title: A nanobody binding to non-amyloidogenic regions of the protein human lysozyme enhances partial unfolding but inhibits amyloid fibril formation.
abstract: We report the effects of the interaction of two camelid antibody fragments, generally called nanobodies, namely cAb-HuL5 and a stabilized and more aggregation-resistant variant cAb-HuL5G obtained by protein engineering, on the properties of two amyloidogenic variants of human lysozyme, I56T and D67H, whose deposition in vital organs including the liver, kidney, and spleen is associated with a familial non-neuropathic systemic amyloidosis. Both NMR spectroscopy and X-ray crystallographic studies reveal that cAb-HuL5 binds to the α-domain, one of the two lobes of the native lysozyme structure. The binding of cAb-HuL5/cAb-HuL5G strongly inhibits fibril formation by the amyloidogenic variants; it does not, however, suppress the locally transient cooperative unfolding transitions, characteristic of these variants, in which the β-domain and the C-helix unfold and which represents key early intermediate species in the formation of amyloid fibrils. Therefore, unlike two other nanobodies previously described, cAb-HuL5/cAb-HuL5G does not inhibit fibril formation via the restoration of the global cooperativity of the native structure of the lysozyme variants to that characteristic of the wild-type protein. Instead, it inhibits a subsequent step in the assembly of the fibrils, involving the unfolding and structural reorganization of the α-domain. These results show that nanobodies can protect against the formation of pathogenic aggregates at different stages in the structural transition of a protein from the soluble native state into amyloid fibrils, illustrating their value as structural probes to study the molecular mechanisms of amyloid fibril formation. Combined with their amenability to protein engineering techniques to improve their stability and solubility, these findings support the suggestion that nanobodies can potentially be developed as therapeutics to combat protein misfolding diseases.
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.
authors.forename: Erwin
authors.initials: E
authors.lastname: De Genst
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.|State Key Laboratory of Chirosciences, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, P.R. China.
authors.forename: Pak-Ho
authors.initials: PH
authors.lastname: Chan
authors.affiliation: Department of Structural Biology, Vlaams Interuniversitair Instituut voor Biotechnologie VIB, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium.|Laboratory of Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium.
authors.forename: Els
authors.initials: E
authors.lastname: Pardon
authors.affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua University, 101, Section 2, Kuang-Fu Road, Hsinchu, 30013, Taiwan.|Institute of Biological Chemistry, Academia Sinica, No 128, Section 2, Academia Road, Taipei 11529, Taiwan.|Institute of Biochemical Sciences, National Taiwan University, No 1, Section 4, Roosevelt Road, Taipei 10617, Taiwan.
authors.forename: Shang-Te D
authors.initials: SD
authors.lastname: Hsu
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.
authors.forename: Janet R
authors.initials: JR
authors.lastname: Kumita
authors.affiliation: Institute of Structural and Molecular Biology, University College London and Birkbeck College, Gower Street, London WC1E 6BT, U.K.
authors.forename: John
authors.initials: J
authors.lastname: Christodoulou
authors.affiliation: Laboratory of Enzymology and Protein Folding, Centre for Protein Engineering, Institute of Chemistry, University of Liege, B-4000 Liege (Sart Tilman), Belgium.
authors.forename: Linda
authors.initials: L
authors.lastname: Menzer
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, U.K.
authors.forename: Dimitri Y
authors.initials: DY
authors.lastname: Chirgadze
authors.affiliation: Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3QZ, U.K.
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
authors.affiliation: Department of Structural Biology, Vlaams Interuniversitair Instituut voor Biotechnologie VIB, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium.|Research Unit of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium.
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.affiliation: Laboratory of Enzymology and Protein Folding, Centre for Protein Engineering, Institute of Chemistry, University of Liege, B-4000 Liege (Sart Tilman), Belgium.
authors.forename: André
authors.initials: A
authors.lastname: Matagne
authors.affiliation: Department of Structural Biology, Vlaams Interuniversitair Instituut voor Biotechnologie VIB, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium.|Laboratory of Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium.
authors.forename: Lode
authors.initials: L
authors.lastname: Wyns
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Dobson
authors.affiliation: Laboratory of Enzymology and Protein Folding, Centre for Protein Engineering, Institute of Chemistry, University of Liege, B-4000 Liege (Sart Tilman), Belgium.
authors.forename: Mireille
authors.initials: M
authors.lastname: Dumoulin
mesh_terms: D000682:Amyloid / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018360:Crystallography, X-Ray; D006801:Humans; D009113:Muramidase / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000276:immunology*; D012995:Solubility
chemical_list: D000682:Amyloid; D011994:Recombinant Proteins; D061905:Single-Domain Antibodies; D009113:Muramidase
references.citation: Acta Crystallogr D Biol Crystallogr. 2011 Apr;67(Pt 4):235-42
references.pmid: 21460441
references.citation: Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):2008-13
references.pmid: 8700876
references.citation: J Appl Crystallogr. 2007 Aug 1;40(Pt 4):658-674
references.pmid: 19461840
references.citation: J Mol Biol. 2005 Aug 19;351(3):662-71
references.pmid: 16023673
references.citation: J Mol Biol. 1971 Feb 14;55(3):379-400
references.pmid: 5551392
references.citation: J Mol Biol. 2005 Sep 23;352(3):597-607
references.pmid: 16095608
references.citation: Nature. 1997 Feb 27;385(6619):787-93
references.pmid: 9039909
references.citation: Methods Mol Biol. 2012;911:533-58
references.pmid: 22886275
references.citation: J Biochem. 1996 Dec;120(6):1216-23
references.pmid: 9010773
references.citation: Protein Expr Purif. 1999 Jun;16(1):171-80
references.pmid: 10336875
references.citation: Trends Biochem Sci. 2000 Dec;25(12):611-8
references.pmid: 11116188
references.citation: Proteins. 1998 Sep 1;32(4):515-22
references.pmid: 9726420
references.citation: J Biotechnol. 2001 Jun;74(4):277-302
references.pmid: 11526908
references.citation: Biochim Biophys Acta. 2012 Feb;1822(2):261-85
references.pmid: 22024360
references.citation: Eur J Biochem. 2001 Jan;268(1):155-9
references.pmid: 11121116
references.citation: Nat Struct Biol. 2002 Apr;9(4):308-15
references.pmid: 11887182
references.citation: Curr Opin Struct Biol. 2004 Feb;14(1):70-5
references.pmid: 15102452
references.citation: J Mol Biol. 2010 Sep 17;402(2):326-43
references.pmid: 20620148
references.citation: Acta Crystallogr D Biol Crystallogr. 1997 May 1;53(Pt 3):240-55
references.pmid: 15299926
references.citation: Biochemistry. 2008 Oct 21;47(42):11041-54
references.pmid: 18816062
references.citation: Proc Natl Acad Sci U S A. 1997 Jun 10;94(12):6170-5
references.pmid: 9177189
references.citation: J Am Chem Soc. 2013 Apr 17;135(15):5656-68
references.pmid: 23350654
references.citation: Angew Chem Int Ed Engl. 1998 Apr 20;37(7):868-893
references.pmid: 29711488
references.citation: Biochemistry. 1999 May 18;38(20):6419-27
references.pmid: 10350460
references.citation: Nat Chem Biol. 2009 Jan;5(1):15-22
references.pmid: 19088715
references.citation: Kidney Int. 2003 May;63(5):1652-7
references.pmid: 12675840
references.citation: Dev Comp Immunol. 2006;30(1-2):187-98
references.pmid: 16051357
references.citation: J Am Chem Soc. 2010 Nov 10;132(44):15580-8
references.pmid: 20958028
references.citation: J Biol Chem. 2003 Jul 11;278(28):25910-8
references.pmid: 12709420
references.citation: Trends Biochem Sci. 2000 Jul;25(7):331-9
references.pmid: 10871884
references.citation: J Struct Biol. 2000 Jun;130(2-3):339-51
references.pmid: 10940237
references.citation: Nature. 2003 Aug 14;424(6950):783-8
references.pmid: 12917687
references.citation: J Mol Biol. 1994 May 20;238(5):777-93
references.pmid: 8182748
references.citation: Proc Natl Acad Sci U S A. 1998 Sep 15;95(19):11037-8
references.pmid: 9736683
references.citation: J Am Chem Soc. 2011 May 25;133(20):7737-7743
references.pmid: 21528861
references.citation: Protein Eng Des Sel. 2010 Jul;23(7):499-506
references.pmid: 20382744
references.citation: Protein Eng. 1996 Jun;9(6):531-7
references.pmid: 8862554
references.citation: J Biomol NMR. 1995 Nov;6(3):277-93
references.pmid: 8520220
references.citation: Amyloid. 2012 Dec;19(4):182-5
references.pmid: 22978355
references.citation: Acc Chem Res. 2006 Sep;39(9):603-10
references.pmid: 16981676
references.citation: Nature. 2003 Dec 18;426(6968):884-90
references.pmid: 14685248
references.citation: Curr Opin Struct Biol. 2010 Feb;20(1):54-62
references.pmid: 20133122
references.citation: J Mol Biol. 2005 Sep 30;352(4):823-36
references.pmid: 16126226
references.citation: Science. 1995 Mar 17;267(5204):1619-20
references.pmid: 7886447
references.citation: Nat Struct Mol Biol. 2011 Dec 18;19(1):79-83
references.pmid: 22179788
references.citation: Cell. 2012 May 25;149(5):1048-59
references.pmid: 22632969
references.citation: Acta Crystallogr D Biol Crystallogr. 2003 Nov;59(Pt 11):2031-8
references.pmid: 14573959
references.citation: Nucleic Acids Res. 1998 Feb 15;26(4):1126-7
references.pmid: 9461478
references.citation: Proc Natl Acad Sci U S A. 2013 Jan 29;110(5):1680-5
references.pmid: 23319605
references.citation: Curr Opin Struct Biol. 2000 Feb;10(1):60-8
references.pmid: 10679462
references.citation: J Mol Biol. 1999 Apr 2;287(3):657-74
references.pmid: 10092466
references.citation: Proc Natl Acad Sci U S A. 2011 Feb 22;108(8):3246-51
references.pmid: 21300904
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
references.pmid: 15572765
references.citation: J Mol Biol. 2005 Feb 25;346(3):773-88
references.pmid: 15713462
references.citation: Proc Natl Acad Sci U S A. 2012 Jun 12;109(24):9629-34
references.pmid: 22645360
references.citation: Protein Sci. 2002 Mar;11(3):500-15
references.pmid: 11847273
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):213-21
references.pmid: 20124702
references.citation: Protein Sci. 2001 Dec;10(12):2525-30
references.pmid: 11714920
references.citation: Hum Mutat. 2000 Aug;16(2):180
references.pmid: 10923049
references.citation: Kidney Int. 2002 Mar;61(3):907-12
references.pmid: 11849445
references.citation: J Mol Biol. 2013 Jul 24;425(14):2397-411
references.pmid: 23557833
references.citation: J Mol Biol. 2007 May 25;369(1):157-67
references.pmid: 17407782
references.citation: Nature. 1993 Apr 8;362(6420):553-7
references.pmid: 8464497
references.citation: J Mol Biol. 2006 Aug 18;361(3):551-61
references.pmid: 16859705
references.citation: Semin Cell Dev Biol. 2004 Feb;15(1):3-16
references.pmid: 15036202
references.citation: Hum Mutat. 2006 Jan;27(1):119-20
references.pmid: 16329101
references.citation: Antimicrob Agents Chemother. 2001 Oct;45(10):2807-12
references.pmid: 11557473
references.citation: Trends Biochem Sci. 2001 Apr;26(4):230-5
references.pmid: 11295555
references.citation: Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):13-20
references.pmid: 8552589
references.citation: Annu Rev Biochem. 2006;75:333-66
references.pmid: 16756495
references.citation: Curr Opin Cell Biol. 2011 Apr;23(2):231-8
references.pmid: 21146391
references.citation: Proc Natl Acad Sci U S A. 2012 Oct 30;109(44):17770-1
references.pmid: 23112193
references.citation: Nat Chem Biol. 2011 Jul 31;7(9):602-9
references.pmid: 21804535
references.citation: Sci Transl Med. 2011 Aug 24;3(97):97ra81
references.pmid: 21865539
references.citation: Biochemistry (Mosc). 2004 Nov;69(11):1233-8
references.pmid: 15627377
references.citation: Protein Sci. 1995 Nov;4(11):2411-23
references.pmid: 8563639
references.citation: FEBS Lett. 1997 Sep 15;414(3):521-6
references.pmid: 9323027