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Single molecule force spectroscopy study of calcium regulated mechanical unfolding of the A6 domain of adseverin

Li Peng-Fei Cao Yi Qin Meng Wang Wei

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Single molecule force spectroscopy study of calcium regulated mechanical unfolding of the A6 domain of adseverin

Li Peng-Fei, Cao Yi, Qin Meng, Wang Wei
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  • Adseverin is a member of calcium-regulated gelsolin superfamily existing in secretory cells,which functions as an actin severing and capping protein.Adseverin is comprised of six independently folded domains (A1-A6),sharing high sequence identity (60%) with that of gelsolin (G1-G6).Calcium binding can convert both adserverin and gelsolin from a globular structure into a necklace structure and expose the actin binding sites.However,compared with gelsolin, adseverin lacks a C-terminal extension.Our previous single molecule force spectroscopy studies indicated that the Cterminal helix is critical to the force regulated calcium activation of gelsolin.It remains largely unexplored how the calcium binding to adseverin is regulated by force. Here,using atomic force microscopy based single molecule force spectroscopy,we demonstrate that the mechanical unfolding of the sixth domain of adseverin (A6) can be significantly affected by calcium binding.In order to identify the unfolding events of A6 unambiguously,we construct a hetero-polyprotein (GB1-A6)4,in which A6 is spliced alternatively with well-characterized protein domain GB1.Therefore,in the force-extension traces,GB1 unfolding events can serve as a fingerprint to identify the unfolding signature of A6. In the absence of calcium,the unfolding traces for (GB1-A6)4 show two distinct categories of events.The higher force events with unfolding forces of ~180 pN and contour length increments of ~ 18 nm correspond to the unfolding of GB1.The other category of events with lower unfolding forces of ~ 25 pN and contour length increments of ~35 nm are attributed to the mechanical unfolding of A6.The unfolding force for A6 is similar to that for the structural homological protein,G6. However,in the presence of calcium ion,the unfolding force of A6 is dramatically increased to ~45 pN,indicating that the structure of A6 can be mechanically stabilized by calcium ion-binding.Moreover,we observe a clear mechanical unfolding intermediate state for the unfolding of calcium bound A6(holo A6).Upon stretching,holo A6 is first partially unfolded to an intermediate state with a contour length increment of ~7.2 nm.Then,the intermediate state is unfolded to release a contour length of ~27.8 nm.The total contour length change is the same as that for the calcium free A6 (apo A6).Because each amino acid in the unfolded structure corresponds to a contour length increment of 0.365 nm,according to the contour length change,we infer that in the unfolding intermediate state of A6,its N-terminal regions is partially unfolded.This leads to the exposure of the cryptic actin binding site on A5,which is otherwise buried in the folded structure of A6.The force regulated activation mechanism for A6 is similar to that for G6,except that they use different sequences from those in the force-sensitive region.In G6 the C-terminal helix serves as the force-responsive tail to regulate actin binding,while in A6 the N-terminal sequences are unstructured upon stretching to promote the actin binding for adseverin. Therefore,we infer that force may be an important regulator for the actin-binding of all members in the gelsolin family proteins,including adseverin and gelsolin.Our study represents an important step towards the understanding of the function of adseverin at a molecular level.
      Corresponding author: Qin Meng, qinmeng@nju.edu.cn
    • Funds: Project supported by the National Natural Science Foundation of China (Grant Nos. 21522402, 11674153, 11374148, 11334004) and the National Basic Research Program of China (Grant No. 2013CB834100).
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    Xue Y, Li X, Li H, Zhang W 2014 Nat. Commun. 5 4348

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    Cheng B, Cui S X 2015 Polymer Mechanochemistry 369 97

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    Yuan G, Le S, Yao M, Qian H, Zhou X, Yan J, Chen H 2017 Angew. Chem. Int. Ed. Engl. 56 5490

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    Gao X, Qin M, Yin P, Liang J, Wang J, Cao Y, Wang W 2012 Biophys. J. 102 2149

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    Feng W, Wang Z, Zhang W 2017 Langmuir 33 1826

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    Luo Z, Cheng B, Cui S 2015 Langmuir 31 6107

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    Yang Z J, Yuan G H, Zhai W L, Yan J, Chen H 2016 Science China-Physics Mechanics Astronomy 59 680013

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    Schoeler C, Malinowska K H, Bernardi R C, Milles L F, Jobst M A, Durner E, Ott W, Fried D B, Bayer E A, Schulten K, Gaub H E, Nash M A 2014 Nat. Commun. 5 5635

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    Pfreundschuh M, Alsteens D, Wieneke R, Zhang C, Coughlin S R, Tampe R, Kobilka B K, Muller D J 2015 Nat. Commun. 6 8857

    [22]

    Dudko O K, Hummer G, Szabo A 2006 Phys. Rev. Lett. 96 108101

    [23]

    Dudko O K, Hummer G, Szabo A 2008 Proc. Natl. Acad. Sci. USA 105 15755

    [24]

    Bell G I 1978 Science 200 618

    [25]

    Rodriguez Del Castillo A, Lemaire S, Tchakarov L, Jeyapragasan M, Doucet J P, Vitale M L, Trifaro J M 1990 EMBO J. 9 43

    [26]

    Maekawa S, Sakai H 1990 J. Biol. Chem. 265 10940

    [27]

    Marcu M G, Zhang L, Elzagallaai A, Trifaro J M 1998 J. Biol. Chem. 273 3661

    [28]

    Cao Y, Lam C, Wang M, Li H 2006 Angew Chem. Int. Ed. Engl. 45 642

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    Rief M, Gautel M, Oesterhelt F, Fernandez J M, Gaub H E 1997 Science 276 1109

  • [1]

    Lee J, Pena M M, Nose Y, Thiele D J 2002 J. Biol. Chem. 277 4380

    [2]

    Nag S, Larsson M, Robinson R C, Burtnick L D 2013 Cytoskeleton 70 360

    [3]

    Silacci P, Mazzolai L, Gauci C, Stergiopulos N, Yin H L, Hayoz D 2004 Cell Mol. Life Sci. 61 2614

    [4]

    Chumnarnsilpa S, Lee W L, Nag S, Kannan B, Larsson M, Burtnick L D, Robinson R C 2009 Proc. Natl. Acad. Sci. USA 106 13719

    [5]

    L C, Gao X, Li W, Xue B, Qin M, Burtnick L D, Zhou H, Cao Y, Robinson R C, Wang W 2014 Nat. Commun. 5 4623

    [6]

    Qian H, Chen H, Yan J 2016 Acta Phys. Sin. 65 188706 (in Chinese)[钱辉, 陈虎, 严洁 2016 物理学报 65 188706]

    [7]

    Zhang W K, Wang C, Zhang X 2003 Chin. Sci. Bull. 48 7 (in Chinese)[张文科, 王驰, 张希 2003 科学通报 48 7]

    [8]

    Cui S X 2016 Acta Polymerica Sinica 2016(9) 1160

    [9]

    Feng W, Wang Z, Zhang W 2017 Langmuir 33 1826

    [10]

    Zhang X, Zhang W K, Li H B, Shen J C 2000 Prog. Nat. Sci:Nat. Key Lab. Newsletter 10 385 (in Chinese)[张希, 张文科, 李宏斌, 沈家骢 2000 自然科学进展:国家重点实验室通讯 10 385]

    [11]

    Pang X C, Cheng B, Cui S X 2016 Chinese Journal of Polymer Science 34 578

    [12]

    Yu X T, Yang Z B, Wang X Y, Tang M J, Wang Z Z, Wang H B 2016 Prog. Biochem. Biophys. 43 28 (in Chinese)[于小婷, 杨忠波, 王鑫艳, 汤明杰, 王占忠, 王化斌 2016 生物化学与生物物理进展 43 28]

    [13]

    Xue Y, Li X, Li H, Zhang W 2014 Nat. Commun. 5 4348

    [14]

    Cheng B, Cui S X 2015 Polymer Mechanochemistry 369 97

    [15]

    Yuan G, Le S, Yao M, Qian H, Zhou X, Yan J, Chen H 2017 Angew. Chem. Int. Ed. Engl. 56 5490

    [16]

    Gao X, Qin M, Yin P, Liang J, Wang J, Cao Y, Wang W 2012 Biophys. J. 102 2149

    [17]

    Feng W, Wang Z, Zhang W 2017 Langmuir 33 1826

    [18]

    Luo Z, Cheng B, Cui S 2015 Langmuir 31 6107

    [19]

    Yang Z J, Yuan G H, Zhai W L, Yan J, Chen H 2016 Science China-Physics Mechanics Astronomy 59 680013

    [20]

    Schoeler C, Malinowska K H, Bernardi R C, Milles L F, Jobst M A, Durner E, Ott W, Fried D B, Bayer E A, Schulten K, Gaub H E, Nash M A 2014 Nat. Commun. 5 5635

    [21]

    Pfreundschuh M, Alsteens D, Wieneke R, Zhang C, Coughlin S R, Tampe R, Kobilka B K, Muller D J 2015 Nat. Commun. 6 8857

    [22]

    Dudko O K, Hummer G, Szabo A 2006 Phys. Rev. Lett. 96 108101

    [23]

    Dudko O K, Hummer G, Szabo A 2008 Proc. Natl. Acad. Sci. USA 105 15755

    [24]

    Bell G I 1978 Science 200 618

    [25]

    Rodriguez Del Castillo A, Lemaire S, Tchakarov L, Jeyapragasan M, Doucet J P, Vitale M L, Trifaro J M 1990 EMBO J. 9 43

    [26]

    Maekawa S, Sakai H 1990 J. Biol. Chem. 265 10940

    [27]

    Marcu M G, Zhang L, Elzagallaai A, Trifaro J M 1998 J. Biol. Chem. 273 3661

    [28]

    Cao Y, Lam C, Wang M, Li H 2006 Angew Chem. Int. Ed. Engl. 45 642

    [29]

    Rief M, Gautel M, Oesterhelt F, Fernandez J M, Gaub H E 1997 Science 276 1109

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Publishing process
  • Received Date:  08 May 2017
  • Accepted Date:  30 June 2017
  • Published Online:  05 October 2017

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