“Structural characterization of the minimal segment of TDP-43 competent for aggregation”

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Archives of Biochemistry and Biophysics xxx (2014) xxx–xxx

YABBI 6604 No. of Pages 10, Model 5G

18 January 2014

Contents lists available at ScienceDirect

Archives of Biochemistry and Biophysics

journal homepage: www.elsevier .com/ locate/yabbi

‘‘Structural characterization of the minimal segment of TDP-43competent for aggregation’’

0003-9861/$ - see front matter � 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: [email protected] (F.E. Baralle), [email protected]

(D.V. Laurents).1 Abbreviations used: ALS, amyotrophic lateral sclerosis; CD, circular dichroism;

FTLD, frontotemportal lobar degeneration; MD, molecular dynamics; PCA, principalcomponent analysis; TDP-43, 43 kDa Tar DNA binding protein; ThT, thioflavin T; WT,wild type.

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

Miguel Mompeán a, Emanuele Buratti b, Corrado Guarnaccia b, Rui M.M. Brito c, Avijit Chakrabartty d,Francisco E. Baralle b,⇑, Douglas V. Laurents a,⇑a Instituto de Química Física ‘‘Rocasolano’’ CSIC, Serrano 119, E-28006 Madrid, Spainb International Centre for Genetic Engineering and Biotechnology, I-34149 Trieste, Italyc Chemistry Dept., Faculty of Science and Technology & Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugald Dept. of Biochemistry, University of Toronto, Toronto Medical Discovery Tower 4-305, MaRS Centre, 101 College St., Toronto, ON M5G 1L7, Canada

a r t i c l e i n f o a b s t r a c t

313233343536373839404142

Article history:Received 1 October 2013and in revised form 13 December 2013Available online xxxx

Keywords:Frontotemportal lobar degeneration (FTLD)Amyotrophic lateral sclerosis (ALS)NMRMolecular dynamicsCircular dichroismProtein misfolding & aggregation

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TDP-43 is a nuclear protein whose abnormal aggregates are implicated in ALS and FTLD. Recently, an Asn/Gln rich C-terminal segment of TDP-43 has been shown to produce aggregation in vitro and reproducemost of the protein’s pathological hallmarks in cells, but little is known about this segment’s structure.Here, CD and 2D heteronuclear NMR spectroscopies provide evidence that peptides corresponding tothe wild type and mutated sequences of this segment adopt chiefly disordered conformations that, inthe case of the wild type sequence, spontaneously forms a b-sheet rich oligomer. Moreover, MD simula-tion provides evidence for a structure consisting of two b-strands and a well-defined, yet non-canonicalstructural element. Furthermore, MD simulations of four pathological mutations (Q343R, N345K, G348Vand N352S) occurring in this segment predict that all of them could affect this region’s structure. In par-ticular, the Q343R variant tends to stabilize disordered conformers, N345K permits the formation oflonger, more stable b-strands, and G348V tends to shorten and destabilize them. Finally, N352S acts toalter the b-stand register and when S352 is phosphorylated, it induces partial unfolding. Our results pro-vide a better understanding of TDP-43 aggregation process and will be useful to design effectors capableto modulate its progression.

� 2014 Published by Elsevier Inc.

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Introduction

TDP-43 is a 414 residue protein involved in mRNA processingand transport to the cytoplasm [1,2]. It has two RRM RNA bindingdomains and the N-terminus carries motifs for nuclear localizationas well as nuclear export. These motifs allow TDP-43 to return tothe nucleus after transport of mRNAs. The C-terminal half ofTDP-43 contains an Asn/Gln rich region as well as a Gly rich regionand has been suggested to contain a prion-like domain [3].

In 2006, TDP-43 was discovered to be the main protein compo-nent of abnormal protein aggregates in the cytoplasm of nerve cellsin FTLD and ALS1 [4,5]. In these aggregates, TDP-43 molecules areubiquinated, hyperphosphorylated and often truncated to yield23–27 kDa C-terminal fragments, with the nuclei of cells harboring

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abnormal TDP-43 cytoplasmic aggregates becoming deficient inTDP-43 [6].

Many genetic, cell biological and biochemical studies have pro-vided evidence implicating TDP-43 in neurological disorders [2]. Inparticular, TDP-43 mutations associated with early disease onset orpoor prognosis are linked to increased aggregation of the protein[7]. However, how TDP-43 harms cells is still an open question.The lack of TDP-43 in the nucleus could be detrimental to the prop-er splicing and transport of mRNAs. Alternatively, the hyperphos-phorylated, ubiquitinated aggregates could be cytotoxic. So far,results have been reported Supplementary both these mechanisms,potentially making them both pathologically relevant [2].

Among the various functional domains present in the TDP-43protein, one of the best characterized regions is represented bythe Asn/Gln-rich segment spanning residues 321–366 in the C-ter-minus of this protein. In particular, this region has been previouslydescribed to be involved in the interaction with hnRNP proteins[8], polyglutamine repeats [9], and nuclear ‘‘Gems’’ [10]. In addi-tion to all these potential connections, recent work from one ofour labs (FB) has shown that tandem repetitions of this Asn/Glnrich region of TDP-43 are capable of aggregating in cells and ofrecruiting normal TDP-43 into these aggregates [11]. Furthermore,

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the aggregated TDP-43 displays modifications that resemble mostof the pathological hallmarks associated with the full length pro-tein such as phosphorylation and ubiquitination. Within this re-gion, there are three short segments that are rich in Asn and Glnresidues, especially the first two (Fig. 1). When the residues ofany of these short segments are substituted by alanines, the result-ing variant is incapable of forming aggregates [11]. On the basis ofthese results, this region, referred to as TDP-43 (342–366) wasidentified as a minimal TDP-43 segment capable of recapitulatingits aggregation process [11].

Approximately forty TDP-43 mutations have been implicated inhereditary cases of ALS or FTLD; an up to date list can be found athttps://www.molgen.ua.ac.be/FTDMutations. Almost all thesemutations map to the sixth TDP-43 exon, which includes theminimal TDP-43 aggregative segment. In particular, the pathogenicmutations Q343R [12], N345K [13], G348V [14] and N352S [15,16]have been identified in the 321–366 region, are associated withearly-onset familial ALS without dementia, and usually showdominant-type inheritance.

At present, little information is available regarding the conse-quences of these mutations on TDP-43 biological properties.Recent studies have shown that the G348V and N352S mutationsdo not affect RNA splicing but do increase aggregation within cells[17]. The conservation of the wild type amino acid residue fromman to fish for Q343, N345 and G348 suggests that these residuesare crucial; in contrast, N352 is replaced by Ser in fish [14], whichsuggests that this position may be more tolerant to substitution.Secondly, it has also been recently established that pathologicalmissense substitutions positions Q343R, G348C, and N352S con-siderably increase the half life of the TDP-43 protein [18].

These findings prompted us to explore whether the TDP-43341–366 region could form a structural nucleus for aggregation.To meet this objective, we have characterized peptides corre-sponding to the WT TDP-43 sequence and point mutants Q343R,N345K and G348V using NMR and CD spectroscopies. The resultsshow that these peptides adopt an unfolded ensemble and thatwild type sequence can spontaneously self-associate to form a b-sheet rich oligomer that strongly enhances ThT fluorescence.

We hypothesize that this segment of TDP-43 as a monomercould occasionally adopt a b-sheet rich structure that may serve

Fig. 1. Schematic structure of the TDP-43 (341–366) polypeptide. Schematic diagrams shin an anti-parallel b-sheet. (b) Configured as a mixed anti-parallel/parallel b-sheet. (c) TQ343R and N345K variants, where the out-of-register Q354–N355–Q356 segment is colopurple arrows. The positions of the wild type residues of the point mutations studied hnumbered according to their position in the full length TDP-43 protein (black) or this sestrand 2 and the C-terminal segment are highlighted with purple arrows. The position ofgreen: three lines � almost always present; two green lines = usually present; dotted sinfigure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: M. Mompeán et al., Arch. Biochem. Biophys

as a nucleus for oligomerization. To characterize this conformation,whose population is too low for our experimental methods, wehave modeled TDP-43 (341–366) as a three stranded b-sheet andstudied its structure and stability by molecular dynamics (MD)methods. In parallel, we have also studied how different patholog-ically relevant mutations; namely, Q343R, N345K, G348V andN352S can affect the structure and stability of this b-sheet. The re-sults obtained have been discussed bearing in mind that matureTDP-43 aggregates are structurally diverse and that the aggrega-tion process will involve interactions with other regions of the fulllength TDP-43 protein, as recently reported [19,20].

Materials and methods

Peptides were synthesized and purified as previously described[17]. Their analysis by NMR and CD spectra corroborated their pur-ity and identity. One additional peptide corresponding to residues341–357, referred to here as TDP-43 (341–357), was purchasedfrom Genescript Corp. Its identity and purity (>95%) were con-firmed by mass spectrometry, NMR and HPLC.

NMR spectroscopy

1D 1H and 2D 1H TOCSY, NOESY and 2D 1H–13C HSQC and1H–15N HSQC spectra were recorded on a Bruker 800 MHz AVNMR instrument which was equipped with a triple resonance(1H,13C,15N) cryoprobe and z-axis gradients. The peptide concen-tration was generally 0.7–0.9 mM. All spectra were acquired in10 mM K2HPO4 and 6 mM deuterated acetic acid (final pH rangedfrom 6.2 to 6.7). For more details on the NMR experiments, pleasesee the Supplementary information.

Circular dichroism spectroscopy

A JASCO J-710 spectropolarimeter was used to record far UV-CDspectra of the TDP-43 peptides. Spectra of the appropriate buffer orcosolvent were recorded at 5.0 and 25.0 �C and were subtracted.Most samples were prepared by diluting small aliquots from theNMR samples into 10 mM K2HPO4 buffer (pH 8.2) or 3 mMKH2PO4/K2HPO4 buffer, pH 6.8. In an additional experiment, the

owing the configuration of the 341–366 Asn/Gln rich region of TDP-43. (a) Arrangedhe structure observed during the course of MD for the wild type sequence. (d) Thered gold. (e) The N352S variant, where Ser hydroxyl interactions are represented asere are colored blue and the mutated residues are colored red. Some residues are

gment (bluish-purple), i.e., A341 = 1, S342 = 2, Q343 = 3, etc., interactions linking b-b-secondary structure observed during the MD simulation (panels c–e) is marked ingle line — sometimes present. (For interpretation of the references to colour in this

. (2014), http://dx.doi.org/10.1016/j.abb.2014.01.007

https://www.molgen.ua.ac.be/FTDMutations


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WT lyophilized peptide was dissolved, a series of spectra were re-corded at intervals of 30–60 min over several hours. Furthermore,two additional series of spectra were recorded on the TDP-43peptide variants dissolved in the pH 6.8 buffer with 25% wt/v Ficoll,or alternatively, 0.15, 0.40 and 1.00 M KCl. For a detailed descrip-tion of the CD spectral analysis, please see the Supplementaryinformation.

ThT fluorescence

A 1.0 mM stock solution of ThT (Sigma, St. Louis, USA) was pre-pared in 3 mM KH2PO4/K2HPO4 buffer, pH 6.8. Fluorescence samplescontained 5 lL of ThT stock solution, whose final concentration was50 and 25 lM of TDP-43 peptides. Amyloid Ab1�40 (rPeptide) andRibonuclease A (Sigma type XII-A) were used as positive and nega-tive controls, respectively. Spectra were recorded at 25.0 �C on a Jo-bin–Yvon Fluoromax-4 instrument using 3 nm excitation andemission slit widths. The excitation wavelength was 440 nm andemission was recorded over 460–500 nm at a scan speed of2 nm s�1.

Molecular dynamics simulations

A starting model for TDP’s aggregation promoting region wasbased on the known tendency of Asn, Ser and Gln residues to be lo-cated in b-strands and the high propensity of Gly and Pro for turnpositions. The N-terminus and C-terminus were acetylated andamidated, respectively, to remove end charges and to more realis-tically mimic this segment’s behavior in the context of a long pro-tein. Two configurations, one with an all-antiparallel orientation ofthe b-strands of the b-sheet (Fig. 1a) and a second with a mixedantiparallel/parallel b-strand orientation (Fig. 1b) were obtainedand considered. The all atom models were built with the PyMOLpackage [21]. The force field utilized was AMBER99SB-ILDN [22].MD was performed using GROMACS (version 4.5.5) [23] software.For more information on the Molecular Dynamics Procedures andAnalysis, see the Supplementary information.

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Results

Experimental results

The backbone regions of the 2D 1H–15N, TOCSY & NOESY and1H–13C HSQC spectra of the Q343R variant of TDP-43 (residues342–366) are shown in Fig. 2. Despite the low chemical shift dis-persion and the presence of several Asn and Gln residues, all theresonances could be uniquely assigned except for the 15N ofQ354 and Q356.

Similar spectra were obtained and assigned for the WT, N345Kand G348V variants (Supplementary Fig. 1a). Their chemical shiftsare chiefly similar to those of Q343R, except for the substitutedresidues and for the residues bordering the substitution. The com-plete assignments are listed in Table 1 in the Supplementary infor-mation. The sidechain resonances were also completely assigned(Supplementary Fig. 2). The chemical shift difference of the 13Cband 13Cc is consistent with the trans conformation for the P349and P363 peptide bond. The chemical shift of M359’s Hc is consis-tent with the sulfur being reduced. One interesting difference isthat the Hd of Pro 349, which are degenerate in WT, Q343R andN345K, have unique chemical shift values in G348V (Supplemen-tary Fig. 2). Therefore, these H adopt unique magnetic environ-ments when G348 is replaced by V, which could reflect astiffening of the backbone.

The 1Ha, 13Ca, 13Cb and 1HN chemical shifts also provided resi-due level information on secondary structure content [24] and


dynamics [25]. The analysis of these chemical shifts, using TALOS+[26], indicated that these peptides are moderately flexible disor-dered ensembles (Fig. 2d). For example, most 13Ca deviated<0.2 ppm from the expected coil values [24] (Fig. 2d). In fully pop-ulated a-helices or b-strands, deviations of +3.1 and �1.5 ppm,respectively, are observed [27]. The scarcity of stable H-bondedstructures in our samples was also corroborated by 1HN tempera-ture factors of <�5 ppB K�1 (Supplementary Fig. 3) [28]. Neverthe-less, we cannot rule out small populations (610%) of orderedconformations, such as the b-hairpin characterized by MD (seebelow). The flexibility of the polypeptide chain was predicted tobe highest at the termini and lowest around residues 347–349and 361–363 (Fig 2d). Finally, the rigidity of the 347–349 segmentis somewhat more apparent in the G348V variant.

In NOESY spectra, Hai�1–HNi resonances could be clearly as-signed for all observed, non-overlapped signals. Strong Hai�1–HdPro NOEs were observed which corroborate that the Pro are mainlyin the trans conformation. The absence of medium and long rangeNOEs is consistent with a lack of stable hydrophobic contacts andb-structure. There is one HNi�1–HNi NOE signal betweenN364–Q365 seen in all spectra, and S347–V348, G357–N358 andR361–E362 crosspeaks are present in the G348V variant’s spectrum(Supplementary Fig. 4). These NOEs are indicative of turns.

Whereas the chemical shift values of the resonances of the WTpeptide were similar to those of the other variants, it was evidentthat the WT peptide produces poorer quality NMR spectra (Supple-mentary Fig. 1). In fact, the 1D 1H spectra recorded before and afteracquisition of the TOCSY and NOESY spectra (16 h difference)showed a decrease of approximately 75% in the peptide signalintensity (Supplementary Fig. 5a). The observation of a visible hazein this sample (Supplementary Fig. 5b), suggested that the WT pep-tide had self-associated to form slowly tumbling aggregates thatare invisible to liquid state NMR.

To corroborate the NMR results obtained on the variants and tocharacterize the NMR-invisible WT oligomers, we turned to far-UVCD spectroscopy. At pH 6.8, 25 �C, the variants Q343R, N345K andG348V showed similar CD spectra with minima near 198 nm thatare typical of disordered ensembles (Fig. 3a). In contrast, a freshlyprepared wild type peptide yielded a somewhat different CD spec-trum with increased negative signal near 220 nm; and an aged WTsample showed a remarkably different spectrum with a minimumat 218 nm and a maximum at 199 nm. Similar results were ob-tained from additional experiments preformed at pH 8.2, 25 �C;pH 6.8, 5 �C and pH 8.2, 5 �C. Quantitative algorithmic analysis ofthese spectra shows that the Q343R, N345K and G348V variantsand freshly dissolved WT peptide are dominated by turn and coilconformations. There is a remarkable increase in b-structure anddecrease in coil in the aged WT sample. Whereas the freshly dis-solved WT peptide contains 23% b-structure and 56% coil, the agedWT sample possesses 44% b-structure and 28% coil (SupplementaryTable 2).

Based on these observations, we then prepared another sampleof WT TDP-43 (342–366) and recorded a series of CD spectra overseveral hours. These spectra revealed that peptide transformsstructurally from an unfolded ensemble to conformations rich inb-structure (Fig. 3b). The observation of an isodichroic point, near210 nm, is consistent with there being two main conformationalensembles, one disordered and one rich in b-structure. This exper-iment was repeated once and similar results were observed (datanot shown).

It is surprising that the WT peptide oligomerized in solutionconditions whereas the pathological variants Q343R, N345K andG348V did not show this tendency. The WT peptide has a netcharge of +0 and the Q343R and N345K carry a net charge of +1at pH 6.8. Electrostatic repulsion can significantly affect proteinsolubility [29] and aggregation [30]. Therefore, we recorded



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Fig. 2. NMR structural characterization of TDP-43 (342–366) peptides. (a) Backbone region of the 1H–15N HSQC spectrum (blue peaks). All 1H–15N signals are labeled. Notethat the peak of A366 is folded; its correct 15N chemical shift is 131.0 ppm. (b) The HN–Ha regions of the 2D 1H TOCSY (green, 60 ms mixing time) and NOESY (red, 150 msmixing time) spectra. (c) The 1H–13C HSQC spectrum (black peaks). These spectra were recorded at pH 6.38 & 5 �C. Some signals which link nuclei from the same residue areconnected by brown dashed lines. Within the TOCSY/NOESY panel, two representative series of sequential inter-residual NOESY connectivities are shown as purple ormagenta dotted lines. D. Top panel: The conformational 13Ca chemical shifts (Dd13Ca) for TDP-43 (242–366) peptides. Values of +3.1 ppm and �1.5 ppm are expected forfully populated a-helices and b-sheets, respectively. The values observed here are much smaller and point to a low population of ordered conformations. The N- and C-terminal residues are not shown. Bottom panel: The predicted order parameter (S2) for TDP-43 (242–366) peptides. Values of one indicate rigidity; values of zero indicate fullyflexible chains. The estimated uncertainty in these values is 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web versionof this article.)



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additional CD spectra of the peptides in increasing concentrationsof KCl to test if aggregation occurs when charge–charge repulsionis screened. Such a condition would be more reflecting the physio-logical conditions, where high macromolecule and metabolicconcentrations produce a crowding effect that promotes theassociation of peptides and proteins [31]. We then used CD spec-troscopy to test if TDP-43 peptide variants oligomerize undercrowding conditions. However, neither high salt nor crowding con-ditions were found to promote self association and structure for-mation in the Q343R, N345K and G348V variants (SupplementaryFig. 6).

Next, ThT fluorescence was employed to obtain insight into thenature of the oligomers formed by the wild type TDP-43 (342–366)peptide. The variants Q343R, N345K and G348V and also RNase A,a well-folded monomeric protein, did not enhance ThT fluores-cence. In contrast, the enhancement of the ThT fluorescence pro-voked by the WT TDP-43 peptide over 20-fold- was almost asstrong as that caused by the amyloid Ab polypeptide implicatedin Alzheimer’s disease (Fig. 3c). It is interesting that aggregatingtandem repeats of the 342–366 segment do not enhance ThS fluo-rescence in vivo (unreported observations), which supports theidea that the context of this segment within the complete proteinversus isolated in a peptide, or in cell versus in vitro is key to itsaggregates’ superstructure.


Finally, we considered the possibility that a peptide correspond-ing to just the segment 341–357, which is particularly rich in Asnand Gln (Fig. 1), might form a preferred conformation. Although thispeptide is sparingly soluble in aqueous buffer, it could be dissolvedin DMSO, methanol or TFE and then diluted into aqueous buffer forspectroscopic analysis. We chose to study the peptide pre-dissolvedin DMSO by NMR, as this solvent is believed to have the smallest ef-fect on peptide conformations. Despite some haze which is typicalof partial aggregated samples, the soluble species of TDP-43(341–357) dissolved in 8% deuterated DMSO and 92% D2O detectedby NMR showed chemical shift values which are very similar to thelonger peptides and to the standard ‘‘coil’’ values for short unstruc-tured peptides (Supplementary Fig. 7 and Table 1G). For CD, it wasnecessary to study the samples pre-dissolved in methanol or TFEdue to the high absorbance of DMSO in the far UV. The analysis ofCD spectra of this peptide using the CDSSTR algorithm gave 6%helix, 31% b-sheet, 21% turn and 42% coil for TDP-43 (341–357) in10% methanol and 2% helix, 41% b-sheet, 16% turn and 43% coilfor TDP-43 (341–357) in 10% TFE. These results indicate popula-tions of b-structure similar to those seen for aggregating WT TDP-43 (342–366) (Fig. 3 & Supplementary Table 2). Lastly, we foundthat TDP-43 (341–357) samples strongly enhance ThT fluorescence,which suggest that its aggregates have amyloid-like characteristics.Taking these NMR, CD and fluorescence results together, we



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Fig. 3. Detection and characterization of WT TDP-43 (343–366) oligomer. (a) Far-UV CD spectra of TDP-43 peptides at pH 6.8, 25 �C. Aged WT peptide showshallmarks of b-sheet rich conformations. (b) Kinetics of the structural transforma-tion of WT TDP-43 (343–366) peptide followed by Far-UV CD. The time (in minutes)elapsed after dissolving the peptide is given in the legend. The spectra are coloredusing a rainbow scale from violet (freshly dissolved) to red (aged). (c) Thioflavin Tfluorescence of 25 lM peptide samples with 50 lM ThT at pH 6.8, 25 �C. The errorbars show the range of values from measurements on three samples. (Forinterpretation of the references to colour in this figure legend, the reader isreferred to the web version of this article.)



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conclude that the TDP-43 (341–357) segment can adopt an aggre-gating b-conformation. This conformation probably correspondsto the b-hairpin that is stable throughout the course of the MD sim-ulation runs (vide infra) and could be the minimal aggregative seg-ment of the protein in vitro.

Because these results raised several questions that could not besatisfactorily answered by these experimental approaches, we thendecided to use Molecular Dynamics (MD) to gain further insightinto the structure of this TDP-43 segment. The objective of thesecalculations was to characterize the putative structure and stabiliz-ing interactions of the putative aggregative nucleus of TDP-43formed by a monomer segment (residues 342–366) arranged in ab-sheet configuration. It is important to stress that these calcula-tions neither provide information on the putative intramolecularinteractions between this segment and other parts of the TDP-43protein nor on the possible intermolecular interactions formingas the structural nucleus grows.

Computational results

The TDP-43 segment 341–366 can adopt a well-defined anti-parallelsheet structure

The MD simulation of TDP-43 (341–366) provides evidence thatit adopts a set of preferred conformations. The structural deviationof a representative MD run is shown in Fig. 4a. The structure’s RMSDis low (<2 Å) and stable throughout the simulation, suggesting thatthis structure could be stable in physiological conditions. The resi-due-level secondary structure of TDP-43 (341–366) during the sim-ulation is shown in Fig. 5a. This analysis shows that the first andsecond b-strands, which are richer in Asn and Gln than the thirdstrand, conserve their b-strand conformation up to 75 ns. Comparedto the starting model, the register of the strands is shifted one posi-tion so that residues Q343, Q344 and N345 of b-strand 1 pair withN352, N353 and Q354 of b-strand 2 (Figs. 1a and c & 6a and b). Thisset of six residues forming two b-strands seems to be the most sta-ble element of structure, as it is conserved in almost all the confor-mations. Both strands can elongate to incorporate residues Q346and S347 (b-strand 1) and S350 and G351 (b-strand 2). In addition,the third strand rapidly loses its canonical b-strand conformation toadopt a well-defined, but non-standard structure that interacts inti-mately with the second b-strand. We therefore think that this con-formation is present at low population in the conditions of thespectroscopic experiments and may seed the formation of the b-sheet rich oligomeric structure by the WT peptide that is detectedby our experimental CD analysis (Fig. 3b).

To ensure a proper sampling of this structure, three additional100 ns MD runs were performed with the all anti-parallel b-strandtopology (Fig. 1a) in which each atom had a distinct starting veloc-ity. A Principle Component Analysis and the observations that allthree MD runs reach stable RMSD values over time and all success-fully converge to the same fold (Supplementary Fig. 8), stronglysupports the validity of this structure. When three independentsimulations were started from the alternative configuration(Fig. 1b), with the 3rd b-strand positioned in parallel to the 2nd,the 2nd and 3rd b-strands rapidly separate (SupplementaryFig. 9). The 3rd b-strand then adopts a series of conformations,while the first two b-strands eventually separate and unfold inall three MD runs. Based on these observations, we conclude thatthe first (all anti-parallel) configuration obtained in Fig. 1a is muchmore likely to be physiologically and pathologically relevant. Forthis reason, this all anti-parallel configuration was utilized to studythe effects of the mutations.

In Gln343 ? Arg, the Arg side chain promotes non-native interactionsMD simulations reveal large structural deviations (Fig. 4b) after

48 ns for the Q343R variant. The register of b-strands 1 and 2 is



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Fig. 4. RMSD (nm) from the starting model for wt anti-parallel TDP(341–366). RMSD (nm, y-axis) from the starting model for wt anti-parallel TDP(341–366) versus time (ns,x-axis). (a) wt TDP-43 (341–366), (b) Q343R, (c) N345K, (d) G348V, (e) N352S, (f) N352-N-Q-N-Q356 ? A-A-A-A-A. In green, residues 341–354, which correspond to the firsttwo strands and their connecting loop; red, residues 355–366 which correspond to the second loop and the C-terminal residues. The overall average is shown in black. (Forinterpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)



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usually shifted relative to the WT structure. After only 63 ns, the b-hairpin structure unfolds (Figs. 1d & 5b). The Q343 side chain pro-jects into solvent in the model of WT TDP-43 (341–366), suggest-ing that the loss of this side chain will not significantly affect thissegment’s conformation. However, the Arg’s side chain that substi-tutes Q343 contains a guanidinum moiety that is closely related toguanidinum chloride, a potent chemical denaturing agent. Guanid-inium chloride denatures proteins by interacting strongly withtheir aromatic and carbonyl groups. Structural snapshots revealthat after 60 ns, Arg343’s guanidinium group frequently formsnon-native H-bonds to carbonyl groups; some of which formedstabilizing interactions in the starting structural model (Fig. 6c).These aberrant interactions would tend to hold the segment innon b-hairpin conformations; in fact, the number of H-bondsformed by the Arg343 side chain increase notably after the nativeH-bond network breaks down (Figs. 5b & 6d). As a result, theseaberrant interactions could account for why the spectroscopicexperiments did not detect the formation of an oligomer rich inb-structure by the Q343R variant.

The variant Asn345 ? Lys shows straighter b-strands with a betternetwork of H-bonds

By MD simulation, b-strands 1 and 2 in the N345K variant werefound to be longer on average than those of the WT sequence andmore stable, as they do not unfold even after 100 ns (Fig. 5c). Thesestrands are also longer on average than they are in WT (Fig. 5c) andadopt the alternative strand register relative to WT that was alsoseen in the Q343R variant (Fig. 1d). This alternative strand registerseems to cause this variant’s RMSD values to be moderately large(Fig. 4c).

In wild type TDP-43 (341–366), the b-strands show a significantdegree of twist. This distortion could be attributed, at least partially,


to the formation of H-bonds between the Asn 345 side chain andthe backbone amide groups of the adjacent residues (Fig. 6e). Inthe N345 K mutant of TDP-43 (342–366), however, the lysine sidechain is unable to maintain these H-bonds and, as a consequence,straight b-strands with an increased number of H-bonds of im-proved geometry are observed (Fig. 6f). These structural changescould account for why the N345K variant’s b-stand structure is ex-pected to be longer and more stable. Considering the higher stabil-ity of the N345K variant’s b-sheet, it seems puzzling thatspectroscopic experiments showed that the WT peptide forms ab-sheet rich aggregate but the N345K variant does not. It is there-fore possible that N345K’s altered b-strand register and less twistedb-strand structure could be incompatible with oligomerization.

Mutating Gly348 ? Val keeps b-stands 1 and 2 short and theirconnecting loop long

b-strands 1 and 2 in the WT TDP-43 (341–366) can grow toencompass residues S342–S347 and S350–N355 (see Fig. 5a from�35 to 75 ns, for example) at the expense of their connecting loop.In fact, in the WT segment, this loop can be as short as just two res-idues: Gly348 and Pro 349. Whereas the effect of the Gly348 ? Valmutation on the overall structural deviation is small (Fig. 4d), it isremarkable that the conformers with longer b-strand observed inWT are never seen along the trajectory of the G348V variant(Fig. 5d). Indeed, with Val at position 348, the b-strands remainshort and their connecting loop is always long, as illustrated bythe consistently large separation between the Ca’s of Ser347 andSer350 (Fig. 6g and h). This behavior, and some signs from NMRof altered flexibility in G348 V, could be related to this variant’sinability to form a multimer with increased b-structure in ourspectroscopic experiments.



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Fig. 5. Secondary structure of TDP-43 residues 341–354 during simulation. Secondary structure of TDP-43 residues 341–354 during the 100 ns simulation, which correspondto the first two b-strands and their connecting loop and are numbered 1–16 as shown in bluish-purple in Fig. 1, over 100 ns of MD simulation (x-axis). (a) wt TDP-43, (b)Q343R, (c) N345K, (d) G348V, (e) N352S and (f) N352-N-Q-N-Q356 ? A-A-A-A-A. (For interpretation of the references to colour in this figure legend, the reader is referred tothe web version of this article.)



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Asn352 ? Ser favors an alternative b-hairpin structureMany TDP-43 missense substitutions involve the creation of

potentially novel phosphorylation sites. In our sequence of inter-est, such an occurrence could involve Asn352 and such an occur-rence could have serious consequences on the structure. First ofall, even in the absence of phosphorylation, it has to be noted thatthe N352S variant substitutes an Asn residue that actively partic-ipates in the structure of the second b-strand for Ser. During thecourse of the simulation and in the presence of this variant theb-strands 1 and 2 maintain a WT-like conformation for about27 ns; afterwards this structure unfolds. It then refolds around32 ns to an alternative conformation featuring a distinctive b-stand register (Figs. 1e & 3e) and a different b-hairpin structure(Fig. 4e) in which strand 1, composed of residues S342–Q346 istwo residues longer than b-strand 2, formed by residues N353–N355. It is notable that S352, the mutated residue, is excludedfrom the short b-strand. In fact, this residue is observed to formH-bonding interactions with the side chains of S347 and S350and with the charged carboxylate group of E362 (Fig. 6i and j).These interactions may therefore account for this variant’s singu-lar b-strand register and loop conformation.

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Phosphorylation of Ser 352 in the N352S variant strongly perturbs thestructure

The effect of adding a phosphate group with a single negativechange to Ser 352 was also studied (Supplementary Fig. 10). Thenegative charge of the phosphate group, and its increased bulk,led to unfavorable interactions with the side chains of S347, S350and E362. Along the course of the simulation, the b-strand struc-ture adopted by residues 342–352 remains short and eventuallyis lost. The side chain of E362 turns outward and by the end ofthe simulation, it is positioned to form a salt bridge with the sidechain of R361.


Multiple Ala substitutions are severely destabilizingSubstituting the residues of each of the three Q/N rich stretches

in TDP-43 (341–366) to Ala has been shown to decisively disruptaggregation [11]. We therefore studied the behavior of theTDP-43 (341–366) with residues Asn352-Asn-Gln-Asn-Gln356substituted to Ala. As shown in Figs. 4f & 5f, it was observed thatthis variant rapidly deviates from the WT structure to adopt a pairof short b-strands composed of just two residues each; namely,Q343 & Q344 in strand 1 and G351 & N352 in strand 2. The strandregister is like the one observed for the Q343R and N345K variants(Fig. 1d). This minimal b-hairpin unfolds after just 15 ns and thechain then adopts a broad variety of conformations.

Two 100 ns MD runs were also performed to study the effect ofsubstituting P363, N364 and Q365 by Ala on the structure. In thesesimulations, the well-defined non-canonical structure breaksdown and in one of the two runs, and the entire b-hairpin structureunfolds (Supplementary Fig. 11). Based on these results and on theobserved instability when the last segment is arranged in parallelto the second (Supplementary Fig. 9) we propose that P363,N364 and Q365 also contribute to the overall conformational sta-bility, at least at this first stage in the formation of the nucleusfor aggregation. However, the ability of the TDP-43 (341–357) pep-tide to adopt substantial populations of b-structure and to precip-itate suggests that these stabilizing interactions are not essentialfor aggregation, at least in vitro.

Discussion

Pathological mutations in TDP-43 destabilize the b structure or shiftthe strand register in the 341–366 Gln/Asn-rich region

Amyloid structures have been implicated in many dementiaand neurological disorders [32]. These structures consist of long



Fig. 6. Representative 3D structures of TDP-43 (341–366) and its variants: (a) Starting structure of wt TDP-43 (341–366), showing the starting configuration. (b) wt TDP-43(341–366), after 20 ns, showing changes in the b-strand register. (c) Snapshot taken along the course of the simulation of Q343R highlighting ‘‘non-native’’ contacts. (d)Number of H-bonds formed by residue 343: black = WT; red = Arg variant (e) Snapshot showing H-bonds formed by N345. (f) Mutation of N345 ? Lys increases the numberof H-bonds and the straightness of the b-sheet. (g) Loop 1 size as gauged by the distance between the Ca of S347 and S350; black = wt; red = G348V variant. (h) The size ofloop 1 is always large in the G348 ? Val mutation. (i) Formation of contacts among the hydroxyl groups of S346, S350, S352 and E362 in the variant N352S. (j) The number ofcontacts (<0.3 nm) among residues S346, S350, N352 and E362 in the WT segment (black) and the N352S variant (red). (For interpretation of the references to colour in thisfigure legend, the reader is referred to the web version of this article.)



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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


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fibrils composed of b-strands running perpendicular to the longfibril axis that are typically stabilized by efficient networks ofH-bonds and by exceptionally tight chain packing. Recently,amyloids with out-of-register b-strands exposing unsatisfiedH-bonding groups [33] or sporting exposed hydrophobic surface[34] have been proposed to be the most toxic and pathologicallyrelevant forms. In this respect, therefore, it is interesting to pointout that all the pathological mutations studied here: Q343R,N345K, G348V and N352S tend to be either structurally destabiliz-ing or to shift the register of the b-strands of the 341–366Gln/Asnregion so as to expose Asn and Gln residues. These changes,detected by MD, could be related to the inability of the NMR andCD experiments to detect the formation of b-sheet rich aggregatesby the Q343R, N345K and G348V variants.

Of the approximately forty pathological mutations described todate in TDP-43, nine, including N352S, yield a new Ser or Thr resi-due, with many of these residues representing potential phosphor-ylation sites. Considering that the abnormal TDP-43 aggregates arehyper-phosphorylated in cells, it will be important to study thephosphorylation state of TDP-43 and their effects on its aggregation.In particular, we tentatively advance that eventual phosphorylationof the Ser hydroxyl group in the N352S variant breaks a network ofH-bonds and as a consequence strongly perturbs the structure.

TDP-43 deposits are commonly described as amorphous or gran-ular aggregates and are generally found to be non-amyloid, sincethey neither bind amyloid specific dyes nor reveal amyloid fibrilswhen visualized by EM [6]. In vitro, of four peptides correspondingto segments from the C-terminal half of TDP-43, one formed amy-loid-like fibrils, whereas the other three formed amorphous aggre-gates [35]. Another study of a series of peptides corresponding tothe C-terminus of TDP-43 did not detect amyloid formation in the341–366 region, although the peptides chosen; namely 337–349and 350–362, would have separated the b-strand 1 from b-strand2 in TDP-43 (341–366) [36], and we expect that this would havehad a detrimental effect on b-sheet structural stability. Our ThTfluorescence results suggest that the b-sheet rich aggregates formedby the WT TDP-43 (342–366) and TDP-43 (341–357) peptide maybe amyloid-like. Very recently, it has been shown that some formsof TDP-43 aggregates implicated in FTLD–TDP and ALS occasionallyadopt amyloid-like structures in some conditions [37,38], althoughthese findings’ significance for TDP-43 pathology is presentlyunclear. Finally, the dichotomy between Asn-rich sequences, thattend to fold quickly into stable, less toxic amyloids, and Gln-richsequences, that tend to form toxic aggregative intermediates andmay not form stable amyloid [39], could be relevant for the TDP-43(341–366) segment, which is slightly Gln-rich (7 Gln vs. 6 Asn).

How does the rest of TDP-43 affect the 341–366 segment’s behavior?

Although previous results [17,11], and the spectroscopic andcomputational findings reporting here, indicate that TDP-43 seg-ment 341–366 is sufficient to form a stable b-structure that couldtrigger aggregation, it is well known that other TDP-43 segments orexternal factors affect its tendency to aggregate. For example, pres-ence of cleaved C-terminal fragments, N-terminal residues, thered/ox status of various Cys residues, and stress conditions canall contribute to trigger aggregation of this factor; for a recent re-view, see: [2].

In particular, just to remain within the considerations made inthis work that mostly concern missense mutations, a large numberof pathology-linked mutations beside the four reported in thisanalysis have been mapped to the 54 residues preceding this seg-ment and the 24 residues following it {for a most recent update,see [40]}. This extended region is rich in Gly residues that adoptunusual backbone angles and are good at making turns, two char-acteristics that are important in aggregate and amyloid formation


in yeast prions [39]. Interestingly, a peptide corresponding to thisextended segment of TDP-43 has also been reported to form amy-loid fibrils [35]. Nevertheless, these amyloid fibrils do not enhanceThT fluorescence, which was suggested to be due the presence of arelatively high proportion of Gly residues. For a mechanistic expla-nation for the formation of these fibrils, the short side chain of Glycan constitute a ‘‘hole’’ that aids efficient side chain packing be-tween b-strands within globular proteins, at protein–protein inter-action interfaces and in amyloid [40]. Glys are also abundant inGly-(Ala/Pro/Arg) dipeptide repeats aggregates that have been re-cently discovered and linked to FTLD/ALS neurological diseases[41,42].

The segment of TDP-43 composed of residues 341–366 is ableto aggregate and drive the aggregation of full-length TDP-43 insidecells. Here, we provide spectroscopic evidence for the formation ofa b-sheet rich oligomeric structure from a mostly disorderedconformational ensemble by the WT peptide. Furthermore, MDevidence supports the observation that this segment forms ab-hairpin composed by residues 342–355 that is followed by anon-standard, although well-defined structural motif. As a result,these observations represent the first step aiming to provide astructural framework for interrogating the effects of pathogenicmutations and for formulating new hypotheses to guide futureresearch in the aggregation process of TDP-43.

Finally, as highlighted here, protein chains may contain aggre-gation-prone or amyloidogenic segments that are normally ‘‘self-chaperoned’’ by other parts of the same protein chain [43] or byinteractions with other partners. In these proteins, structured re-gions and binding to other factors can lock an aggregation-pronesegment in a ‘‘safe’’ conformation. However, unfolding conditionsor missense mutations can break the structured region’s hold andunleash aggregative segments. Here, we show that the 341–366residues in the C-terminus of TDP-43 may well represent one suchelement within this protein and suggest that more work to charac-terize the protein’s structure at high resolution should be priori-tized by future research trends. This will reveal not only theconformation of the TDP-43 341–366 segment in the context ofthe full length protein, but also interactions which could promoteor hinder its aggregation. Such studies could provide furtherinsight into how in-cell conditions or mutations increase TDP-43aggregation and to design small effector molecules aimed atpreventing, slowing down, or even reverting this process.

Acknowledgments

We thank Dr. D. Pantoja-Uceda for assistance with NMRspectrometers and Prof. M. Bruix for critically reading the MS.The authors state no conflict of interests. This work was fundedby Grants CTQ 2010-21567-C02-02 to DVL from the SpanishMinisterio de Ciencia e Innovación and AriSLA (TARMA) to FEBand the Theirry-Latran Foundation (REHNPALS). The fundersplayed no role in the experimental design or execution.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.abb.2014.01.007.

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“Structural characterization of the minimal segment of TDP-43 competent for aggregation”

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