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Cryo-EM structures reveal the dynamic transformation of human alpha-2-macroglobulin working as a protease inhibitor

Science China Life Sciences volume 65, pages 2491–2504 (2022)Cite this article

Abstract

Human alpha-2-macroglobulin is a well-known inhibitor of a broad spectrum of proteases and plays important roles in immunity, inflammation, and infections. Here, we report the cryo-EM structures of human alpha-2-macroglobulin in its native state, induced state transformed by its authentic substrate, human trypsin, and serial intermediate states between the native and fully induced states. These structures exhibit distinct conformations, which reveal the dynamic transformation of alpha-2-macro-globulin that acts as a protease inhibitor. The results shed light on the molecular mechanism of alpha-2-macroglobulin in entrapping substrates.

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References

  • Arimura, Y., and Funabiki, H. (2021). Structural mechanics of the alpha-2-macroglobulin transformation. J Mol Biol 434, 167413.

    Article  PubMed  Google Scholar 

  • Asplin, I.R., Wu, S.M., Mathew, S., Bhattacharjee, G., and Pizzo, S.V. (2001). Differential regulation of the fibroblast growth factor (FGF) family by α2-macroglobulin: evidence for selective modulation of FGF-2-induced angiogenesis. Blood 97, 3450–3457.

    Article  CAS  PubMed  Google Scholar 

  • Barrett, A.J., Brown, M.A., and Sayers, C.A. (1979). The electrophoretically “slow” and “fast” forms of the α2-macroglobulin molecule. Biochem J 181, 401–418.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Blacker, D., Wilcox, M.A., Laird, N.M., Rodes, L., Horvath, S.M., Go, R. C.P., Perry, R., Watson Jr. B., Bassett, S.S., McInnis, M.G., et al. (1998). Alpha-2 macroglobulin is genetically associated with Alzheimer disease. Nat Genet 19, 357–360.

    Article  CAS  PubMed  Google Scholar 

  • Borth, W., and Luger, T.A. (1989). Identification of α2-macroglobulin as a cytokine binding plasma protein. J Biol Chem 264, 5818–5825.

    Article  CAS  PubMed  Google Scholar 

  • Bu, G., Williams, S., Strickland, D.K., and Schwartz, A.L. (1992). Low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor is an hepatic receptor for tissue-type plasminogen activator. Proc Natl Acad Sci USA 89, 7427–7431.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cáceres, L.C., Bonacci, G.R., Sánchez, M.C., and Chiabrando, G.A. (2010). Activated α2 macroglobulin induces matrix metalloproteinase 9 expression by low-density lipoprotein receptor-related protein 1 through MAPK-ERK1/2 and NF-κB activation in macrophage-derived cell lines. J Cell Biochem 111, 607–617.

    Article  PubMed  Google Scholar 

  • Cater, J.H., Wilson, M.R., and Wyatt, A.R. (2019). Alpha-2-macroglobulin, a hypochlorite-regulated chaperone and immune system modulator. Oxid Med Cell Longev 2019, 1–9.

    Article  Google Scholar 

  • Chuang, W.H., Liu, P.C., Hung, C.Y., and Lee, K.K. (2014). Purification, characterization and molecular cloning of alpha-2-macroglobulin in cobia, Rachycentron canadum. Fish Shellfish Immunol 41, 346–355.

    Article  CAS  PubMed  Google Scholar 

  • Dodds, A.W., Ren, X.D., Willis, A.C., and Law, S.K.A. (1996). The reaction mechanism of the internal thioester in the human complement component C4. Nature 379, 177–179.

    Article  CAS  PubMed  Google Scholar 

  • Dolmer, K., Huang, W., and Gettins, P.G.W. (2000). NMR solution structure of complement-like repeat CR3 from the low density lipoprotein receptor-related protein. J Biol Chem 275, 3264–3269.

    Article  CAS  PubMed  Google Scholar 

  • Dursun, E., Gezen-Ak, D., Hanağasi, H., Bilgiç, B., Lohmann, E., Ertan, S., Atasoy, İ.L., Alaylioğlu, M., Araz, Ö.S., Önal, B., et al. (2015). The interleukin 1 alpha, interleukin 1 beta, interleukin 6 and alpha-2-macroglobulin serum levels in patients with early or late onset Alzheimer’s disease, mild cognitive impairment or Parkinson’s disease. J Neuroimmunol 283, 50–57.

    Article  CAS  PubMed  Google Scholar 

  • Emsley, P., Lohkamp, B., Scott, W.G., and Cowtan, K. (2010). Features and development of Coot. Acta Crystlogr D Biol Crystlogr 66, 486–501.

    Article  CAS  Google Scholar 

  • Fredslund, F., Jenner, L., Husted, L.B., Nyborg, J., Andersen, G.R., and Sottrup-Jensen, L. (2006). The structure of bovine complement component 3 reveals the basis for thioester function. J Mol Biol 361, 115–127.

    Article  CAS  PubMed  Google Scholar 

  • Fyfe, C.D., Grinter, R., Josts, I., Mosbahi, K., Roszak, A.W., Cogdell, R.J., Wall, D.M., Burchmore, R.J.S., Byron, O., and Walker, D. (2015). Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment. Acta Crystlogr D Biol Crystlogr 71, 1478–1486.

    Article  CAS  Google Scholar 

  • Galliano, M.F., Toulza, E., Gallinaro, H., Jonca, N., Ishida-Yamamoto, A., Serre, G., and Guerrin, M. (2006). A novel protease inhibitor of the α2-macroglobulin family expressed in the human epidermis. J Biol Chem 281, 5780–5789.

    Article  CAS  PubMed  Google Scholar 

  • Garcia-Ferrer, I., Arêde, P., Gómez-Blanco, J., Luque, D., Duquerroy, S., Castón, J.R., Goulas, T., and Gomis-Rüth, F.X. (2015). Structural and functional insights into Escherichia coli α2-macroglobulin endopeptidase snap-trap inhibition. Proc Natl Acad Sci USA 112, 8290–8295.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gettins, P.G.W., Beechem, J.M., and Crews, B.C. (1993). α2-Macroglobulin bait region integrity. FEBS Lett 325, 267–270.

    Article  CAS  PubMed  Google Scholar 

  • Gettins, P.G.W., Crews, B., Beth, A.H., and Hideg, K. (1995). Bait region-thiol ester mapping in human α2-macroglobulin. FEBS Lett 367, 137–140.

    Article  CAS  PubMed  Google Scholar 

  • Harthun, N.L., Weaver, A.M., Brinckerhoff, L.H., Deacon, D.H., Gonias, S. L., and Slingluff Jr., C.L. (1998). Activated α2-macroglobulin reverses the immunosuppressive activity in human breast cancer cell-conditioned medium by selectively neutralizing transforming growth factor-β in the presence of interleukin-2. J Immunother 21, 85–94.

    Article  CAS  PubMed  Google Scholar 

  • Harwood, S.L., Lyngsø, J., Zarantonello, A., Kjøge, K., Nielsen, P.K., Andersen, G.R., Pedersen, J.S., and Enghild, J.J. (2021). Structural investigations of human A2M identify a hollow native conformation that underlies its distinctive protease-trapping mechanism. Mol Cell Proteomics 20, 100090.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • He, H., McCartney, D.J., Wei, Q., Esadeg, S., Zhang, J., Foster, R.A., Hayes, M.A., Tayade, C., Van Leuven, F., and Croy, B.A. (2005). Characterization of a murine alpha 2 macroglobulin gene expressed in reproductive and cardiovascular tissue1. Biol Reprod 72, 266–275.

    Article  CAS  PubMed  Google Scholar 

  • Hibbetts, K., Hines, B., and Williams, D. (1999). An overview of proteinase inhibitors. J Vet Internal Med 13, 302–308.

    Article  CAS  Google Scholar 

  • Holtet, T.L., Nielsen, K.L., Etzerodt, M., Moestrup, S.K., Gliemann, J., Sottrup-Jensen, L., and Thøgersen, H.C. (1994). Receptor-binding domain of human α2-macroglobulin expression, folding and biochemical characterization of a high-affinity recombinant derivative. FEBS Lett 344, 242–246.

    Article  CAS  PubMed  Google Scholar 

  • Janssen, B.J.C., Huizinga, E.G., Raaijmakers, H.C.A., Roos, A., Daha, M. R., Nilsson-Ekdahl, K., Nilsson, B., and Gros, P. (2005). Structures of complement component C3 provide insights into the function and evolution of immunity. Nature 437, 505–511.

    Article  CAS  PubMed  Google Scholar 

  • Kidmose, R.T., Laursen, N.S., Dobó, J., Kjaer, T.R., Sirotkina, S., Yatime, L., Sottrup-Jensen, L., Thiel, S., Gál, P., and Andersen, G.R. (2012). Structural basis for activation of the complement system by component C4 cleavage. Proc Natl Acad Sci USA 109, 15425–15430.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kimanius, D., Forsberg, B.O., Scheres, S.H., and Lindahl, E. (2016). Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. eLife 5, e18722.

    Article  PubMed  PubMed Central  Google Scholar 

  • Kolodziej, S.J., Wagenknecht, T., Strickland, D.K., and Stoops, J.K. (2002). The three-dimensional structure of the human α2-macroglobulin dimer reveals its structural organization in the tetrameric native and chymotrypsin α2-macroglobulin complexes. J Biol Chem 277, 28031–28037.

    Article  CAS  PubMed  Google Scholar 

  • LaMarre, J., Wollenberg, G.K., Gonias, S.L., and Hayes, M.A. (1991). Cytokine binding and clearance properties of proteinase-activated alpha 2-macroglobulins. Lab Invest 65, 3–14.

    CAS  PubMed  Google Scholar 

  • Le, B.V., Williams, M., Logarajah, S., and Baxter, R.H.G. (2012). Molecular basis for genetic resistance of Anopheles gambiae to Plasmodium: structural analysis of TEP1 susceptible and resistant alleles. PLoS Pathog 8, e1002958.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liebschner, D., Afonine, P.V., Baker, M.L., Bunkóczi, G., Chen, V.B., Croll, T.I., Hintze, B., Hung, L.W., Jain, S., McCoy, A.J., et al. (2019). Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystlogr D Struct Biol 75, 861–877.

    Article  CAS  Google Scholar 

  • Liu, Q., Ling, T.Y., Shieh, H.S., Johnson, F.E., Huang, J.S., and Huang, S.S. (2001). Identification of the high affinity binding site in transforming growth factor-β involved in complex formation with α2-macroglobulin. J Biol Chem 276, 46212–46218.

    Article  CAS  PubMed  Google Scholar 

  • Luque, D., Goulas, T., Mata, C.P., Mendes, S.R., Gomis-Rüth, F.X., and Castón, J.R. (2022). Cryo-EM structures show the mechanistic basis of pan-peptidase inhibition by human α2-macroglobulin. Proc Natl Acad Sci USA 119, e2200102119.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Marrero, A., Duquerroy, S., Trapani, S., Goulas, T., Guevara, T., Andersen, G.R., Navaza, J., Sottrup-Jensen, L., and Gomis-Rüth, F.X. (2012). The crystal structure of human α2-macroglobulin reveals a unique molecular cage. Angew Chem Int Ed 51, 3340–3344.

    Article  CAS  Google Scholar 

  • Meyer, C., Hinrichs, W., and Hahn, U. (2012). Human α2-macroglobulin-another variation on the venus flytrap. Angew Chem Int Ed 51, 5045–5047.

    Article  CAS  Google Scholar 

  • Misra, U.K., and Pizzo, S.V. (2015). Activated α2-macroglobulin binding to human prostate cancer cells triggers insulin-like responses. J Biol Chem 290, 9571–9587.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nielsen, K.L., Holtet, T.L., Etzerodt, M., Moestrup, S.K., Gliemann, J., Sottrup-Jensen, L., and Thogersen, H.C. (1996). Identification of residues in α-macroglobulins important for binding to the α2-macroglobulin receptor/low density lipoprotein receptor-related protein. J Biol Chem 271, 12909–12912.

    Article  CAS  PubMed  Google Scholar 

  • Okubo, H., Ishibashi, H., Shibata, K., Tsuda-Kawamura, K., and Yanase, T. (1984). Distribution of α2-macroglobulin in normal, inflammatory, and tumor tissues in rats. Inflammation 8, 171–179.

    Article  CAS  PubMed  Google Scholar 

  • Panyutich, A., and Ganz, T. (1991). Activated α2-macroglobulin is a principal defensin-binding protein. Am J Respir Cell Mol Biol 5, 101–106.

    Article  CAS  PubMed  Google Scholar 

  • Peslova, G., Petrak, J., Kuzelova, K., Hrdy, I., Halada, P., Kuchel, P.W., Soe-Lin, S., Ponka, P., Sutak, R., Becker, E., et al. (2009). Hepcidin, the hormone of iron metabolism, is bound specifically to α-2-macroglobulin in blood. Blood 113, 6225–6236.

    Article  CAS  PubMed  Google Scholar 

  • Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D. M., Meng, E.C., and Ferrin, T.E. (2004). UCSF Chimera—A visualization system for exploratory research and analysis. J Comput Chem 25, 1605–1612.

    Article  CAS  PubMed  Google Scholar 

  • Pettersen, E.F., Goddard, T.D., Huang, C.C., Meng, E.C., Couch, G.S., Croll, T.I., Morris, J.H., and Ferrin, T.E. (2021). UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci 30, 70–82.

    Article  CAS  PubMed  Google Scholar 

  • Pochon, F., Tourbez, M., Favaudon, V., and Delain, E. (1987). Covalent and non-covalent interaction of chymotrypsin with α2-macroglobulin. FEBS Lett 217, 101–105.

    Article  CAS  PubMed  Google Scholar 

  • Punjani, A., Rubinstein, J.L., Fleet, D.J., and Brubaker, M.A. (2017). cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods 14, 290–296.

    Article  CAS  PubMed  Google Scholar 

  • Rehman, A.A., Ahsan, H., and Khan, F.H. (2013). Alpha-2-macroglobulin: a physiological guardian. J Cell Physiol 228, 1665–1675.

    Article  CAS  PubMed  Google Scholar 

  • Sottrup-Jensen, L., Gliemann, J., and Van Leuven, F. (1986). Domain structure of human α2-macroglobulin. FEBS Lett 205, 20–24.

    Article  CAS  PubMed  Google Scholar 

  • Sottrup-Jensen, L., Petersen, T.E., and Magnusson, S. (1980). A thiol-ester in α2-macroglobulin cleaved during proteinase complex formation. FEBS Lett 121, 275–279.

    Article  CAS  PubMed  Google Scholar 

  • Sottrup-Jensen, L., Petersen, T.E., and Magnusson, S. (1981). Trypsin-induced activation of the thiol esters in α2-macroglobulin generates a short-lived intermediate (“nascent” α2-M) that can react rapidly to incorporate not only methylamine or putrescine but also proteins lacking proteinase activity. FEBS Lett 128, 123–126.

    Article  CAS  PubMed  Google Scholar 

  • Sottrup-Jensen, L., Sand, O., Kristensen, L., and Fey, G.H. (1989). The α-macroglobulin bait region. J Biol Chem 264, 15781–15789.

    Article  CAS  PubMed  Google Scholar 

  • Van Leuven, F., Cassiman, J.J., and Van den Berghe, H. (1981a). Functional modifications of α2-macroglobulin by primary amines. I. Characterization of α2 M after derivatization by methylamine and by factor XIII. J Biol Chem 256, 9016–9022.

    Article  CAS  PubMed  Google Scholar 

  • Van Leuven, F., Cassiman, J.J., and Van den Berghe, H. (1981b). Functional modifications of α2-macroglobulin by primary amines. II. Inhibition of covalent binding of trypsin to α2 M by methylamine and other primary amines. J Biol Chem 256, 9023–9027.

    Article  CAS  PubMed  Google Scholar 

  • Van Leuven, F., Cassiman, J.J., and Van den Berghe, H. (1982). Functional modifications of α2-macroglobulin by primary amines. Kinetics of inactivation of α2-macroglobulin by methylamine, and formation of anomalous complexes with trypsin. Biochem J 201, 119–128.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Van Leuven, F., Marynen, P., Sottrup-Jensen, L., Cassiman, J.J., and Van den Berghe, H. (1986). The receptor-binding domain of human α2-macroglobulin. Isolation after limited proteolysis with a bacterial proteinase. J Biol Chem 261, 11369–11373.

    Article  CAS  PubMed  Google Scholar 

  • Vandooren, J., and Itoh, Y. (2021). Alpha-2-macroglobulin in inflammation, immunity and infections. Front Immunol 12, 803244.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wong, S.G., and Dessen, A. (2014). Structure of a bacterial α2-macroglobulin reveals mimicry of eukaryotic innate immunity. Nat Commun 5, 4917.

    Article  CAS  PubMed  Google Scholar 

  • Wyatt, A.R., Constantinescu, P., Ecroyd, H., Dobson, C.M., Wilson, M.R., Kumita, J.R., and Yerbury, J.J. (2013). Protease-activated alpha-2-macroglobulin can inhibit amyloid formation via two distinct mechanisms. FEBS Lett 587, 398–403.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wyatt, A.R., Kumita, J.R., Farrawell, N.E., Dobson, C.M., and Wilson, M. R. (2015). Alpha-2-macroglobulin is acutely sensitive to freezing and lyophilization: implications for structural and functional studies. PLoS ONE 10, e0130036.

    Article  PubMed  PubMed Central  Google Scholar 

  • Yang, B., Wu, Y.J., Zhu, M., Fan, S.B., Lin, J., Zhang, K., Li, S., Chi, H., Li, Y.X., Chen, H.F., et al. (2012). Identification of cross-linked peptides from complex samples. Nat Methods 9, 904–906.

    Article  CAS  PubMed  Google Scholar 

  • Zhang, K. (2016). Gctf: real-time CTF determination and correction. J Struct Biol 193, 1–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zheng, S.Q., Palovcak, E., Armache, J.P., Verba, K.A., Cheng, Y., and Agard, D.A. (2017). MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat Methods 14, 331–332.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhong, E.D., Bepler, T., Berger, B., and Davis, J.H. (2021). CryoDRGN: reconstruction of heterogeneous cryo-EM structures using neural networks. Nat Methods 18, 176–185.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (31971154, 31730023, 31521002), the Chinese Ministry of Science and Technology (2017YFA0504700, 2021YFA1300100), the Chinese Academy of Sciences (CAS) (XDB37010100), and the National Laboratory of Biomacromolecules of China (2019KF07). We thank Jianhui Li, Zhenwei Yang, Yuanyuan Chen, and Xiaoxia Yu at the Core Facility for Protein Research (Institute of Biophysics, Chinese Academy of Sciences) for assistance with CD and ITC experiments; Gang Ji, Xiaojun Huang, Boling Zhu, Xujing Li at the Center for Biological Imaging (CBI), Core Facility for Protein Sciences, Chinese Academy of Sciences for assistance with EM data collection; and Jifeng Wang, Zhensheng Xie, Xiang Ding, Mengmeng Zhang (Proteomics, Institute of Biophysics, Chinese Academy of Sciences) for assistance with the Q-Exactive mass spectrometer analysis.

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Authors and Affiliations

  1. National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China

    Xiaoxing Huang, Youwang Wang, Cong Yu, Hui Zhang, Xinxin Li, Kai Song, Min Zhou & Ping Zhu

  2. The Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), Beijing, 100193, China

    Qiang Ru

  3. University of Chinese Academy of Sciences, Beijing, 100049, China

    Youwang Wang, Cong Yu, Hui Zhang, Xinxin Li & Ping Zhu

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  1. Xiaoxing Huang
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Huang, X., Wang, Y., Yu, C. et al. Cryo-EM structures reveal the dynamic transformation of human alpha-2-macroglobulin working as a protease inhibitor. Sci. China Life Sci. 65, 2491–2504 (2022). https://doi.org/10.1007/s11427-022-2139-2

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