rongsheng jin lab in stock

Jahid S, Ortega JA, Vuong LM, Acquistapace IM, Hachey SJ, Flesher JL, La Serra MA, Brindani N, La Sala G, Manigrasso J, Arencibia JM, Bertozzi SM, Summa M, Bertorelli R, Armirotti A, Jin R, Liu Z, Chen CF, Edwards R, Hughes CCW, De Vivo M, Ganesan AK. PMID: 35385746; PMCID: PMC9127750.

Chen P, Zeng J, Liu Z, Thaker H, Wang S, Tian S, Zhang J, Tao L, Gutierrez CB, Xing L, Gerhard R, Huang L, Dong M, Jin R. PMID: 34145250; PMCID: PMC8213806.

Chen P, Lam KH, Liu Z, Mindlin FA, Chen B, Gutierrez CB, Huang L, Zhang Y, Hamza T, Feng H, Matsui T, Bowen ME, Perry K, Jin R. PMID: 31308519; PMCID: PMC6684407.

Lam, K. H., Guo, Z., Krez, N., Matsui, T., Perry, K., Weisemann, J., Rummel, A., Bowen, M. E. & Jin, R. A viral-fusion-peptide-like molecular switch drives membrane insertion of botulinum neurotoxin A1. Nat Commun 9, 5367 (2018) doi: 10.1038/s41467-018-07789-4.

Chen, P., Tao, L., Liu, Z., Dong, M. & Jin, R. Structural insight into Wnt signaling inhibition by Clostridium difficile toxin B. FEBS J (2018) doi: 10.1111/febs.14681.

Chen, P., Tao, L., Wang, T., Zhang, J., He, A., Lam, K. H., Liu, Z., He, X., Perry, K., Dong, M*. & Jin, R*. Structural basis for recognition of frizzled proteins by Clostridium difficile toxin B. Science 360, 664-669 (2018) (*corresponding authors) doi: 10.1126/science.aar1999. PMCID: PMC6231499

Lam, K. H., Sikorra, S., Weisemann, J., Maatsch, H., Perry, K., Rummel, A., Binz, T. & Jin, R. Structural and biochemical characterization of the protease domain of the mosaic botulinum neurotoxin type HA. Pathog Dis 76 (2018) doi: 10.1093/femspd/fty044. PMCID: PMC5961070

Silva, D. A., Stewart, L., Lam, K. H., Jin, R. & Baker, D. Structures and disulfide cross-linking of de novo designed therapeutic mini-proteins. FEBS J 285, 1783-1785 (2018) doi: 10.1111/febs.14394. PMCID: PMC6001749

Lam, K. H., Qi, R., Liu, S., Kroh, A., Yao, G., Perry, K., Rummel, A. & Jin, R. The hypothetical protein P47 of Clostridium botulinum E1 strain Beluga has a structural topology similar to bactericidal/permeability-increasing protein. Toxicon 147, 19-26 (2018) doi: 10.1016/j.toxicon.2017.10.012. PMCID: PMC5902665

Chevalier, A., Silva, D.A., Rocklin, G.J., Hicks, D.R., Vergara, R., Murapa, P., Bernard, S.M., Zhang, L., Lam, K.H., Yao, G., Bahl, C.D., Miyashita, S.I., Goreshnik, I., Fuller, J.T., Koday, M.T., Jenkins, C.M., Colvin, T., Carter, L., Bohn, A., Bryan, C.M., Fernández-Velasco, D.A., Stewart, L., Dong, M., Huang, X., Jin, R., Wilson, I.A., Fuller, D.H. & Baker, D. Massively parallel de novo protein design for targeted therapeutics. Nature 550(7674):74-79 (2017) doi: 10.1038/nature23912. PMCID: PMC5802399

Yao, G., Lam, K.H., Weisemann, J., Peng, L., Krez, N., Perry, K., Shoemaker, C.B., Dong, M., Rummel, A. & Jin, R. A camelid single-domain antibody neutralizes botulinum neurotoxin A by blocking host receptor binding. Sci Rep. 7;7(1):7438. (2017) doi: 10.1038/s41598-017-07457-5. PMCID: PMC5547058

Yao, G., Lam, K.H., Perry, K., Weisemann, J., Rummel, A. & Jin, R. Crystal Structure of the Receptor-Binding Domain of Botulinum Neurotoxin Type HA, Also Known as Type FA or H. Toxins (Basel) 9, 93 (2017) doi: 10.3390/toxins9030093. PMCID: PMC5371848

Yao, G., Zhang, S., Mahrhold, S., Lam, K. H., Stern, D., Bagramyan, K., Perry, K., Kalkum, M., Rummel, A.*, Dong, M.* & Jin, R.* N-linked glycosylation of SV2 is required for binding and uptake of botulinum neurotoxin A. Nat Struct Mol Biol 23 (7):656-662 (2016) (*corresponding authors) doi: 10.1038/nsmb.3245. PMCID: PMC5033645

Lee, K., Lam, K. H., Kruel, A. M., Mahrhold, S., Perry, K., Cheng, L. W., Rummel, A. & Jin, R. Inhibiting oral intoxication of botulinum neurotoxin A complex by carbohydrate receptor mimics. Toxicon 107, 43-49 (2015) doi: 10.1016/j.toxicon.2015.08.003. PMCID: PMC4658216

Lam, K.H. & Jin, R. Architecture of the botulinum neurotoxin complex: a molecular machine for protection and delivery. Current Opinion in Structural Biology 31:89-95 (2015) doi: 10.1016/j.sbi.2015.03.013. PMCID: PMC4476938

Lam, K.H., Yao, G. & Jin, R. Diverse binding modes, same goal: The receptor recognition mechanism of botulinum neurotoxin. Progress in Biophysics and Molecular Biology 117(2-3):225-31 (2015) doi: 10.1016/j.pbiomolbio.2015.02.004. PMCID: PMC4417461

Lam, T.I., Stanker, L.H., Lee, K., Jin, R. & Cheng, L.W. Translocation of botulinum neurotoxin serotype A and associated proteins across the intestinal epithelia. Cellular Microbiology 17(8):1133-1143 (2015) doi: 10.1111/cmi.12424. PMCID: PMC4610714

Matsui, T.*, Gu, S., Lam, K.H., Carter, L.G., Rummel, A., Mathews, II. & Jin, R.* Structural Basis of the pH-Dependent Assembly of a Botulinum Neurotoxin Complex. J. Mol. Biol. 426(22):3773-3782 (2014) doi: 10.1016/j.jmb.2014.09.009. (*corresponding authors) PMCID: PMC4252799

Lee, K., Zhong, X., Gu, S., Kruel, A.M., Dorner, M.B., Perry, K., Rummel, A., Dong, M. & Jin, R. Molecular basis for disruption of E-cadherin adhesion by botulinum neurotoxin A complex. Science 344(6190):1405-1410 (2014) doi: 10.1126/science.1253823. PMCID: PMC4164303

Lee, K., Lam, K.H., Kruel, A.M., Perry, K., Rummel, A. and Jin, R. High-resolution crystal structure of HA33 of botulinum neurotoxin type B progenitor toxin complex. Biochem. Biophys. Res. Commun. 446(2):568-573 (2014) doi: 10.1016/j.bbrc.2014.03.008. PMCID: PMC4020412

Yao, Y., Lee, K., Gu, S., Lam, K.H. & Jin, R. Botulinum Neurotoxin A Complex Recognizes Host Carbohydrates through Its Hemagglutinin Component, Toxins (Basel) 6(2):624-635 (2014) doi: 10.3390/toxins6020624. PMCID: PMC3942755

Lee, K., Gu, S., Jin, L., Le, T.T.N., Cheng, L.W., Strotmeier, J., Kruel, A.M., Yao, G., Perry, K., Rummel, A.* & Jin, R.* Structure of a Bimodular Botulinum Neurotoxin Complex Provides Insights into Its Oral Toxicity. PLoS Pathog. 9(10): e1003690 (2013) doi:10.1371/journal.ppat.1003690. (*corresponding authors) PMCID: PMC3795040

Zong, Y. and Jin, R. Structural mechanisms of the agrin-LRP4-MuSK signaling pathway in neuromuscular junction differentiation. Cell. Mol. Life Sci. 70(17):3077-88 (2013) doi: 10.1007/s00018-012-1209-9. PMCID: PMC4627850

Gu, S. and Jin, R. Assembly and function of the botulinum neurotoxin progenitor complex. Curr. Top. Microbiol. Immunol. 364:21-44 (2013) doi: 10.1007/978-3-642-33570-9_2. PMCID: PMC3875173

Gu, S., Rumpel, S., Zhou, J., Strotmeier, J., Bigalke, H., Perry, K., Shoemaker, C.B., Rummel, A. & Jin, R. Botulinum neurotoxin is shielded by NTNHA in an interlocked complex. Science 335(6071):977-81 (2012) doi: 10.1126/science.1214270. PMCID: PMC3545708

Zong, Y., Zhang, B., Gu, S., Lee, K., Zhou, J., Yao, G., Figueiredo, D., Perry, K., Mei, L.* & Jin, R.* Structural basis of neuron-specific regulation of postsynaptic differentiation. Gene & Development 26:247-258 (2012) doi: 10.1101/gad.180885.111. (*corresponding authors) PMCID: PMC3278892

Yao, G., Zong, Y., Gu, S., Zhou, J., Xu, H., Mathews, II. & Jin, R. Crystal structure of the glutamate receptor GluA1 amino-terminal domain. Biochem. J. 438(2):255-63 (2011) doi: 10.1042/BJ20110801. PMCID: PMC3296483

Strotmeier, J., Gu, S., Jutzi, S., Mahrhold, S., Zhou, J., Pich, A., Eichner, T., Bigalke, H., Rummel, A.*, Jin, R.* & Binz, T*. The biological activity of botulinum neurotoxin type C is dependent upon novel types of ganglioside binding sites. Mol. Microbiol. 81(1):143-56 (2011) doi: 10.1111/j.1365-2958.2011.07682.x. Epub 2011 Jun 2. (*corresponding authors)

Strotmeier, J., Lee, K., Völker, A.K., Mahrhold, S., Zong, Y., Zeiser, J., Zhou, J., Pich, A., Bigalke, H., Binz, T., Rummel, A.* & Jin, R.* Botulinum neurotoxin serotype D attacks neurons via two carbohydrate-binding sites in a ganglioside-dependent manner. Biochem. J. 431(2):207-16 (2010) (*corresponding authors)

Jin, R.*, Singh, S.K., Gu, S., Furukawa, H., Sobolevsky, A.I., Zhou, J., Jin, Y. & Gouaux E.* Crystal structure and association behavior of the GluR2 amino-terminal domain. EMBO J. 28(12):1812-23 (2009) (*corresponding authors) PMCID: PMC2699365

Kumar, J., Schuck. P., Jin, R. & Mayer, M.L. The N-terminal domain of GluR6-subtype glutamate receptor ion channels. Nat. Struct. Mol. Biol. 16(6):631-8 (2009) PMCID: PMC2729365

Jin, R., Rummel, A., Binz, T. & Brunger, A.T. Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity. Nature 444:1092-5 (2006)

Jin, R., Clark, S., Weeks, A.M., Dudman, J.T., Gouaux, E. & Partin, K.M. Mechanism of positive allosteric modulators acting on AMPA receptors. J. Neurosci. 25(39):9027-36 (2005)

Jin, R., Junutula, J.R., Matern, H.T., Ervin, K.E., Scheller, R.H. & Brunger, A.T. Exo84 and Sec5 are competitive regulatory Sec6/8 effectors to the RalA GTPase. EMBO J. 24:2064-74 (2005)

Jin, R., Bank, T., Mayer, M. L., Traynelis, S. & Gouaux, E. Structural basis for partial agonist action at ionotropic glutamate receptors. Nat. Neurosci. 6(8):803-10 (2003)

Wild type and mutated recombinant full-length activated BoNT/A1 were produced under biosafety level 2 containment (project number GAA A/Z 40654/3/123) recombinantly in K12 E. coli strain in Dr. Rummel’s lab6-tag were purified on Co2+-Talon matrix (Takara Bio Europe S.A.S., France) and eluted with 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 250 mM imidazole. For proteolytic activation, BoNT/A1 was incubated for 16 h at room temperature with 0.01 U bovine thrombin (Sigma-Aldrich Chemie GmbH, Germany) per µg of BoNT/A1. Subsequent gel filtration (Superdex-200 SEC; GE Healthcare, Germany) was performed in phosphate buffered saline (pH 7.4).

The purified ciA-D12 (S124C) was labeled with Alexa Fluor C2 647 maleimide (Molecular Probes) according to the manufacturer’s instructions. The labeled ciA-D12 was further purified by MonoQ ion-exchange chromatography in 10 mM Hepes (pH 8.0) and eluted with a NaCl gradient. The calculated dye to protein ratio was ~1 mole of dye per mole of ciA-D12. BoNT/A1i-ciA-D12 complex were prepared by mixing BoNT/A1i and ciA-D12 in 1:1.5 molar ratio and the complex was purified by size-exclusion chromatography using Superdex-200.

Liposomes containing 10% GT1b, 69% DOPC, 20% DOPS, 0.5% rhodamine-PE, and 0.5% biotin-PE were prepared by extrusion through a 100 nm pore membrane. To form proteoliposomes, 10 nM BoNT/A1i–D12* or oxidized BoNT/A1iDS–D12* was incubated with 0.5 mg/ml lipid at room temperature for 1 h at the pH indicated. The mixture was then diluted 1000-fold and incubated for 5 minutes in a passivated, quartz microscope chamber functionalized with streptavidin. The biotinylated liposomes were retained and unbound proteins are washed away by exhaustive rinsing with buffer. At the low densities needed for optical resolution of individual liposomes, we could observe sufficient liposomes for statistical analysis, while minimizing the probability that a diffraction-limited spot would contain multiple liposomes. Samples were imaged using a prism-based Total Internal Reflection Fluorescence (TIRF) microscope. Samples were first excited with a laser diode at 640 nm (Newport Corporation, Irvine, CA) to photobleach Alexa 647-labeled BoNT/A1i–D12* or BoNT/A1iDS-D12* molecules followed by excitation with a diode pumped solid state laser at 532 nm (Newport Corporation, Irvine, CA) to probe for Rhodamine-labeled liposomes. Emission from protein and lipids was separated using an Optosplit ratiometric image splitter (Cairn Research Ltd., Faversham UK) containing a 645 nm dichroic mirror, a 585/70 band pass filter for Rhodamine, and a 670/30 band pass filter for Alexa 647 (all Chroma, Bellows Falls, VT). The replicate images were relayed to a single iXon EMCCD camera (Andor Technologies, Belfast, UK) at a frame rate of 10 Hz. Data were processed in MATLAB to cross-correlate the replicate images and extract time traces for diffraction-limited spots with intensity above baseline. Single-molecule traces were hand selected based on the exhibition of single-step decays to baseline during 640 illumination and the appearance of Rhodamine emission during 532 illumination.

Citation:Lee K, Gu S, Jin L, Le TTN, Cheng LW, Strotmeier J, et al. (2013) Structure of a Bimodular Botulinum Neurotoxin Complex Provides Insights into Its Oral Toxicity. PLoS Pathog 9(10):

Structural information of HAs is available for serotypes C and D, such as the crystal structures of HA33 of serotype C (HA33-C) [12], [13], a complex composed of HA17 and HA33 of serotype D [14], and HA70 of serotype C (HA70-C) [15], [16]. However, BoNT/C and D rarely cause human botulism but are known to cause the syndrome in cattle, poultry, and wild birds. For BoNT/A, the major cause of human botulism, only the structure of HA33 (HA33-A), which displays an amino-acid identity of ∼38% to HA33-C and D, has been solved [17]. We have recently determined the crystal structure of the BoNT/A–NTNHA complex [5]. However, it remains largely unclear how the HAs assemble with one another and how they interact with BoNT and NTNHA.

Interacting residues are labeled in open-book views of the interfaces. (A) Interface between HA70 and HA17. (B) Interfaces between HA17 and the two HA33s are indicated by purple and blue. The HA33 residues involved in both interfaces are in green. (C) Interface between two HA33s attached to the same HA17. See Fig. S5 and S6 in Text S1 for stereo versions of the detailed interactions.

To determine the physiological relevance of these HA–glycan interactions during toxin absorption, we examined their ability to interfere with the HA complex-mediated disruption of Caco-2 TER. Lac, Gal, and IPTG markedly inhibited the TER reduction induced by the HA complex, and showed higher potencies when applied to the apical than to the basolateral compartment (Fig. 5A–B and Fig. S11A–D in Text S1). In contrast, α2,3- and α2,6-SiaLac, and to a lesser extent Neu5Ac, inhibited the decrease in TER only when applied apically, albeit more weakly than Lac (Fig. 5A–B and Fig. S11E–F in Text S1). We then examined the transport of the HA complex across the Caco-2 monolayer using a fluorescence-labeled HA complex (HA*) (Fig. 5C). Lac and IPTG efficiently inhibited the transport of HA* when it was applied to the apical or basolateral chamber. Blocking the transport of HA* via α2,3- and α2,6-SiaLac was more potent toward the basolateral compartment than toward the apical side. Neu5Ac at 50 mM did not inhibit transport of HA* from either side of the Caco-2 cell monolayer. These data are consistent with the ability of these carbohydrates to inhibit TER reduction induced by the HA complex. Collectively, these results suggest that the binding of HAs to Neu5Ac- and Gal-containing glycans on epithelial cells is essential for the transport of BoNT across the intestinal wall. Moreover, the carbohydrate receptors may play a more important role in the initial L-PTC absorption in the intestinal lumen, whereas other host receptors (e.g., E-cadherin) are involved once it gains access to the basolateral side.

(A, B) TER of Caco-2 monolayers was measured when Alexa-488-labeled HA complex (HA*) pre-incubated with Lac, IPTG, α2,3-SiaLac, or α2,6-SiaLac was applied to the apical (A; 58 nM) or basolateral (B; 17 nM) chambers. Values are means ± SD (n = 4–12). (C) HA* (with or without carbohydrates) or the Alexa-488-labeled HA33-DAFA complex (HA33-DAFA *) was applied to the apical (at 58 nM) or basolateral (at 17 nM) chamber. The fluorescence signals in both chambers were quantified after 24 hours and the amount of transported HA*/HA33-DAFA * was expressed as a percentage of the total HA*/HA33-DAFA * used. Values are means ± SD (n = 3–22).

The loose linkage between the M-PTC and the HA complex clearly suggests divided functions. We previously reported that the M-PTC"s compact structure protects BoNT against digestive enzymes and the extreme acidic environment of the GI tract [5], [23]. We now show that the HA complex is mainly responsible for BoNT absorption in the small intestine, through binding to specific host carbohydrate receptors. This new finding permitted the identification of IPTG as a prototypical oral inhibitor that extends survival following lethal oral BoNT/A intoxication of mice. Multivalent interactions involving nine binding sites for Neu5Ac- and Gal-containing glycans increase the overall avidity of binding between the L-PTC and glycans on the epithelial cell surface, and thus compensate for the modest glycan-binding affinities at individual binding sites (Fig. 6C). Similarly, the potency of carbohydrate receptor mimics could be improved by optimizing the HA–glycan interactions as revealed here or by introducing new HA–inhibitor interactions at individual binding sites based on rational design, as well as by designing multivalent inhibitors. Although such inhibitors cannot be used to treat fully developed food-borne botulism, they could provide temporary protection upon pre-treatment and could also be useful for cases of intestinal colonization with C. botulinum spores such as in cases of infant or adult intestinal botulism. Our results also suggest that the L-PTC could be exploited for alternative applications. For example, protein-based therapeutics could be coupled to the modified non-toxic L-PTC to allow oral delivery by improving drug stability, absorption efficiency, and bioavailability.

The purified HA70 was labeled with Alexa Fluor® 488 carboxylic acid, succinimidyl ester (Life Technologies) according to the manufacturer"s instructions. The labeled HA70 was further purified by Superdex 200 chromatography in 20 mM Tris (pH 8.0) and 50 mM NaCl. The calculated dye to protein ratio was ∼2 moles of dye per mole of monomeric HA70.

The HA17–HA33, the HA70–HA17, and the HA70D3–HA17 complexes were produced by co-expression and co-purification as described above. To assemble the mini-HA complex (HA70D3–HA17–HA33), the purified HA33 and the HA70D3–HA17 complex were mixed at a molar ratio of ∼2.5∶1 and incubated at 4°C overnight. The excess HA33 was removed by Superdex 200 chromatography with 20 mM Tris (pH 7.6) and 50 mM NaCl. The fully assembled HA complex was reconstituted by mixing the purified HA70 and the HA17–HA33 complex at a molar ratio of ∼1∶1.3. The mixture was incubated at 4°C overnight and the excess HA17–HA33 complex was removed from the mature HA complex by Superdex 200 chromatography with 20 mM Tris (pH 7.6) and 50 mM NaCl. The fluorescence-labeled HA complex was prepared with Alexa Fluor® 488-labeled HA70 and unlabeled HA17–HA33 complex (HA*) or HA17–HA33DAFA complex (HA33DAFA*) using a similar protocol.

Sedimentation equilibrium (SE) experiments were performed in a ProteomeLab XL-I (BeckmanCoulter) analytical ultracentrifuge. Purified HA samples were dialyzed extensively against a buffer containing 50 mM Tris (pH 7.6) and various NaCl concentrations, or 50 mM citric acid (pH 2.3) and various NaCl concentrations. Protein samples at concentrations of 0.4, 0.2, and 0.1 unit of OD280 were loaded in 6-channel equilibrium cells and centrifuged at 20°C in an An-50 Ti 8-place rotor at the first speed indicated until equilibrium was achieved and thereafter at the second speed. HA33 was analyzed at rotor speeds of 19,000 and 22,000 rpm. The HA17–HA33 and the HA70D3–HA17–HA33 complexes were analyzed at 12,000 and 14,000 rpm. The HA70–HA17 and the HA70–HA17–HA33 complexes were run at speeds of 6,000 and 8,000 rpm. For each sample, data sets for the two different speeds were analyzed independently using HeteroAnalysis software (by J.L. Cole and J.W. Lary, University of Connecticut). Three independent experiments were performed for each sample.

The L-PTC of BoNT/A was obtained from List Biological Laboratories, Inc. (Campbell, California) and Miprolab GmbH (Göttingen, Germany). The recombinant HA complex was reconstituted in vitro as described above. Negatively stained EM specimens were prepared following a previously described protocol [54]. Briefly, 3 µl of the L-PTC (∼0.02 mg/ml in 20 mM MES, pH 6.2, and 100 mM NaCl) or the HA complex (∼0.01 mg/ml in 20 mM Tris, pH 7.6, and 50 mM NaCl) was placed on a freshly glow-discharged carbon-coated EM grid, blotted with filter paper after 40 seconds, washed with two drops of deionized water, and then stained with two drops of freshly prepared 1% uranyl formate, which also served to fix the proteins.

Carbohydrate inhibition assays: Lac, Gal, IPTG, Neu5Ac, α2,6- and α2,3-SiaLac were dissolved in IMDM, sterile filtered and stored at −20°C. Neu5Ac stock solution was adjusted to pH 7.4. The wild type HA complex (HA wt), fluorescence-labeled HA complex (HA*), or the HA70-TPRA complex were pre-incubated with the corresponding carbohydrate over night at 4°C in IMDM and diluted to the final concentration with IMDM prior to administration. The TER upon administration of each carbohydrate in the highest concentrations used was checked in the absence of HA and was virtually identical to that of the control without sugars.

Transport measurement: For paracellular transport studies, filters were incubated in IMDM added to the apical (0.5 ml) and basolateral (1.5 ml) reservoir. As marker substance Alexa Fluor® 488 labeled HA* or HA33-DAFA* was administered to the apical or basolateral reservoirs at final concentrations of 58 nM and 17 nM, respectively. After 24 hour of incubation, 200 µl of samples were taken from the apical and the basolateral reservoir. The marker substance was measured in a BioTek Synergy 4 fluorescence spectrophotometer at 495 nm excitation and 519 nm emission wavelengths.

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abstract = {Mixed-chirality peptide macrocycles such as cyclosporine are among the most potent therapeutics identified to date, but there is currently no way to systematically search the structural space spanned by such compounds. Natural proteins do not provide a useful guide: Peptide macrocycles lack regular secondary structures and hydrophobic cores, and can contain local structures not accessible with L-amino acids. Here, we enumerate the stable structures that can be adopted by macrocyclic peptides composed of L- and D-amino acids by near-exhaustive backbone sampling followed by sequence design and energy landscape calculations. We identify more than 200 designs predicted to fold into single stable structures, many times more than the number of currently available unbound peptide macrocycle structures. Nuclear magnetic resonance structures of 9 of 12 designed 7- to 10-residue macrocycles, and three 11- to 14-residue bicyclic designs, are close to the computational models. Our results provide a nearly complete coverage of the rich space of structures possible for short peptide macrocycles and vastly increase the available starting scaffolds for both rational drug design and library selection methods.},

Mixed-chirality peptide macrocycles such as cyclosporine are among the most potent therapeutics identified to date, but there is currently no way to systematically search the structural space spanned by such compounds. Natural proteins do not provide a useful guide: Peptide macrocycles lack regular secondary structures and hydrophobic cores, and can contain local structures not accessible with L-amino acids. Here, we enumerate the stable structures that can be adopted by macrocyclic peptides composed of L- and D-amino acids by near-exhaustive backbone sampling followed by sequence design and energy landscape calculations. We identify more than 200 designs predicted to fold into single stable structures, many times more than the number of currently available unbound peptide macrocycle structures. Nuclear magnetic resonance structures of 9 of 12 designed 7- to 10-residue macrocycles, and three 11- to 14-residue bicyclic designs, are close to the computational models. Our results provide a nearly complete coverage of the rich space of structures possible for short peptide macrocycles and vastly increase the available starting scaffolds for both rational drug design and library selection methods.

Aaron Chevalier*, Daniel-Adriano Silva*, Gabriel J. Rocklin*, Derrick R. Hicks, Renan Vergara, Patience Murapa, Steffen M. Bernard, Lu Zhang, Kwok-Ho Lam, Guorui Yao, Christopher D. Bahl, Shin-Ichiro Miyashita, Inna Goreshnik, James T. Fuller andMerika T. Koday, Cody M. Jenkins, Tom Colvin, Lauren Carter, Alan Bohn, Cassie M. Bryan, D. Alejandro Fernández-Velasco, Lance Stewart, Min Dong, Xuhui Huang, Rongsheng Jin, Ian A. Wilson, Deborah H. Fuller, David Baker

author = {Aaron Chevalier* and Daniel-Adriano Silva* and Gabriel J. Rocklin* and Derrick R. Hicks and Renan Vergara and Patience Murapa and Steffen M. Bernard and Lu Zhang and Kwok-Ho Lam and Guorui Yao and Christopher D. Bahl and Shin-Ichiro Miyashita and Inna Goreshnik and James T. Fuller andMerika T. Koday and Cody M. Jenkins and Tom Colvin and Lauren Carter and Alan Bohn and Cassie M. Bryan and D. Alejandro Fernández-Velasco and Lance Stewart and Min Dong and Xuhui Huang and Rongsheng Jin and Ian A. Wilson and Deborah H. Fuller and David Baker},

Marcos, Enrique*, Basanta, Benjamin*, Chidyausiku, Tamuka M., Tang, Yuefeng, Oberdorfer, Gustav, Liu, Gaohua, Swapna, G. V. T., Guan, Rongjin, Silva, Daniel-Adriano, Dou, Jiayi, Pereira, Jose Henrique, Xiao, Rong, Sankaran, Banumathi, Zwart, Peter H., Montelione, Gaetano T., Baker, David

author = {Marcos, Enrique* and Basanta, Benjamin* and Chidyausiku, Tamuka M. and Tang, Yuefeng and Oberdorfer, Gustav and Liu, Gaohua and Swapna, G. V. T. and Guan, Rongjin and Silva, Daniel-Adriano and Dou, Jiayi and Pereira, Jose Henrique and Xiao, Rong and Sankaran, Banumathi and Zwart, Peter H. and Montelione, Gaetano T. and Baker, David},

abstract = {Nature provides many examples of self- and co-assembling protein-based molecular machines, including icosahedral protein cages that serve as scaffolds, enzymes, and compartments for essential biochemical reactions and icosahedral virus capsids, which encapsidate and protect viral genomes and mediate entry into host cells. Inspired by these natural materials, we report the computational design and experimental characterization of co-assembling, two-component, 120-subunit icosahedral protein nanostructures with molecular weights (1.8 to 2.8 megadaltons) and dimensions (24 to 40 nanometers in diameter) comparable to those of small viral capsids. Electron microscopy, small-angle x-ray scattering, and x-ray crystallography show that 10 designs spanning three distinct icosahedral architectures form materials closely matching the design models. In vitro assembly of icosahedral complexes from independently purified components occurs rapidly, at rates comparable to those of viral capsids, and enables controlled packaging of molecular cargo through charge complementarity. The ability to design megadalton-scale materials with atomic-level accuracy and controllable assembly opens the door to a new generation of genetically programmable protein-based molecular machines.},

Nature provides many examples of self- and co-assembling protein-based molecular machines, including icosahedral protein cages that serve as scaffolds, enzymes, and compartments for essential biochemical reactions and icosahedral virus capsids, which encapsidate and protect viral genomes and mediate entry into host cells. Inspired by these natural materials, we report the computational design and experimental characterization of co-assembling, two-component, 120-subunit icosahedral protein nanostructures with molecular weights (1.8 to 2.8 megadaltons) and dimensions (24 to 40 nanometers in diameter) comparable to those of small viral capsids. Electron microscopy, small-angle x-ray scattering, and x-ray crystallography show that 10 designs spanning three distinct icosahedral architectures form materials closely matching the design models. In vitro assembly of icosahedral complexes from independently purified components occurs rapidly, at rates comparable to those of viral capsids, and enables controlled packaging of molecular cargo through charge complementarity. The ability to design megadalton-scale materials with atomic-level accuracy and controllable assembly opens the door to a new generation of genetically programmable protein-based molecular machines.

What we know thus far from in vivo and ex vivo assays is that BoNT itself can transcytose across the intestinal epithelium (Fujinaga, 2010; Couesnon et

What role do NAPs play in translocation? Previous data suggested that NAPs, specifically HA33, can disrupt tight junctions, compromising the epithelial cell barrier and allowing BoNT complex to enter through a paracellular pathway (Fujinaga, 2010). The HA-C was also shown to bind E-cadherin, a cell adhesion protein that likely plays a major role in toxin cell surface binding (Sugawara et

Our results also contrast with ex vivo studies where upper intestinal sections were treated with fluorescent-labelled BoNT/A heavy chain at 4°C for 30–120

Cheng LW, Onisko B, Johnson EA, Reader JR, Griffey SM, Larson AE, et al. Effects of purification on the bioavailability of botulinum neurotoxin type A. Toxicology.2008;249:123–129. [Abstract]

Fujinaga Y, Matsumura T, Jin Y, Takegahara Y. Sugawara Y. A novel function of botulinum toxin-associated proteins: HA proteins disrupt intestinal epithelial barrier to increase toxin absorption. Toxicon.2009;54:583–586. [Abstract]

Inoue K, Fujinaga Y, Watanabe T, Ohyama T, Takeshi K, Moriishi K, et al. Molecular composition of Clostridium botulinum type A progenitor toxins. Infect Immun.1996;64:1589–1594. Abstract]

Jin Y, Takegahara Y, Sugawara Y, Matsumura T. Fujinaga Y. Disruption of the epithelial barrier by botulinum haemagglutinin (HA) proteins – differences in cell tropism and the mechanism of action between HA proteins of types A or B, and HA proteins of type C. Microbiology.2009;155:35–45. [Abstract]

Lee K, Gu S, Jin L, Le TT, Cheng LW, Strotmeier J, et al. Structure of a bimodular botulinum neurotoxin complex provides insights into its oral toxicity. PLoS Pathog.2013;9:e1003690. Abstract]

Matsumura T, Jin Y, Kabumoto Y, Takegahara Y, Oguma K, Lencer WI. Fujinaga Y. The HA proteins of botulinum toxin disrupt intestinal epithelial intercellular junctions to increase toxin absorption. Cell Microbiol.2008;10:355–364. [Abstract]

Sugawara Y, Matsumura T, Takegahara Y, Jin Y, Tsukasaki Y, Takeichi M. Fujinaga Y. Botulinum hemagglutinin disrupts the intercellular epithelial barrier by directly binding E-cadherin. J Cell Biol.2010;189:691–700. Abstract]

Yao G, Lee K, Gu S, Lam KH. Jin R. Botulinum neurotoxin A complex recognizes host carbohydrates through its hemagglutinin component. Toxins.2014;6:624–635. Abstract]

A free program for multiple sequence alignment editing, visualisation and analysis that is available in two forms: a lightweight Java applet for use in web applications, and a powerful desktop application that employs web services for sequence alignment, secondary structure prediction and the retrieval of alignments, sequences, annotation and structures from public databases and any DAS 1.53 compliant sequence or annotation server. Use it to view and edit sequence alignments, analyse them with phylogenetic trees and principal components analysis (PCA) plots and explore molecular structures and annotation. Jalview has built in DNA, RNA and protein sequence and structure visualisation and analysis capabilities. It uses Jmol to view 3D structures, and VARNA to display RNA secondary structure.

The IR8a CREL functions in subcellular trafficking. a Immunofluorescence with antibodies against GFP (green), IR8a (blue) and IR64a (red) on antennal sections of animals expressing the indicated transgenes in Ir8a neurons. Genotypes are of the form Ir8a-Gal4/UAS-EGFP:Ir8ax. The white asterisks (in this and other panels) indicate the central cavity of sacculus chamber 3, into which the IR64a+IR8a-expressing OSN ciliated dendrites project (see also the merged panels, in which bright-field images are overlaid to provide anatomical landmarks). In the top left panel, the arrowhead marks the ciliated ending of one neuron; the soma and inner segment of this neuron are also indicated (the outer segment—before the cilium—is difficult to see because only trace levels of receptors are detected in this region). Scale bar (for all panels in this figure): 10 μm. For each genotype, the phenotype was assessed in multiple sections of antennae from at least 20 animals from two independent genetic crosses, allowing observation of several hundred different neurons. We quantified the localisation properties by counting the number of sensory cilia with detectable EGFP signal as a percentage of the total number of cell bodies in the imaged samples; this is not expected to be 100% because sensory endings for each OSN soma are not necessarily present in the thin tissue sections (see ‘Methods’ section on imaging): EGFP:IR8awt = 75% (83 labelled cilia/111 soma), EGFP:IR8a∆CREL = 0% (0/93), EGFP:IR8aN669Q = 61% (65/106). b Immunofluorescence with antibodies against GFP (green), IR8a (blue) and IR64a (red) on antennal sections of animals expressing the indicated transgenes in Ir8a neurons in an Ir8a mutant background. Genotypes are of the form Ir8a1/Y;Ir8a-Gal4/UAS-EGFP:Ir8ax. EGFP:IR8a∆CREL and EGFP:IR8aN669Qare impaired in localisation to the cilia in the absence of endogenous IR8a (the occasional projections from the soma represent protein within the inner segment only). In addition, both proteins appear to be destabilised; consequently, endogenous IR64a is also detected at substantially lower levels in these two genotypes (but see Additional file 4: Figure S4). OSNs that express EGFP:IR8a∆CREL also display signs of sickness (e.g. smaller soma). For each genotype, the phenotype was assessed in multiple sections of antennae from at least 20 animals from two independent genetic crosses. Quantifications: EGFP:IR8awt = 79% (177/225), EGFP:IR8a∆CREL = 0% (0/198), EGFP:IR8aN669Q = 35% (78/220)

Our observation that wild-type IR8a can promote cilia transport of IR8aN669Q (Fig. 2a, b) raised the question of whether a tuning IR that is targeted to cilia with IR8aN669Q can facilitate the localisation of a tuning IR that cannot, if they are incorporated into a common complex. We tested this possibility in two ways: first, we examined the distribution of IR64a in its own neurons expressing EGFP:IR8aN669Q (but not endogenous IR8a) together with a control receptor (IR75a, which cannot localise to cilia with IR8aN669Q) or test receptors (IR75c or IR84a, which can localise with IR8aN669Q). While co-expression of IR75c or IR84a (but not IR75a) promoted cilia targeting of EGFP:IR8aN669Q, in no case did this lead to localisation of IR64a to the sensory compartment (Additional file 6: Figure S6A). Rather, the levels of IR64a were substantially reduced upon co-expression of an additional tuning IR. This might be because these ectopically expressed IRs preferentially combine with EGFP:IR8aN669Q, thereby excluding IR64a from associating with the co-receptor resulting in its destabilisation (as observed previously [28, 32]). Second, in Or22a neurons, we misexpressed IR75a together with EGFP:IR8awt or EGFP:IR8aN669Q, in the absence or presence of IR75c. Unexpectedly, we observed that addition of IR75c led to lower levels and abolished cilia localisation of IR75a when co-expressed with EGFP:IR8awt (Additional file 6: Figure S6B), suggesting that IR75c outcompetes—rather than collaborates—with IR75a to form stable, transport-competent complexes. Together, these results indicate that different tuning IRs do not readily assemble into a common complex with IR8a.

To determine where in the endomembrane system the trafficking of IR8a∆CREL and IR8aN669Q is blocked, we visualised the distribution of these EGFP-tagged receptors relative to markers for different organelles: endoplasmic reticulum (ER) (labelled with tdTomato:Sec61β [34]), Golgi apparatus (labelled with γCOP:mRFP [35]) and the cilia transition zone (labelled with antibodies against B9d1 [36]). We used genetically encoded markers for the ER and Golgi in order to express them only in the OSNs of interest, thereby avoiding confounding signal from the organelle-rich epidermal cells in the antenna [37].

The IR8a CREL N-glycosylation site is not essential for odour-evoked IR signalling. a Representative traces of the responses of Or22a neurons—those exhibiting the larger of the two spike amplitudes within this sensillum (black arrowhead)—expressing the indicated combinations of IRs, exposed to a 1-s pulse (black bar) of phenylacetic acid (1% v/v). Genotypes are of the form UAS-EGFP:Ir8ax/UAS-Ir84a;Or22a-Gal4/+, except for the control (Or22a-Gal4/+). b Quantification of the odour-evoked responses of the genotypes shown in a. Mean solvent corrected responses ±SEM are shown (n (number of sensilla) are indicated beneath each bar; mixed genders). Bars labelled with different letters are statistically different from each other (p < 0.05; Student’s t test with Benjamini and Hochberg correction for multiple comparisons). c Representative traces of the responses of Or22a neurons expressing the indicated combinations of IRs, exposed to a 1-s pulse (black bar) of propionic acid (1% v/v). Genotypes are of the form UAS-EGFP:Ir8ax/+;Or22a-Gal4/UAS-Ir75c, except for the control (Or22a-Gal4/+). d Quantification of the odour-evoked responses of the genotypes shown in c, presented as in b

We extended this analysis with a second tuning receptor, IR75c, which detects propionic acid [11]. Although Or22a neurons displayed weak endogenous responses to this odour, these are much lower than those exhibited upon expression of IR75c+EGFP:IR8awt (Fig. 5c, d). As expected, IR75c+EGFP:IR8a∆CREL-expressing neurons had similar propionic acid sensitivity to those lacking IRs (Fig. 5c, d). In contrast to the observations with IR84a, IR75c+EGFP:IR8aN669Q co-expression yielded responses that are decreased compared to IR75c+EGFP:IR8awt (Fig. 5c, d), which might reflect defects in function or diminished levels of cilia localisation not discernable with available reagents. Together, however, these observations indicate that the CREL glycosylation is not essential for IR signalling.

—Pfizer Invests In Bind Therapeutics “” Pfizer and Bind Therapeutics will collaborate on the development of Bind’s Accurin therapeutics. The Cambridge, Mass.-based biotech firm uses “medicinal nanoengineering” to create drug-carrying particles targeted for specific tissues and cells. For an exclusive option to develop and commercialize selected Accurins, Pfizer will pay about $50 million in up-front and development fees along with another $160 million for achieving regulatory and sales milestones for each Accurin. Bind also has a $180 million deal with Amgen for a kinase inhibitor. Bind’s technology originates from Massachusetts Institute of Technology and Harvard Medical School. /articles/91/i14/Pfizer-Invests-Bind-Therapeutics.html 20130408 Concentrates 91 14 /magazine/91/09114.html Pfizer Invests In Bind Therapeutics pharmaceuticals, nanotechnology, collaboration con bus Ann M. Thayer business Pfizer Invests In Bind Therapeutics Chemical & Engineering News Pfizer Invests In Bind Therapeutics Pfizer Invests In Bind Therapeutics