Native State Hydrogen Exchange Study of Suppressor and Pathogenic Variants of Transthyretin Kai Liu 1 , Jeffery W. Kelly 1 * and David E. Wemmer 2,3 * 1 Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, BCC260, La Jolla CA 92037, USA 2 Department of Chemistry MC-1460, University of California, Berkeley, CA 94720 USA 3 Physical Biosciences Division Lawrence Berkeley National Laboratory, Berkeley, CA 94720 USA Transthyretin (TTR) is an amyloidogenic protein whose aggregation is responsible for numerous familial amyloid diseases, the exact phenotype being dependent on the sequence deposited. Many familial disease var- iants display decreased stability in vitro, and early onset pathology in vivo. Only subtle structural differences were observed upon crystallo- graphic comparison of the disease-associated variants to the T119M inter- allelic trans-suppressor. Herein three human TTR single amino acid variant homotetramers including two familial amyloidotic polyneuropa- thy (FAP) causing variants (V30M and L55P), and a suppressor variant T119M (known to protect V30M carriers from disease by trans-suppres- sion) were investigated in a residue-specific fashion by monitoring 2 H– 1 H exchange employing NMR spectroscopy. The measured protection factors for slowly exchanging amide hydrogen atoms reveal destabiliza- tion of the protein core in the FAP variants, the core consisting of strands A, B, E and G and the loop between strands A and B. The same core exhi- bits much slower exchange in the suppressor variant. Accelerated exchange rates were observed for residues at the subunit interfaces in L55P, but not in the T119M or V30M TTR. The correlation between desta- bilization of the TTR core strands and the tendency for amyloid formation supports the view that these strands are involved in amyloidogenicity, consistent with previous 2 H– 1 H exchange analysis of the WT-TTR amyloi- dogenic intermediate. q 2002 Elsevier Science Ltd. All rights reserved Keywords: transthyretin; amyloid; hydrogen exchange; NMR spectroscopy; protection factor *Corresponding authors Introduction Transthyretin (TTR) exists as a homotetramer of 127-residue subunits (55 kDa as the tetramer) in human plasma (0.2 – 0.3 mg ml 21 ) and cerebral spinal fluid (CSF) (0.02 mg ml 21 ). It is the primary transporter of thyroxine in the CSF and the second- ary transporter in blood plasma, where it also binds holo-retinol binding protein (RBP) whether or not thyroxine is bound, 1 preventing rapid clear- ance of RBP in the kidney. In some people the normally soluble TTR is transformed into amyloid fibrils. In this process the tetramer dissociates into folded monomers that undergo a substantial conformational change owing to a denaturation stress (such as low pH), allowing self-assembly into an insoluble cross-b- sheet quaternary structure in vivo. TTR amyloid deposition occurs in several organs, including the heart, peripheral nerves and ocular vitreous. Depo- sition of wild-type (WT) TTR is implicated as the cause of senile systemic amyloidogenesis (SSA) (onset around 80 years of age). Amyloidogenicity of the majority of the 85 characterized sequence variants appears to cause a spectrum of diseases classified as familial amyloid polyneuropathy (FAP), which have an earlier onset, with the exact age and pathology dictated by the mutation. It is likely that the interplay between genetic, tissue-dependent environmental and structural factors play a crucial role in the TTR amyloidogeni- city. Numerous TTR mutations associated with FAP are conservative surface mutations that 0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved E-mail addresses of the corresponding authors: [email protected]; [email protected]Abbreviations used: TTR, transthyretin; WT-TTR, wild-type transthyretin; 2 H– 1 H exchange, deuterium – proton exchange; SSA, senile systemic amyloidogenesis; FAP, familial amyloid polyneuropathy; GdnHCl, guanidinium hydrochloride; CSF, cerebral spinal fluid; T 4 , thyroxine; RBP, retinol binding protein; EM, electron microscopy; HSQC, heteronuclear single quantum coherence; HNCA, hydrogen nitrogen C-alpha correlation triple resonance experiment. doi:10.1061/S0022-2836(02)00471-0 available online at http://www.idealibrary.com on B w J. Mol. Biol. (2002) 320, 821–832
Transcript
Native State Hydrogen Exchange Study of Suppressorand Pathogenic Variants of Transthyretin
Kai Liu1, Jeffery W. Kelly1* and David E. Wemmer2,3*
1Department of Chemistry andSkaggs Institute for ChemicalBiology, The Scripps ResearchInstitute, 10550 North TorreyPines Road, BCC260, La JollaCA 92037, USA
2Department of ChemistryMC-1460, University ofCalifornia, Berkeley, CA 94720USA
3Physical Biosciences DivisionLawrence Berkeley NationalLaboratory, Berkeley, CA 94720USA
Transthyretin (TTR) is an amyloidogenic protein whose aggregation isresponsible for numerous familial amyloid diseases, the exact phenotypebeing dependent on the sequence deposited. Many familial disease var-iants display decreased stability in vitro, and early onset pathology invivo. Only subtle structural differences were observed upon crystallo-graphic comparison of the disease-associated variants to the T119M inter-allelic trans-suppressor. Herein three human TTR single amino acidvariant homotetramers including two familial amyloidotic polyneuropa-thy (FAP) causing variants (V30M and L55P), and a suppressor variantT119M (known to protect V30M carriers from disease by trans-suppres-sion) were investigated in a residue-specific fashion by monitoring2H–1H exchange employing NMR spectroscopy. The measured protectionfactors for slowly exchanging amide hydrogen atoms reveal destabiliza-tion of the protein core in the FAP variants, the core consisting of strandsA, B, E and G and the loop between strands A and B. The same core exhi-bits much slower exchange in the suppressor variant. Acceleratedexchange rates were observed for residues at the subunit interfaces inL55P, but not in the T119M or V30M TTR. The correlation between desta-bilization of the TTR core strands and the tendency for amyloid formationsupports the view that these strands are involved in amyloidogenicity,consistent with previous 2H– 1H exchange analysis of the WT-TTR amyloi-dogenic intermediate.
Transthyretin (TTR) exists as a homotetramer of127-residue subunits (55 kDa as the tetramer) inhuman plasma (0.2–0.3 mg ml21) and cerebralspinal fluid (CSF) (0.02 mg ml21). It is the primarytransporter of thyroxine in the CSF and the second-ary transporter in blood plasma, where it alsobinds holo-retinol binding protein (RBP) whetheror not thyroxine is bound,1 preventing rapid clear-ance of RBP in the kidney.
In some people the normally soluble TTR istransformed into amyloid fibrils. In this processthe tetramer dissociates into folded monomersthat undergo a substantial conformational changeowing to a denaturation stress (such as low pH),allowing self-assembly into an insoluble cross-b-sheet quaternary structure in vivo. TTR amyloiddeposition occurs in several organs, including theheart, peripheral nerves and ocular vitreous. Depo-sition of wild-type (WT) TTR is implicated as thecause of senile systemic amyloidogenesis (SSA)(onset around 80 years of age). Amyloidogenicityof the majority of the 85 characterized sequencevariants appears to cause a spectrum of diseasesclassified as familial amyloid polyneuropathy(FAP), which have an earlier onset, with the exactage and pathology dictated by the mutation.
It is likely that the interplay between genetic,tissue-dependent environmental and structuralfactors play a crucial role in the TTR amyloidogeni-city. Numerous TTR mutations associated withFAP are conservative surface mutations that
0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved
doi:10.1061/S0022-2836(02)00471-0 available online at http://www.idealibrary.com onBw
J. Mol. Biol. (2002) 320, 821–832
influence tetramer stability and/or tertiary struc-tural stability. They also strongly influence the vel-ocity of the required tetramer dissociation, whichhas been shown to be rate limiting in amyloid fibrilformation.
The FAP-associated mutations are concentratedin the two b-sheets, wherein mutations at any ofabout 40% of the residues leads to FAP. Previousstudies demonstrate that wild-type TTR formsamyloid over a 72 hour time course when placedunder acidic conditions that mediate tetramer dis-sociation into an alternatively folded monomericamyloidogenic intermediate.2,3 The FAP variantspopulate the monomeric amyloidogenic intermedi-ate faster and to a greater extent at a given dena-turation stress relative to wild-type, leading toaccelerated amyloid formation. Given the stabilityof WT-TTR, it is not yet clear how and where dis-sociation and partial denaturation occur in vivo.One hypothesis is that partial denaturation takesplace within a cell in the lysosomes, where the pHis <5. Another hypothesis is that the amyloido-genic form of TTR is formed in the extracellularmilieu as a result of tetramer dissociation. This pro-cess yields a concentration of a folded monomer,dictated by tetramer stability and rate of dis-sociation that can misfold as a result of a denatur-ing microenvironment. The existence of atetramer-folded monomer equilibrium at pH 7 hasbeen demonstrated by subunit exchangeexperiments.4 Another hypothesis put forward isthat tetramer dissociation leads to a misfoldedmonomer incompatible with reforming the tetra-mer but capable of forming amyloid.5 The obser-vation of a tetramer-folded monomer equilibriumis consistent with our previous 2H– 1H exchangestudy on WT-TTR, which demonstrated exchangewithin 72 hours for amides comprising residues atthe subunit interfaces.6 The transient existence ofTTR monomers raises the possibility that TTRamyloid formation could also take place undernative conditions in vivo, via subunit dissociationand transient conformational change during the18–72 hour half-life of TTR in the body;7 however,under these conditions the concentration of theamyloidogenic intermediate would be extremelylow, making this mechanism seem least likely.
X-ray crystal structural analysis of TTR variantsfocusing on a ground state explanation for amyloi-dogenicity has proven difficult.8 – 13 Numerous X-ray crystallographic studies on non-amyloidogenicand amyloidogenic TTR variants reveal that nearlyall FAP variant homotetramers crystallize in thesame space group (P21212) as wild-type TTR, withthe exception of L55P (C2), and display only subtlestructural changes in the vicinity of the mutation.10
Some of these changes have been interpreted asbeing important for amyloidogenesis.11,12 Furtherstudies will clarify the relevance of these differ-ences. It has also proven difficult to explain theamyloidogencity of the human lysozyme variantsbased on their ground state structures.14 Thus, itappears that single amino acid substitutions in
TTR affect the denaturation kinetics and thermo-dynamics to a much greater extent than the dis-cernable details of its tetrameric structure.Furthermore, the observations that amyloid fibrilshave a common core structure and form from avariety of proteins suggest that amyloidogenic pro-teins are likely to be conformationally flexibleunder amyloidogenic conditions, allowing necess-ary structural changes during the process ofamyloidogenesis.15 – 17 Thus, the changes inmutated proteins that make amyloidogenic confor-mationally altered forms easier to populate are noteasy to identify by crystallography.
Deuterium–proton exchange experiments wereused as part of our quest to understand TTR struc-tural changes that produce the TTR amyloidogenicintermediate(s) responsible for fibril formation.3
These studies identified the regions in WT-TTRthat are destabilized under amyloidogenic con-ditions (pH 4.5), most notably the CBEF sheet. Pre-vious 2H– 1H exchange studies on WT-TTR at pH 7revealed that the slowest amide exchange rates arein strands A, B, E and G and the loop connectingstrand A and B, hereinafter referred to as the stablecore. Here we report native state 2H– 1H exchangestudies on three TTR variants (pH 7) which revealthe effect of the single-site substitutions on the qua-ternary and tertiary structural flexibility of TTR.Our results demonstrate significant destabiliza-tion/flexibility of the stable core in the two dis-ease-causing variants, L55P and V30M TTR,whereas increased core stability was observed inthe T119M suppressor variant.
Results
Backbone amide resonance assignments
The three bacterially expressed single site TTRvariants all contain an additional methionine attheir N terminus (128 residue sequence) relative tothe human sequences. Biophysical studies demon-strate that neither stability nor amyloid formationis affected by the presence of the N-terminal Metresidue. At pH 6 (40 8C), where the non-amyloido-genic tetramer is the predominant structure,1H– 15N HSQC peaks from all residues wereobserved for the T119M and V30M TTR variants.In the case of L55P, peaks from V14 and V16 werenot detected. Increasing the pH from 6 to 7 resultsin line broadening for some 1H–15N resonances.Those exhibiting broadening include—Leu12,Val14, Lys15, Gly57, Thr75, Ser117, Thr118 in thecase of V30M; and Leu12, Met13, Lys15, Gly22,Thr49, Gly57, Thr118, Ala120 in the case of L55P;and Leu12, Gly57 for T119M TTR. Most of theseresidues were not well protected in WT-TTRaccording to a previous analogous study.6 Thus,the loss of the signal is likely to be caused by anincrease in the intrinsic solvent exchange ratesassociated with the pH increase from pH 6 to 7.
822 2H-H Exchange of TTR Variants
One of the prerequisites for NMR based 2H– 1Hexchange experiments is reliable 1H– 15N backboneresonance assignments. In the neutral pH range,the TTR variants studied are tetramers, with 2,2,2molecular symmetry, which makes resonancesfrom all subunits equivalent. The effective molecu-lar mass is ca 55 kDa. At this molecular mass mak-ing backbone assignments via routine triple-resonance NMR experiments on 15N, 13C uniformlyenriched samples is hindered by the rapid 1H and13C transverse relaxation. However, deuteration ofthe protein at fairly high levels removes many ofthe efficient 1H–1H and 1H–13C relaxation path-ways, narrowing the lines. For doing the backboneassignments, 2H, 15N and 13C triply enriched TTRsamples were used to collect HNCA spectra withdeuterium decoupling.18 About 90% of the possibleHNCA cross-peaks (both intraresidue and sequen-tial) were observed for the T119M and V30M var-iants at pH 6 (40 8C), and 85% of the expectedconnectivities were observed in the case of L55P.As in WT-TTR, expected sequential peaks werenot observed in the AB loop and the H strand. Res-onance assignments associated with V30M, L55Pand T119M at pH 6 were facilitated by comparingwith the wild-type assignments.6 The majority ofthe chemical shifts for HSQC signals in the threesingle-site variants are identical to those found forthe wild-type protein at pH 6. Residues that exhibitaltered 1H– 15N chemical shifts relative to WT-TTRare highlighted in the ribbon diagram represen-tation in Figure 1. HNCA spectra were used toestablish new assignments for shifted peaks aswell as to confirm assignments for the unshiftedpeaks. The resonance assignments for HSQC spec-tra collected at pH 7 were extrapolated from thoseat pH 6 by recording several HSQC spectra at
intervening pH values, and following the move-ment of individual peaks carefully.
Native-state hydrogen exchange studies on thetetramer (high protein concentration, pH 7)
In these experiments, the sample of deuteratedTTR (exchanged at pD 2 at low ionic strength fol-lowed by a pH-jump to pH 7 to refold and tetra-merize) was diluted into 1H2O to initiate the2H– 1H exchange. Since the protein is initially deut-erated (2H–15N), any 1H– 15N signals in the HSQCspectra (V30M, L55P, T119M) result from theexchange-in of protons from the solvent. The peakintensities reflect the extent of exchange. After thesample is diluted into H2O (about sixfold), it takesabout an hour to concentrate the diluted sample tomake a 600 ml NMR sample (,3 mg, which is,0.3 mM), and then an additional hour to recordan HSQC spectrum. Hence the dead time in our2H– 1H exchange monitored by NMR is about twohours. The subsequent time course of 2H– 1Hexchange was monitored by recording HSQC spec-tra periodically.
We first attempted the 2H–1H exchange at pH 6for all three variants. However, aggregation wasproblematic in the case of V30M and L55P result-ing in irreversible sample loss. Extensive protofila-ment formation was observed by electronmicroscopy (EM) in the sample precipitate. Due tothe sample loss, protection factors could not beaccurately measured for these variants. However,at pH 7, no significant aggregation was observedfor V30M, L55P and T119M. This is demonstratedby the summed intensities of selected unprotectedresidues utilized as internal standards to monitorthe sample condition. Figure 2 demonstrates that
Figure 1. Chemical shift mapping of the three TTR variants. Point mutations are highlighted in magenta in the rib-bon diagram representation of WT-TTR. For the FAP variants (V30M and L55P) orange-red is used to highlight theresidues exhibiting significant chemical shift changes, whereas the color blue is used to identify residues exhibitingchemical shift perturbations in the suppressor variant T119M.
2H-H Exchange of TTR Variants 823
the summed intensities of the selected residuesfluctuated only modestly without significant long-term drift, which would be observed if aggregationwere occurring. Thus, the protection factorsmeasured at pH 7 reflect the entire protein popu-lation. However, as a result of the pH increase, thelower protection factor detection limit is increasedto #105, compared to #104 in our previous 2H– 1Hexchange study on wild-type TTR at pH 5.7.
The 2H–1H exchange rates of the backboneamides were extracted by fitting the observedintensities, I(t ), to the formula:
IðtÞ ¼ I0ð1 2 expð2kobstÞÞ
where I0 is the signal intensity of a specific amidewhen it is fully protonated; t is the elapsed timeafter the initial dilution; I(t ) is the signal intensityof the cross peak at time t and kobs is the 2H– 1Hexchange rate of the amide. In Figure 3, residuesfrom different structural regions of TTR with non-overlapped 1H– 15N resonances were chosen asexamples of the intensity versus time plots.
The process of hydrogen exchange can bedescribed by a kinetic model with terms for theopening and closing rates of the exchangeablestates (kopen and kclose with the equilibrium constantfor the opening process Keq ¼ kopen=kclose), and therate of chemical transfer (kchemical) in the open state.In the limit that kclose q kchemical; known as EX2conditions, the observed exchange rate kobs ¼Keqkchemical: In EX2 condition differences in protec-tion factors, which are directly proportional to theobserved exchange rates, reflect differences in theequilibrium constants for opening, and can be useto evaluate the energetics of the opening process.In the limit that kclose p kchemical; known as EX1conditions, all opening events lead to exchangeand hence kobs ¼ kopen: Such conditions prevail athigh pH, where kchemical is large, or if the proteincloses (folds) slowly. A majority of proteins fold athigh rates so that EX2 conditions prevail at neutralpH. However, the folding kinetics for TTR have notbeen characterized, in large part due to compli-cations from the formation of tetramers, and hencewe cannot verify that exchange for TTR at pH 7occurs in the EX2 limit. However, regardless ofwhether the exchange rates are reflecting a changein the equilibrium constant or the opening rate,the differences in patterns of exchange in thesequence variants provides information about theinitial stages of unfolding that are required to gen-erate the amyloidogenic form of the protein.
The measured exchange rates and the calculatedprotection factors (kobs/kintrinsic) for the majority ofthe residues are listed in Tables 1–3. The upperlimit in our measurement of kobs, imposed by theinherent dead time, is 1023 min21. In principlethere is no lower limit for the detection of k, pro-vided the sample remains stable. The slowestamide exchange rates observed in TTR variantsare ,1027 min21. In order to calculate the protec-tion factor, the intrinsic exchange rates kintrinsic, for
Figure 2. The concentration of soluble T119M (a), L55P(b) and V30M TTR (c) was monitored as a function oftime by the summed signal intensities of selected resi-dues (identified in the box). Residues were selected bytwo criteria, the amide groups must display wellresolved NMR signals and exchange completely beforethe first time point is recorded.
824 2H-H Exchange of TTR Variants
unprotected amides at pH 7 and 25 8C, wereobtained using the program SPHERE†.19,20 Thetypical values of kintrinsic vary from approximately100–1000 min21. The protection factors determinedare in the range of 105 to .107. A summary of thenumber of amide groups with protection factorsover 105 for WT-TTR and the three variants is illus-trated in Figure 4. It is worth noting that the 2H– 1Hexchange for the WT-TTR was carried out at pH5.75.6 The total number of amides with protectionfactors over 105 are 43, 41, 40 and 27 for WT,T119M, V30M and L55P, respectively.
There are two types of subunit interfaces in TTR:the hydrogen-bonded interface, also referred to asthe monomer–monomer interface formed by anintermolecular antiparallel b-sheet interaction; andthe hydrophobic interface, also referred to as thedimer–dimer interface. At both types of interfacesthere are residues with amide groups engaged inintermolecular hydrogen bonding which can beused as 2H– 1H exchange probes to study the sub-unit interface 2H– 1H exchange behavior.6 Themeasured protection factors for these residues aresummarized in Table 4.
Discussion
Conservative mutations are unlikely to alterstructure dramatically as demonstrated by theplethora of almost identical lysozyme variantstructures.21 X-ray crystallography reveals that thestructures of FAP and suppressor TTR variants arevery similar to that of WT-TTR.10 In contrast, thevariants differ dramatically in their amyloidogeni-city both in vitro and in vivo, and in their hydrogenexchange behavior as well. In the study describedhere, 2H– 1H exchange is employed on a residue-specific basis to analyze the capability of individ-ual amides to participate in 2H–1H exchange. Theexperiments were carried out on TTR sequencesdiffering by only one amino acid residue from thewild-type. 2H– 1H exchange was carried out underphysiological conditions in which fibril formationis very inefficient. In fact in samples from theseconditions only a very small amount of protofila-ment can be detected, and only by EM. The proto-filements presumably arise from the normaltetramer to folded monomer to misfolded mono-mer equilibrium. Thus, the measured protectionfactors do not provide a direct probe of the struc-tural changes involved in the process of fibril for-mation, but rather provide residue-specificinformation on the regions of each TTR variantthat undergo transient unfolding, leading to2H– 1H exchange. Both quaternary and tertiarystructure unraveling can affect the exchange kin-etics. The protection factors reflect the likelihoodthat a particular region in a given variant of TTRundergoes conformational fluctuations. The lack
Figure 3. Intensity versus time plots of strategicallychosen residues in TTR variants undergoing 2H–Hexchange. The observed intensities are normalized tothe average intensities of selected amides indicated inFigure 2. The time courses are fit to a single exponentialusing the program KaleideGraph.
† Available at www.fccc.edu/research/labs/roder/sphere
of detailed information on the folding kinetics ofTTR prevents a direct thermodynamic interpret-ation of the changes in exchange rates. However,it is unclear in fact whether formation of amyloidis limited by the unfolding equilibrium, or the kin-etics. Recent data have shown that amyloidogenicTTR sequence variants do have altered kinetics oftetramer dissociation that correlates with acceler-ated amyloidosis. As such, data on both kineticsand thermodynamics of the process of TTR unfold-ing are of interest. In the subsequent discussion theterm destabilization will be used to reflect changesdetected in hydrogen exchange without being ableto specify whether this is the traditional thermo-dynamic (equilibrium constant) stability, or akinetic (opening rate) stability.
The results of the native state hydrogenexchange studies of TTR variants are summarizedin Figure 5, using ribbon diagrams and schematicrepresentations, color-coded to reflect the observedprotection factors. There are several features thatare revealed by comparisons of the exchange pat-terns that are interesting.
Inspection of the ribbon diagram representationdiscloses that the overall structures of the subunitsin three variants are very similar, as reportedpreviously.10 Minor differences are exhibited bythe L55P structure, wherein residues 54–55 adopta coil conformation instead of b-strand confor-mation. In addition the helix is a half-a-turnshorter. These are rather localized changes whosedirect relevance to amyloidogenicity is doubtful.
Table 1. Measured backbone amide 2H–H exchange rates and protection factors in T119M-TTR
a The amide signals are exchange-broadened at pH 7, exchange rates cannot be determined accurately due to irregular lineshapeand low signal intensities. We observed that the broadening increases following increasing the pH between pH 6 and 7, whichsuggests that the broadening is most likely to be caused by fast solvent exchange.
b Residues are marginally protected, i.e. completely exchanged between 2 and 24 hours. Due to insufficient time points at the tran-sition-region of the exponential slope, the exchange rates are only estimated.
c The exchange rates cannot be measured accurately due to the low signal intensities.d Signal overlapped with other residues.
826 2H-H Exchange of TTR Variants
On a residue-specific level, the hydrogen bond net-works are identical (except for residues 54 and 55in L55P TTR) in the two b-sheet regions of thefour TTR sequences under comparison (Figure5(b)). The subunit interfaces are also very similar.Comparisons of the total numbers of residueswith protection factors over 105, however, revealthat residues in L55P tend to be much more likelyto participate in amide exchange with the solventas a result of a structured-to-unstructured tran-sition than the rest of the TTR variants. Only 27residues in L55P exhibit protection factors exceed-ing 105 as opposed to 40, 43 and 41 residues inV30M, WT and T119M, respectively, (Figure 4).The structural plasticity reflected in the amideexchange rates of L55P correlates with the clinicalobservation that it is one of the most aggressivedisease-causing variants. V30M, also a disease-causing variant, is more similar to T119M and WTin terms of the total numbers of protected amides.The question then is how the single site substi-tutions affect TTR at the quaternary and tertiarystructural levels.
Examination of Table 4 reveals that T119M andV30M exhibit similar protection factors, 105–106, atthe two unique quaternary structural interfaces,the hydrophobic interfaces and the hydrogen-bonded interfaces. It will be interesting to deter-mine whether this similarity is also reflected in thesubunit exchange rates of these two variantsunder the same conditions, which can be measuredusing a ion exchange chromatography methoddeveloped recently.4 In L55P, most of the residuesat both interfaces are either unprotected, or theprotection factors cannot be measured due tobroad amide signals, which also indicate enhancedmobility. T4 (thyroxine) binding has been shown tostabilize the quaternary structures in TTR, anddifferences in T4 binding affinity have been demon-strated in TTR variants.22 The 2H–1H exchangeexperiments described here were carried out inthe absence of thyroxine (T4), which is physiologi-cally relevant owing to the fact that only 10–15%of the T4 binding capacity is utilized in plasma.2H– 1H exchange studies done on apo-TTR demon-strate that the difference in stability is intrinsic
Table 2. Measured backbone amide 2H–H exchange rates and protection factors in L55P-TTR
a The amide signals are exchange-broadened at pH 7, exchange rates cannot be determined accurately due to irregular lineshapeand low signal intensities. We observed that the broadening increases following increasing pH between pH 6 and 7, which suggeststhat the broadening is most likely to be caused by fast solvent exchange.
b The exchange rates cannot be measured accurately due to the low signal intensities.c Signal overlapped with other residues.d Residues are marginally protected, i.e. completely exchanged between 2 and 24 hours. Due to insufficient time points at the tran-
sition-region of the exponential slope, the exchange rates are only estimated.
2H-H Exchange of TTR Variants 827
rather than being caused by different T4 bindingaffinities in vivo. It is interesting that the L55Pmutation is away from residues directly involvedin the quaternary structural interactions, thus themechanism of destabilization is not obvious. ForWT-TTR at pH 5.75, the measured protection fac-tors are around 104, with the exception of Glu89,,106.6 This is generally lower than either V30M orT119M. A previous crystallographic study suggeststhat the wild-type structure determined at pHvalues as low as pH 5.3 is identical with structuresdetermined at pH 7.0, at least in the presence ofhigh salt.10 The same is probably true of the mutantproteins as well. It will be interesting to determine
whether the stability of the WT-TTR interfaceincreases when the pH is increased to pH 7.
At the tertiary structural level, the disease-caus-ing variants exhibit consistent destabilization rela-tive to the suppressor subunit T119M (Figures 4and 5). In T119M, there are seven residues exhibit-ing protection factors of 107 and 12 residues charac-terized by protection factors greater than 107. Forcomparison there are three residues in V30M andtwo residues in L55P exhibiting protection factorsof 107, and no residues characterized by protectionfactors exceeding 107 in V30M and L55P. InT119M, the majority of residues with protectionfactors over 107 are located in b-strands B and E,
Table 3. Measured backbone amide 2H–H exchange rates and protection factors in V30M-TTR
a The amide signals are exchange-broadened at pH 7, exchange rates cannot be determined accurately due to irregular lineshapeand low signal intensities. We observed that the broadening increases following increasing pH between pH 6 and 7, which suggeststhat the broadening is most likely to be caused by fast solvent exchange.
b Residues are marginally protected, i.e. completely exchanged between 2 and 24 hours. Due to insufficient time points at the tran-sition-region of the exponential slope, the exchange rates are only estimated.
c Signal overlapped with other residues.d The exchange rates cannot be measured accurately due to the low signal intensities.
828 2H-H Exchange of TTR Variants
which are part of the front sheet in which most ofthe disease-causing substitutions are found (Figure5(b)). The BCEF sheet is also the region thatbecomes disordered in the WT amyloidogenicintermediate formed at pH 4.5. In contrast, thesame residues are ten times less protected inV30M, and 100 times less protected in L55P thanin T119M. Given the observation that the protec-tion factors of residues at the subunit interfacesare similar for V30M and T119M, it seems that theV30M mutation destabilizes the front sheet, whichin turn also destabilizes Lys15, Ile107 and Leu111from the back sheet which are part of the stablecore in WT-TTR and the suppressor variant. Inaddition, Val20 and Gly22 from the b-turn betweenb-strands A and B also become more labile, whichmight be the result of reduced interaction betweenthe two sheets. For L55P, it is not clear whetherdestabilization of the tertiary structure is a direct
consequence of Pro substitution, or indirect conse-quence of increased subunit exchange,4 or both.The destabilization of the front sheet in the twodisease-associated variants is consistent with2H– 1H exchange studies carried under WT-TTR atamyloidogenic conditions (pH 4.5), in which desta-bilization of the front sheet was also observed.3
Interestingly, even though residue accessibilityto exchange varies from variant to variant, thesame group of residues (in b-strands A, B, E andG, and the b-turn between b-strands A and B)emerge as the most protected core. Similar to WT,the outside strands of both sheets in the TTR sub-unit (strands C and D) have many residues withlow protection factors, the exception being Glu42in the TTR variants. Since strand D is anchored toA with only three hydrogen bonds (two hydrogenbonds in L55P) its lability is not surprising. Thepattern observed for residues in strand C suggeststhat exchange for most residues occurs through anunzipping of the strand from the C-terminal end.Beyond that, the slowly exchanging E42 seems toserve as an anchor point for the N-terminal part ofthe strand.
Calorimetric unfolding data have been reportedfor the same set of sequence variants studiedhere23. In their work the WT and T119M varianthad indistinguishable stability, but L55P andV30M were found to be less stable by 1.5 and2.2 kcal mol21, respectively. This order of stabilitycontrasts with the changes in hydrogen exchange,in which L55P is found to be much more labilethan V30M. L55P is also more prone to formationof amyloid both in vitro and in vivo. Since thecalorimetric measurements are done with fairly
Figure 4. Bar graph represen-tation of the number of residuesseparated into three groups basedon measured protection factors forWT, T119M, V30M and L55P. The2H–1H exchange for WT was car-ried out at pH 5.75, whereas thethree variants were evaluated atpH 7.0.
Table 4. Measured protection factors for residues at thesubunit interfaces
Monomer–monomer interfaceDimer–dimer
interface
E89 Y116 T118 A120 Y114 V122
WT 106 – ,104 ? ,104 ,104
T119M – – 106 106 105 106
V30M – – ? 106 106 105
L55P – – ? ? 105 –
–, Completely exchanged after two hours. ?, The protectionfactors cannot be measured due to broadened signals.
2H-H Exchange of TTR Variants 829
rapid temperature scans, there is some concernthat they may not be equilibrium measurements,reversibility of the transitions was not mentioned.However, at the high unfolding temperatures (93–102 8C) the kinetics may be rapid, and it may bethat the kinetic rather than thermodynamic stab-ility of TTR variants is more relevant to under-standing amyloid formation. Furthermeasurements are required to address this issue,the better correlation of hydrogen exchange differ-ences with amyloidogenicity suggests both areworth following.
We have also mapped the chemical shift changesrelative to the WT-TTR in the three variants, andfound the pattern of residues exhibiting significantchemical shift changes are the same as for the resi-dues exhibiting differences in protection factors(Figure 1). This indicates that the regions of thestructure in which the stability or flexibility isaltered can be detected by chemical shift changes,even though the diffraction studies indicate thatthe structural changes are quite small.
In conclusion, the native state 2H–1H exchangestudy for the three TTR variants probes the influ-ence of single site substitutions on the stability/flexibility at both the quaternary and tertiary struc-tural levels. In particular, the destabilization of theprotein core, which is comprised of strands C, B, Eand G (Figure 5), in V30M and L55P compared toT119M is consistent with both previous 2H– 1Hexchange studies and supports the idea set forthpreviously that destabilization of the folded statecontributes to the amyloidogenicity of TTR17.There are .85 sequence variants known for TTR.Most amino acid changes lead to increased amyloi-dogenicity and pathogenesis in people producingsuch proteins. Sites of pathogenic changes are dis-tributed throughout the molecule, with a particu-
larly high frequency in the front sheet (strandsCBEF). However, there are also pathogenicmutations in the regions of the molecule that arenot part of the core, and are labile as judged byhydrogen exchange. It is possible that those resi-dues affect different processes important for TTRfibril formation. Further studies on the subunitexchange behavior of FAP-associated variants com-pared to nonpathogenic mutations may helpexplain the differences in structural fluctuationsbetween the variants, and shed light on the differ-ences in their amyloidogenicity in vivo.
Materials and Methods
Per-deuteration of TTR amide groups
2H and 15N doubly labeled TTR was produced for thedifferent variants by growing Escherichia coli in 2H2Oand 15NH4Cl minimal media, using the same expressionsystem described previously.24 During protein purifi-cation, the less protected amide deuterons are replacedby protons from the solvent, but in order to be sure thatall amides are fully deuterated, the lyophilized TTR wasdissolved in 2H2O at 0.25 mg ml21and the solution wasadjusted to p2H 2.0 with 2HCl. At very low pH TTR isin a molten globule state which allows for relativelyrapid complete exchange of amide hydrogen atomswith the solvent. The TTR variants were kept undersuch conditions for more than 12 hours at room tempera-ture to assure complete replacement of amide hydrogenatoms by deuterium. The protein solutions were thendiluted fivefold in 2H2O, and then neutralized (p2H 7.0)with NaO2H to initiate refolding. Dilution of the solutionwas needed to reduce protein aggregation during therefolding process. The solutions after refolding, whichcontain renatured TTR, were centrifuged and filtered toremove any aggregated material before being used inhydrogen exchange experiments.
Figure 5 (legend opposite)
830 2H-H Exchange of TTR Variants
Deuterium–proton exchange at pH 7 at highTTR concentration
Samples of 2H and 15N doubly labeled TTR were keptat 20 mg ml21 in 2H2O at pH 7 until use in exchangeexperiments. The proton–deuterium exchange processwas initiated by diluting labeled protein in 2H2O tenfoldinto 1H2O buffer at pH 7, containing 50 mM phosphateand 100 mM NaCl. The solution of protein was concen-trated in MiniKros and Centricon systems startingimmediately after dilution, followed by NMR exper-iments, which were started about two hours after theinitiation of exchange. The NMR samples were thenkept at room temperature, and HSQC spectra (with caone hour acquisition times) were collected at 40 8Cperiodically thereafter.
NMR spectroscopy
We used 2H, 15N and 13C triply labeled TTR variantsamples to record HNCA spectra for assignments.18 All3D spectra were collected on a Bruker DRX-500 spec-trometer at 40 8C, and used deuterium decoupling.Samples were about 20 mg ml21 TTR (,1.5 mM mono-mer concentration), maintained at pH 6 with 50 mMK3PO4 and 100 mM NaCl.
Supporting information available
The backbone assignments for WT, L55P, V30M andT119M TTR are available free of charge via the Internet†.
Acknowledgments
This work was supported in part by NIH grantsAG10770 (to D.E.W.) and DK46335 (to J.W.K).
Figure 5. (a) Ribbon diagram representation of the X-ray crystal structures of the TTR subunits using the pro-gram MOLMOL. The eight b-strands are labeled byletters A–H and hydrogen bonds involving backboneamide groups are depicted by blue bars for L55P, V30M,and T119M. (b) Schematic representations of exchangerates are depicted. Arrows are used to indicate hydrogenbonds, pointing from the hydrogen bond donor to thehydrogen bond acceptor. The color code for the residuesis the same as for (a), the color representing themeasured protection factor: navy blue, protection factors.107; blue, ,107; violet, ,106; orange red, ,105; orange,,104, yellow, fully exchanged within two hours (protec-tion factor not determined); gray, the protection factorscannot be measured due to lack of probes. The H strandfrom a neighboring subunit that is hydrogen bonded tothe monomer is also shown.
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Edited by C. R. Matthews
(Received 28 November 2001; received in revised form 7 May 2002; accepted 8 May 2002)
