quantitative method to estimate the resolution used here was that, we bought the strategy of gold standard method to estimate our IPET map resolution, however, the frequency at which the FSC curve fell below 0.5 (instead of 0.143), i.e. ~20 Å, was used as the resolution (The resolution of IPET reconstruction was estimated by the following four methods: i) a conventional single-particle method1919solution .ab/Fc domains. The 120 reconstructions  at two contour levels were displayed after alignment based on their Fc domains, and then put into arrays  for Fab to Fc  Histogram of the distances among the domains. The distances between two domains in each molecule were measured based on the mass center of each domain. The results showed that all distances were within a range of 60 to 120 Å. This distance was consistent with that measured by liquid AFM based on the progression of antibody moving on a virus covered flat surfaceab to Fc , suggestab1-Fc-Fab2 domains showed that over 90% of the antibodies had an angle within a range from ~40° to ~72°, in which the angle at peak population (~28.8%) was ~56.6 ± 4°  Histogram of the angles of F6.6 ± 4° .This finulation ~.8% was ~ab domains was ~135.4° ± 5°, whereas over 70% of the angles were distributed within the full width, starting from the half maximal point of 90o (a range from ~90° to ~160°). This range was consistent with that of AFM measurements from 3D structures of ten antibody particlesab-Fc domains was ~124.7° ± 5°  Analysis of the angles between any two domain directions . The dom.7° ± 5°  and ~135ab domains could not be distingushed within the current resolution, the dihedral angle was measured only up to 90°). A drop in distribution below 10° ((iv) Histogram of the dihedral angles between the normal planes of direction of two domains . The norelow 10°  suggesteTo achieve a 3D structure of a non-crystalized protein, a single-particle reconstruction method was often used. In this process, images from thousands to hundreds of thousands of randomly orientated particles that are assumed to have an identical structure were classified based on projections of a 3D model and then averaged together before being back-projected into a 3D density map via an iterative refinement processab1-Fc-Fab2) to their initial models  and fuzzy, which was likely due to the flexibility of the domains in position and orientation.To demonstrate the limitations of single-particle reconstruction on a structurally highly flexible protein, we attempted using the single-particle reconstruction to study antibody structure with the EMAN software packagel models , suggestab domains  also showed a “Y” shaped structure . These mSimilar artifacts, such as the presence of blurry domains in reference-free class averagesab domains were obviously smaller in dimension or fuzzy in density  density . Based oStructural determination of an individual protein molecule was first attempted about four decades ago6−/Å21241434547ab-inito 3D reconstruction without requiring a pre-given initial model.We, however, believed in the possibility of achieving a structure of an individual protein structure at near nanometer resolution because: i) nearly all TEM instruments have a resolution capability better than 3 Å; ii) ~100 low-dose images  are theoretically sufficient to reconstruct a 3D density map of sub-nanometer resolution6 filament) and the micrographs were acquired by a traditional CCD , our achieved resolution was benefited by i) IPET/FETR reconstruction strategy/algorithms; ii) sample preparation by high-resolution OpNS protocol14−/Å2).In this research, we obtained 120 structures from each individual antibody particle at an intermediate resolution. Despite that the fact that the TEM instrument used in this work was one of lowest models of TEM  certain level of inaccuracy in identifying Fab and Fc domains; viii) time consuming data acquisition (~2–3 hours spent on each tomographic set of ~100 images); ix) challenges in tomographic data collection under high magnification (~80 K × ); and x) low efficiency in 3D reconstructions. For example, in this study, a total of 30 tilting series were acquired, in which more than half of the tilting series was excluded due to an uneven stain or jumps in defocus . Only particles visualized with a clear boundary were selected. As a result, each tilting series contributed 5~40 antibody particles for IPET 3D reconstruction. In total, ~60% of IPET 3D density maps were excluded for structural analyses due to uneven staining, buffer solvent aggregation, tilting images that were missing at certain angles, protein overlapping, deformation, missing alignment, and failure to identify Fab and Fc domains in a molecule. Eventually, only ~40% of the IPET maps  were submitted for conformational determination by TMD and statistical analyses. In the case of domain identification, IPET 3D reconstructions showed that the Fc domain had an overall size similar to that of the crystal structure, but presented a larger center hole and stronger densities than that from the crystal structure. One of the best tilting series showed ~60 particles presented in the 0° tilting micrograph  certain preferred orientation of particles; ii) certain level of protein flatness due to the drying process; iii) uneven staining distribution; iv) unidentified artifacts through negative-staining; v) missing wedge effects (tilted only up to ± 60° instead of ± 90°); vi) inability to identify the heavy and light chains within Fcrograph . Of thesIn spite of the above weaknesses, the success in obtaining over a hundred 3D structures at an intermediate resolution from each individual antibody particle (one of most highly dynamics and flexible proteins) demonstrates that IPET could be a useful approach towards achieving a meaningful resolution structure for studying the highly dynamic, heterogeneous and flexible protein particles without involving any averaging of different particles2PO4, 136.9 mM NaCl, and 8.1 mM Na2HPO4; Invitrogen), and then prepared using the optimized NS protocol by 1% (w/v) uranyl formate (UF)14Monoclonal antibody IgG subclass 1 , was expressed in HEK-293 cells, and purified using protein A/G chromatographyab for 40Å, Fc for 50 Å) with a different separation angle  with a high-sensitivity 4,096 × 4,096 pixel Gatan Ultrascan CCD camera, and at 80,000 x magnification, using a Zeiss Libra 120 TEM. Raw images were low-pass filtered to 10 Å to reduce noise, and high-pass filtered to 600 Å to remove large area background fluctuation. Particles were selected by EMAN Boxer−/Å2 The tilt series of tomographic data under defocus ~1.0 μm were controlled and imaged by manual operation using Gatan tomography software (for Zeiss Libra 120 TEM) that had been preinstalled in the microscopes. Micrographs were initially aligned together with the IMOD software packagectfit (EMAN software package)Electron tomography data of all antibody specimens were acquired under similar conditions by same TEM and CCD as described above. The specimens mounted on a Gatan room temperature high-tilt holder were tilted at angles ranging from −60° to 60° in steps of 1.5°. The total dose of electron illumination was ~200 eEach micrograph of antibody particles was Gaussian low-pass filtered and examined before selection for 3D reconstruction. A small image containing only one targeted particle was selected, and marked with a window size of 220 × 220 pixels (~326 × 326 Å) on the 0° tilt image micrographIS−Ib)/Nb, in which Is was the average power inside particle, and Ib was the average power outside the particle. The standard deviation of noise, Nb, was calculated from background standard deviation outside the particle. The particle area was defined by using particle shaped mask generated from IPET final 3D with low pass filtered to 40 Å and set as 3 times its molecular weight. This same method was used to calculate 2D SNR. 2D mask was generated based on the 3D projection at each tilt angle.In IPET reconstruction, an initial model was obtained via direct back-projection of these small-images into a 3D map, followed by 3D reconstruction refinements performed via three rounds of refinement loops with an FETR algorithmTo analyze tomographic 3D reconstructions, the raw ET images were split into two groups based on having an odd- or even-numbered index in the order of tilt anglesab and Fc domains of the mouse IgG2 antibody were therefore truncated from the crystal structure 1IGTThe current resolution (~1–3 nm) of IPET 3D reconstructions were not sufficient to determine the secondary structure of each individual antibody; however, the reconstructions were sufficient to provide the information about the domain orientations and positions, which may reveal the domain fluctuation. We chose 1IGT as an example to reflect the antibody dynamics by docking its three domains into each domain in the reconstruction. We chose 1IGT is because there is no full length human IgG1 crystal structure available so far. Three available crystal structures of antibodies, 1IGT, 1HZH and 1IGY are very similar in the lengths of sequences. This small difference would not make a significant difference at a ∼1–3 nm resolution of 3D reconstructions in the analysis of the domain locations and orientations. The Fab and Fc domains in the 3D reconstruction, the following criteria was used: i) the hole size of Fc domain was distinguishably larger than that of Fab domain, and ii) under high contour levels, Fc domain density was more like a “C” or “O” shape, while Fab resembled more of a “dumb-bell” shape  simulation techniqueThe TMD simulation was performed using the program NAMD2.7bc domains using the VMD commandab domains were measured and analyzed to study the equilibrium fluctuation and structural dynamic character of antibody particles. In brief, we introduced nine vectors to represent the domain location and orientation with respect to each other  the length of each domain-domain vectors; ii) the angle, αabc, between two Fc-Fab domain-domain vectors; iii) the angle between two Fab domain direction vectors; iv) the angle between two domain direction vectors of Fab and Fc domains; v) the angle between two normal direction vectors of two Fab domains plane; and vi) the angle between two normal directions of Fab and Fc domains plane. These measurements were used to calculate the angles between the domains. The distances and angles between antibody domains were plotted onto histograms and fitted with sixth degree polynomial curves by MATLAB. These histograms represented the conformational space of each antibody molecule, which in turn indicated the extent of its dynamic flexibility.To determine the structural dynamics of the antibody, all pseudo structures were aligned based on their Fch other . The domThis project was designed and initiated by G.R. and L.Z., and the E.M. specimens were prepared by P.B. and H.T. X.Z., L.Z. and H.T. acquired the E.M. images. X.Z. and L.Z. conducted the image process and 3D reconstruction by IPET, G.R conducted the single-particle reconstruction. X.Z. performed the conformation determination by TMD., and statistically analyzed the distributions. X.Z., S.Z. and G.R. conducted the resolution analysis. X.Z., L.Z., M.J.R. and G.R. interpreted and manipulated the data. G.R. and X.Z. drafted the initial manuscript, which was revised by M.J.R., Z.L., B.P. and H.T.Accession Codes: Density maps of all 120 antibodies have been deposited at the Electron Microscopy Data Bank (EMDB) and assigned reference codes EMDB-6099.How to cite this article: Zhang, X. et al. 3D Structural Fluctuation of IgG1 Antibody Revealed by Individual Particle Electron Tomography. Sci. Rep.5, 09803; doi: 10.1038/srep09803 (2015).


Lymphocyte function-associated antigen 1 (LFA-1) is an integrin that transmits information in two directions across the plasma membrane of leukocytes, in so-called outside-in and inside-out signaling mechanisms. To investigate the structural basis of these mechanisms, we studied the conformational space of the apo I-domain using replica-averaged metadynamics simulations in combination with nuclear magnetic resonance chemical shifts. We thus obtained a free energy landscape that reveals the existence of three conformational substates of this domain. The three substates include conformations similar to existing crystallographic structures of the low-affinity I-domain, the inactive I-domain with an allosteric antagonist inhibitor bound underneath α helix 7, and an intermediate affinity state of the I-domain. The multiple substates were validated with residual dipolar coupling measurements. These results suggest that the presence of three substates in the apo I-domain enables the precise regulation of the binding process that is essential for the physiological function of LFA-1.  •We identified three substates in the apo LFA-1 I-domain using RAM simulations•Independent RDC measurements were accurately reproduced using the three substates•The three substates correspond to the inactive, low, and intermediate affinity states•The three substates are associated with the precise regulation of the LFA-1 binding Kukic et al. identified three substates of the apo LFA-1 I-domain using replica-averaged metadynamics simulations with NMR restraints. The presence of the inactive, low, and intermediate affinity substates in the apo LFA-1 I-domain represents an intrinsic property that can regulate the complex allosteric mechanism of the protein. Integrins are a family of transmembrane receptor proteins that play key roles in mediating the attachment and communication of cells with their environments, including other cells and the extracellular matrix . They ca2+ ion. The distal bottom face of the I-domain bears the N- and C-terminal interface to another domain of the αL subunit, called the β-propeller domain  of the αL subunit . The I-dr domain .2+ ion of MIDAS induces significant structural rearrangements in the loops forming MIDAS, namely the loops between β sheet 1 and α helix 1, α helix 3 and α helix 4, and β sheet 4 and α helix 5  for ICAM-1. However, during the inside-out signaling, intracellular signals received by the LFA-1 cytoplasmic domains make the I-domain much more competent for ligand binding. The conformational changes from the cytoplasmic domains are further transferred to the β I-like domain of the αL subunit. The β I-like domain MIDAS has been proposed to bind Glu310, located in the linker following α helix 7, and exerts a pull on α helix 7 that further induces the rearrangements at the I-domain MIDAS . So far,Both the inside-out and the outside-in signaling mechanisms can be inhibited by the binding of the I-domain inhibitors to the pocket underneath α helix 7 , which rThe structure-affinity relationship of the I-domain led to the suggestion that its affinity is regulated by its conformation and that the allosteric link between MIDAS and the motion of α helix 7 is critical in the binding regulation and the transmission of the outside-in and inside-out signals. Although the general mechanism that governs this conformational dependence of affinity has been laid out by structural and biophysical studies, a complete understanding of the intrinsic dynamics of the LFA-1 I-domain is currently lacking. To investigate this problem, we set out to characterize the dynamics of the I-domain in the apo form and the way it facilitates the complex signaling and allosteric modes of LFA-1.R1 and R2 relaxation rates and the model-free analysis, showed very restricted backbone motions on the pico- to nanosecond timescales. Surprisingly, almost all reported S2 values of the backbone amides were above 0.9. On the contrary, residual dipolar coupling (RDC) measurements from the same study unequivocally showed large discrepancies with all crystallographic structures determined to date, which were particularly large for the residues that belong to α helix 7. These results led to a proposal that α helix 7 is in dynamic exchange between many conformations on the microsecond to millisecond timescales and that the motion of α helix 7 in the apo I-domain can be crucial for the function of LFA-1.In a previous study, the apo I-domain dynamics were studied on a broad range of timescales . Short tIn this study, we obtained a conformational ensemble that represents the dynamics of the apo I-domain and illustrates the way these dynamics facilitate the complex signaling and allosteric modes of LFA-1. In particular, the low-frequency motions of the apo I-domain were characterized with a recently developed replica-averaged metadynamics (RAM) methodology. The RAM methodology integrates experimentally measured nuclear magnetic resonance (NMR) chemical shifts with bias exchange metadynamics, which is an advanced sampling method particularly effective in exploring the conformational space of proteins. The NMR chemical shifts help decode the chemical environment of atomic nuclei on timescales similar to those obtained from RDC measurements, and the RAM simulations are able to explore low-probability regions of the free energy landscape by allowing free diffusion along particular reaction coordinates (or collective variables [CVs]). The results obtained from this work strongly support the idea that not only the LA state but also the AI and IA states are embedded in the dynamics of the apo form of the I-domain. The existence of multiple states was then further validated with the previously measured RDCs. The presence of the multiple affinity states in the apo I-domain allows more precise regulation of ligand binding and is most probably essential for the physiological function of LFA-1.Experimentally measured backbone chemical shifts of LFA-1  were useFor each replica in the RAM simulations, we use a different metadynamics history-dependent potential  acting o2+ of MIDAS.The RAM simulations of the LFA-1 I-domain reached convergence after 150 ns when the bias potentials acting on all four replicas started to become stationary . The simThe free energy landscape clearly reveals the existence of three distinct minima . The twoUnrestrained simulations, unlike the chemical shift restrained RAM simulations, were only able to sample values of the angle θ similar to those obtained in the LA-like state B. On theThe conformations identified in the highest energy minimum can be separated from the rest of the structures in the RAM ensemble by examining the distance between the side chain of residue Phe292 and the MIDAS site B. WhereaDisplacement of the side chain of residue Phe292 out of the hydrophobic pocket in the IA-like state is followed by the formation of the salt bridge between Glu146 and Lys263 B, which The conformational rearrangements in the IA-like state do not cause significant backbone conformational changes elsewhere in the I-domain and hence leave MIDAS in the closed conformation, in accordance with the conclusions of the mutagenesis studies . HoweverThe conformations from the IA-like state appear in the free energy landscape with a free energy of at least 2.8 kJ/mol higher than that of the global minimum of the LA-like state. From these relative free energies, we can calculate the populations of the three states at 300 K, which are 48% for the LA-like state, 36% for the AI-like state, and 16% for IA-like states.The convergence of the RAM simulations was confirmed by the comparison between the experimentally measured chemical shift values and the values back-calculated from the RAM ensemble . The cheα helix = 0.18, rmsdβ sheet = 0.19). This was not the case when the secondary structure populations were obtained from the unrestrained ensemble . In particular, the populations of α helices 5, 6, and 7 were not accurately reproduced. Most importantly, the RAM ensemble was able to reproduce the complex secondary structure composition of α helix 7 derived from experimentally measured chemical shifts: the N-terminal residues (292–295) in the 310 conformation similar to the conformation observed in the crystallographic structures . This structure was initially solvated in a water box extending 12 Å from its surface and then energy minimized. All simulations were carried out in the canonical ensemble by keeping the volume fixed and by thermosetting the system with the modified Berendsen thermostat  and to the ith residue in the protein  specifies the position of the centroid of the Gaussian, and W and σi are the Gaussian height and width, respectively. The history-dependent bias potential defined by Equation In this equation, We performed RAM simulations of the LFA-1 I-domain at 300 K, using four replicas, one for each of the following four CVs:CV1: dihedral correlation. CV1 acts on 374 ϕ and φ dihedral angles of all residues in the protein. Parameters: Gaussian width σ = 0.1.CV2: dihedral correlation. CV2 acts on 35 χ1 angles that belong to all heavy side chain residues  except for residues in α helices 1 and 7. Parameters: Gaussian width σ = 0.1.CV3: dihedral correlation. CV3 acts on 25 χ1 angles that belong to the heavy side chain residues of α helices 1 and 7. Parameters: Gaussian width σ = 0.1.CV4: angle. CV4 uses the angle θ that α helix 7 forms with the hydrophobic core of the I-domain . The θ ateq∼150 ns , IA state , HA state , and AI state .In addition to the RAM and unrestrained ensembles, we created ensembles of the LFA-1 I-domain from the structures available in PDB. This ensemble includes structures of the I-domain in the LA state (PDB ID, All authors participated in the research. All authors participated in writing and editing the final manuscript.


The SIRP family of myeloid-paired receptors are characterized by having both activating and inhibiting members with extracellular regions that are relatively similar. Making good reagents to these receptors is not straightforward, particularly as they are relatively polymorphic. We describe the production of a monoclonal antibody (MAb) called OX130 that recognizes both common alleles of the human activating SIRPβ1 receptor but also cross-reacts with one of the common alleles of the inhibitory human SIRPα receptor. Thus one might get different outcomes when this MAb is used in assays from different individuals and shows the importance of characterizing SIRP MAb in this way. The SIRP family consists of three members with three Ig-like domains: SIRPα gives inhibitory signals, SIRPβ1 gives activating signals, and SIRPγ probably does not signal. In addition there are some more distantly related proteins with different numbers of extracellular domains. Like many paired receptors, these proteins are diverging rapidly in evolution with most sequence differences in the N-terminal domain. There are two common alleles of SIRPα that differ markedly in sequence  and two SIRPβ1 (12 differences). Thus reagents that are specific for each class of proteins are not straightforward to produce. In this report, we describe the production of the OX130 MAb that recognizes both common alleles of SIRPβ1 but also binds one SIRPα1 allele.P6 tag was expressed using the pEE14 vector in the CHO Lec3.2.8.1 cell line. Recombinant SIRPβ1(1) was purified by nickel affinity chromatography and used to immunize mice. Monoclonal antibodies were produced by standard procedures using the NS1 cell line. The MAbs were screened by ELISA on recombinant SIRP proteins, and those reactive with SIRP1 were cloned. One clone OX130 (mouse IgG) that also labeled SIRPβ1 cells by flow cytometry was characterized in detail by surface plasmon resonance analysis using a BIAcore™ 3000 , employing previously described methods. For this analysis, the SIRP proteins were expressed as three domain constructs together with rat CD4d3 + 4 as an antigenic tag to enable binding to a rat CD4 MAb (OX68) that had been immobilized on the BIAcore CM5 chip ( SIRPβ1 proteins (accession nos. NP_006056 and CAI21700), and SIRPγ (accession no. NP_061026), as described previously. Approximately 1000 response units (RU) of each recombinant SIRP protein were immobilized, including rat CD4 as a negative control protein. Human CD47 at 22 μM (prepared as described earlier) was passed over the immobilized proteins, followed by OX130 MAb and by CD47 again to see if OX130 MAb binding prevented CD47 binding to SIRPα.Recombinant human SIRPβ1 protein comprising three extracellular immunoglobulin superfamily (IgSF) domains (termed SIRPβ1(1) with accession numbers NP_006056, residues 1-360) with a C-terminal STRHCM5 chip . The SIRELISA results showed that OX130 MAb bound recombinant SIRPβ1  but not SIRPα  was tested—see below) or SIRPγ (data not shown). In order to give a more quantitative comparison of the reactivity of OX130, the MAb was tested for binding using surface plasmon resonance (SPR) to various SIRP proteins that had been immobilized to a BIAcore chip through a CD4 tag with an overlay of the BIAcore traces . Examination of the amino acid sequences of the SIRP domain 1 illustrated the many differences between the SIRPα alleles and SIRPβ1 alleles (The OX130 MAb recognized both common alleles of SIRPβ1 but not SIRPγ. However it reacted with one of the two common alleles of SIRPα but not the other. This binding prevented the subsequent binding of CD47, showing that the binding site of the MAb is in the N-terminal domain near the ligand binding domain. alleles . One reg