title: The importance of a gatekeeper residue on the aggregation of transthyretin: implications for transthyretin-related amyloidoses.
abstract: Protein aggregation into β-sheet-enriched amyloid fibrils is associated with an increasing number of human disorders. The adoption of such amyloid conformations seems to constitute a generic property of polypeptide chains. Therefore, during evolution, proteins have adopted negative design strategies to diminish their intrinsic propensity to aggregate, including enrichment of gatekeeper charged residues at the flanks of hydrophobic aggregation-prone segments. Wild type transthyretin (TTR) is responsible for senile systemic amyloidosis, and more than 100 mutations in the TTR gene are involved in familial amyloid polyneuropathy. The TTR 26-57 segment bears many of these aggressive amyloidogenic mutations as well as the binding site for heparin. We demonstrate here that Lys-35 acts as a gatekeeper residue in TTR, strongly decreasing its amyloidogenic potential. This protective effect is sequence-specific because Lys-48 does not affect TTR aggregation. Lys-35 is part of the TTR basic heparin-binding motif. This glycosaminoglycan blocks the protective effect of Lys-35, probably by neutralization of its side chain positive charge. A K35L mutation emulates this effect and results in the rapid self-assembly of the TTR 26-57 region into amyloid fibrils. This mutation does not affect the tetrameric protein stability, but it strongly increases its aggregation propensity. Overall, we illustrate how TTR is yet another amyloidogenic protein exploiting negative design to prevent its massive aggregation, and we show how blockage of conserved protective features by endogenous factors or mutations might result in increased disease susceptibility.
authors.affiliation: From the Instituto de Bioquímica Médica Leopoldo de Meis, Programa de Biologia Estrutural.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Sant'Anna
authors.affiliation: From the Instituto de Bioquímica Médica Leopoldo de Meis, Programa de Biologia Estrutural.
authors.forename: Carolina
authors.initials: C
authors.lastname: Braga
authors.affiliation: From the Instituto de Bioquímica Médica Leopoldo de Meis, Programa de Biologia Estrutural.
authors.forename: Nathalia
authors.initials: N
authors.lastname: Varejão
authors.affiliation: From the Instituto de Bioquímica Médica Leopoldo de Meis, Programa de Biologia Estrutural.
authors.forename: Karinne M
authors.initials: KM
authors.lastname: Pimenta
authors.affiliation: the Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Graña-Montes
authors.affiliation: From the Instituto de Bioquímica Médica Leopoldo de Meis, Programa de Biologia Estrutural.
authors.forename: Aline
authors.initials: A
authors.lastname: Alves
authors.affiliation: the Instituto de Microbiologia Professor Paulo de Goés, Universidade Federal do Rio de Janeiro, Rio de Janeiro CEP 21941-590, Brazil and.
authors.forename: Juliana
authors.initials: J
authors.lastname: Cortines
authors.affiliation: the Faculdade de Farmácia, and.
authors.forename: Yraima
authors.initials: Y
authors.lastname: Cordeiro
authors.affiliation: the Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain salvador.ventura@uab.es.
authors.forename: Salvador
authors.initials: S
authors.lastname: Ventura
authors.affiliation: From the Instituto de Bioquímica Médica Leopoldo de Meis, Programa de Biologia Estrutural, foguel@bioqmed.ufrj.br.
authors.forename: Debora
authors.initials: D
authors.lastname: Foguel
mesh_terms: D000682:Amyloid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D028227:Amyloid Neuropathies, Familial / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D015870:Gene Expression; D006493:Heparin / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007930:Leucine / Q000737:chemistry* / Q000378:metabolism; D008239:Lysine / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D011228:Prealbumin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D066263:Protein Aggregation, Pathological; D055503:Protein Multimerization; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity
chemical_list: D000682:Amyloid; D011228:Prealbumin; D011994:Recombinant Proteins; D006493:Heparin; D007930:Leucine; D008239:Lysine
keywords: Aggregation Propensity; Amyloid; Gatekeeper Residue; Heparin; Peptides; Protein Aggregation; Protein Misfolding; Rational Mutation; Transthyretin
references.citation: J Mol Biol. 2005 Aug 26;351(4):850-64
references.pmid: 16024039
references.citation: Biophys J. 2007 May 15;92(10):3448-58
references.pmid: 17325005
references.citation: Biophys J. 1995 Aug;69(2):640-51
references.pmid: 8527678
references.citation: FEBS Lett. 2002 Oct 9;529(2-3):193-7
references.pmid: 12372599
references.citation: Anal Biochem. 1978 Nov;91(1):13-31
references.pmid: 9762080
references.citation: Annu Rev Biochem. 1997;66:385-407
references.pmid: 9242912
references.citation: Proc Natl Acad Sci U S A. 2003 Jan 7;100(1):330-5
references.pmid: 12506200
references.citation: Brain. 1995 Aug;118 ( Pt 4):849-56
references.pmid: 7655883
references.citation: Biochemistry. 1996 May 21;35(20):6470-82
references.pmid: 8639594
references.citation: J Mol Biol. 2003 May 9;328(4):963-74
references.pmid: 12729768
references.citation: Structure. 2005 Aug;13(8):1143-51
references.pmid: 16084386
references.citation: Biochem Biophys Res Commun. 1992 Aug 31;187(1):397-403
references.pmid: 1520326
references.citation: Int J Mol Sci. 2013 Mar 06;14(3):5284-311
references.pmid: 23466880
references.citation: Mol Cell. 2011 Jul 8;43(1):8-18
references.pmid: 21726806
references.citation: J Mol Biol. 2012 Aug 10;421(2-3):185-203
references.pmid: 22244854
references.citation: Structure. 2010 Oct 13;18(10):1244-60
references.pmid: 20947013
references.citation: Proc Natl Acad Sci U S A. 1990 Apr;87(7):2843-5
references.pmid: 2320592
references.citation: EMBO Rep. 2007 Aug;8(8):737-42
references.pmid: 17668004
references.citation: Electrophoresis. 1997 Dec;18(15):2714-23
references.pmid: 9504803
references.citation: Protein Eng Des Sel. 2012 Jul;25(7):357-66
references.pmid: 22706763
references.citation: Biochemistry. 2001 Oct 2;40(39):11757-67
references.pmid: 11570876
references.citation: Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):16748-53
references.pmid: 12481032
references.citation: Proc Natl Acad Sci U S A. 2004 May 11;101(19):7258-63
references.pmid: 15123800
references.citation: Biochemistry. 1992 Dec 15;31(49):12345-52
references.pmid: 1463722
references.citation: J Biol Chem. 2004 Feb 13;279(7):5699-707
references.pmid: 14604984
references.citation: Curr Top Med Chem. 2006;6(6):597-608
references.pmid: 16712494
references.citation: Hum Mutat. 2009 Mar;30(3):431-7
references.pmid: 19156839
references.citation: Biochem Soc Trans. 2012 Oct;40(5):1032-7
references.pmid: 22988860
references.citation: Proc Natl Acad Sci U S A. 2002 Mar 5;99(5):2760-5
references.pmid: 11880628
references.citation: J Mol Biol. 2001 Oct 5;312(5):1103-19
references.pmid: 11580253
references.citation: Biochem Biophys Res Commun. 1991 Mar 29;175(3):1159-64
references.pmid: 2025248
references.citation: Proc Natl Acad Sci U S A. 2002 Dec 10;99 Suppl 4:16427-32
references.pmid: 12351683
references.citation: J Mol Biol. 2008 Oct 24;382(5):1157-67
references.pmid: 18662699
references.citation: J Am Chem Soc. 2011 Jan 19;133(2):334-44
references.pmid: 21142149
references.citation: Biophys J. 2006 Aug 1;91(3):957-67
references.pmid: 16698785
references.citation: Nat Rev Neurosci. 2003 Sep;4(9):727-38
references.pmid: 12951565
references.citation: BMC Bioinformatics. 2007 Feb 27;8:65
references.pmid: 17324296
references.citation: J Biol Chem. 2008 Oct 31;283(44):29639-43
references.pmid: 18723507
references.citation: Curr Opin Struct Biol. 2006 Feb;16(1):118-26
references.pmid: 16434184
references.citation: J Cell Biol. 2006 Jul 31;174(3):323-7
references.pmid: 16880267
references.citation: Nature. 2005 Dec 8;438(7069):878-81
references.pmid: 16341018
references.citation: Proc Natl Acad Sci U S A. 2006 Aug 29;103(35):13057-61
references.pmid: 16916929
references.citation: Biochemistry. 2004 Jun 15;43(23):7365-81
references.pmid: 15182180
references.citation: Curr Med Chem. 2012;19(15):2304-11
references.pmid: 22471982
references.citation: J Mol Biol. 2002 May 10;318(4):935-40
references.pmid: 12054791
references.citation: Biochemistry. 2005 Nov 29;44(47):15525-33
references.pmid: 16300401
references.citation: Lancet Neurol. 2003 Aug;2(8):482-92
references.pmid: 12878436
references.citation: J Biol Chem. 2001 Jul 20;276(29):27207-13
references.pmid: 11306576
references.citation: Proc Natl Acad Sci U S A. 2012 Jun 12;109(24):9629-34
references.pmid: 22645360
references.citation: J Mol Biol. 2006 Feb 3;355(5):1037-47
references.pmid: 16359707
references.citation: Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):2817-22
references.pmid: 14981241
references.citation: Proc Natl Acad Sci U S A. 2002 Mar 5;99(5):2754-9
references.pmid: 11880627
references.citation: Ophthalmic Genet. 2012 Mar;33(1):28-33
references.pmid: 21843040
references.citation: Biophys J. 2005 Dec;89(6):4201-10
references.pmid: 16169975
references.citation: Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6445-50
references.pmid: 10841549
references.citation: Acta Biochim Biophys Sin (Shanghai). 2007 Aug;39(8):549-59
references.pmid: 17687489
references.citation: Int J Pept Protein Res. 1994 Oct;44(4):388-98
references.pmid: 7875942
references.citation: J Biol Chem. 2012 Jul 13;287(29):24765-73
references.pmid: 22547072
references.citation: Methods Enzymol. 1999;309:256-74
references.pmid: 10507029
references.citation: PLoS One. 2013;8(1):e54175
references.pmid: 23326595
references.citation: J Biol Chem. 1989 Sep 25;264(27):15863-8
references.pmid: 2777769
references.citation: Nat Biotechnol. 2004 Oct;22(10):1302-6
references.pmid: 15361882
references.citation: Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4224-8
references.pmid: 9539718
references.citation: Biochem J. 1996 Mar 1;314 ( Pt 2):701-7
references.pmid: 8670088
references.citation: Front Physiol. 2012 Nov 20;3:438
references.pmid: 23181023
references.citation: Proc Natl Acad Sci U S A. 2011 Apr 5;108(14):5584-9
references.pmid: 21422279
references.citation: Nat Struct Biol. 1994 Jun;1(6):399-409
references.pmid: 7664054
references.citation: Protein Pept Lett. 2005 Apr;12(3):245-9
references.pmid: 15777273
references.citation: Biochemistry. 1999 Oct 12;38(41):13560-73
references.pmid: 10521263