1 3 ‘‘Structural characterization of the minimal segment of TDP-43 4 competent for aggregation’’ 5 6 7 Miguel Mompeán a Q1 , Emanuele Buratti b , Corrado Guarnaccia b , Rui M.M. Brito c , Avijit Chakrabartty d , 8 Francisco E. Baralle b,⇑ , Douglas V. Laurents a,⇑ 9 a Instituto de Química Física ‘‘Rocasolano’’ CSIC, Serrano 119, E-28006 Madrid, Spain 10 b International Centre for Genetic Engineering and Biotechnology, I-34149 Trieste, Italy 11 c Chemistry Dept., Faculty of Science and Technology & Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal 12 d Dept. of Biochemistry, University of Toronto, Toronto Medical Discovery Tower 4-305, MaRS Centre, 101 College St., Toronto, ON M5G 1L7, Canada 13 14 15 17 article info 18 Article history: 19 Received 1 October 2013 20 and in revised form 13 December 2013 21 Available online xxxx 22 Q2 Keywords: 23 Frontotemportal lobar degeneration (FTLD) 24 Amyotrophic lateral sclerosis (ALS) 25 NMR 26 Molecular dynamics 27 Circular dichroism 28 Protein misfolding & aggregation 29 30 abstract 31 TDP-43 is a nuclear protein whose abnormal aggregates are implicated in ALS and FTLD. Recently, an Asn/ 32 Gln rich C-terminal segment of TDP-43 has been shown to produce aggregation in vitro and reproduce 33 most of the protein’s pathological hallmarks in cells, but little is known about this segment’s structure. 34 Here, CD and 2D heteronuclear NMR spectroscopies provide evidence that peptides corresponding to 35 the wild type and mutated sequences of this segment adopt chiefly disordered conformations that, in 36 the case of the wild type sequence, spontaneously forms a b-sheet rich oligomer. Moreover, MD simula- 37 tion provides evidence for a structure consisting of two b-strands and a well-defined, yet non-canonical 38 structural element. Furthermore, MD simulations of four pathological mutations (Q343R, N345K, G348V 39 and N352S) occurring in this segment predict that all of them could affect this region’s structure. In par- 40 ticular, the Q343R variant tends to stabilize disordered conformers, N345K permits the formation of 41 longer, more stable b-strands, and G348V tends to shorten and destabilize them. Finally, N352S acts to 42 alter the b-stand register and when S352 is phosphorylated, it induces partial unfolding. Our results pro- 43 vide a better understanding of TDP-43 aggregation process and will be useful to design effectors capable 44 to modulate its progression. 45 Ó 2014 Published by Elsevier Inc. 46 47 48 Introduction 49 TDP-43 is a 414 residue protein involved in mRNA processing 50 and transport to the cytoplasm [1,2]. It has two RRM RNA binding 51 domains and the N-terminus carries motifs for nuclear localization 52 as well as nuclear export. These motifs allow TDP-43 to return to 53 the nucleus after transport of mRNAs. The C-terminal half of 54 TDP-43 contains an Asn/Gln rich region as well as a Gly rich region 55 and has been suggested to contain a prion-like domain [3]. 56 In 2006, TDP-43 was discovered to be the main protein compo- 57 nent of abnormal protein aggregates in the cytoplasm of nerve cells 58 in FTLD and ALS 1 [4,5]. In these aggregates, TDP-43 molecules are 59 ubiquinated, hyperphosphorylated and often truncated to yield 60 23–27 kDa C-terminal fragments, with the nuclei of cells harboring 61 abnormal TDP-43 cytoplasmic aggregates becoming deficient in 62 TDP-43 [6]. 63 Many genetic, cell biological and biochemical studies have pro- 64 vided evidence implicating TDP-43 in neurological disorders [2]. In 65 particular, TDP-43 mutations associated with early disease onset or 66 poor prognosis are linked to increased aggregation of the protein 67 [7]. However, how TDP-43 harms cells is still an open question. 68 The lack of TDP-43 in the nucleus could be detrimental to the prop- 69 er splicing and transport of mRNAs. Alternatively, the hyperphos- 70 phorylated, ubiquitinated aggregates could be cytotoxic. So far, 71 results have been reported Supplementary both these mechanisms, 72 potentially making them both pathologically relevant [2]. 73 Among the various functional domains present in the TDP-43 74 protein, one of the best characterized regions is represented by 75 the Asn/Gln-rich segment spanning residues 321–366 in the C-ter- 76 minus of this protein. In particular, this region has been previously 77 described to be involved in the interaction with hnRNP proteins 78 [8], polyglutamine repeats [9], and nuclear ‘‘Gems’’ [10]. In addi- 79 tion to all these potential connections, recent work from one of 80 our labs (FB) has shown that tandem repetitions of this Asn/Gln 81 rich region of TDP-43 are capable of aggregating in cells and of 82 recruiting normal TDP-43 into these aggregates [11]. Furthermore, 0003-9861/$ - see front matter Ó 2014 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.abb.2014.01.007 ⇑ Corresponding authors. Fax: +34 91 564 2431 (D.V. Laurents). E-mail addresses: baralle@icgeb.org (F.E. Baralle), dlaurents@iqfr.csic.es (D.V. Laurents). 1 Abbreviations used: ALS, amyotrophic lateral sclerosis; CD, circular dichroism; FTLD, frontotemportal lobar degeneration; MD, molecular dynamics; PCA, principal component analysis; TDP-43, 43 kDa Tar DNA binding protein; ThT, thioflavin T; WT, wild type. Archives of Biochemistry and Biophysics xxx (2014) xxx–xxx Contents lists available at ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi YABBI 6604 No. of Pages 10, Model 5G 18 January 2014 Please cite this article in press as: M. Mompeán et al., Arch. Biochem. Biophys. (2014), http://dx.doi.org/10.1016/j.abb.2014.01.007