BIOPHYSICS
`
`The V122I cardiomyopathy variant of transthyretin
`increases the velocity of rate-limiting
`tetramer dissociation, resulting in
`accelerated amyloidosis
`
`Xin Jiang*, Joel N. Buxbaum†, and Jeffery W. Kelly*‡
`
`*Department of Chemistry and The Skaggs Institute of Chemical Biology, and †Department of Molecular and Experimental Medicine,
`The Scripps Research Institute, 10550 North Torrey Pines Road, BCC506, La Jolla, CA 92037
`
`Edited by Robert L. Baldwin, Stanford University Medical Center, Stanford, CA, and approved October 16, 2001 (received for review August 9, 2001)
`
`The transthyretin (TTR) amyloid diseases are of keen interest,
`because there are >80 mutations that cause, and a few muta-
`tions that suppress, disease. The V122I variant is the most
`common amyloidogenic mutation worldwide, producing famil-
`ial amyloidotic cardiomyopathy primarily in individuals of Afri-
`can descent. The substitution shifts the tetramer-folded mono-
`mer equilibrium toward monomer (lowers tetramer stability)
`and lowers the kinetic barrier associated with rate-limiting
`tetramer dissociation (pH 7; relative to wild-type TTR) required
`for amyloid fibril formation. Fibril formation is also accelerated
`because the folded monomer resulting from the tetramer-folded
`monomer equilibrium rapidly undergoes partial denaturation
`and self-assembles into amyloid (in vitro) when subjected to a
`mild denaturation stress (e.g., pH 4.8).
`Incorporation of the
`V122I mutation into a folded monomeric variant of transthyretin
`reveals that this mutation does not destabilize the tertiary
`structure or alter the rate of amyloidogenesis relative to the
`wild-type monomer. The increase in the velocity of rate-limiting
`tetramer dissociation coupled with the lowered tetramer sta-
`bility (increasing the mol fraction of folded monomer present at
`equilibrium) may explain why V122I confers an apparent abso-
`lute anatomic risk for cardiac amyloid deposition.
`
`The amyloidoses are a large group of protein misfolding
`
`diseases that are accelerated by certain point mutations and
`suppressed by others (1–10). Familial amyloidotic cardiomyop-
`athy (FAC) does not result from loss of transthyretin (TTR)
`function (because of insolubility);
`instead,
`it appears to be
`caused by tissue-selective TTR amyloid deposition in the heart
`(11–14). Wild-type (WT) TTR can also deposit as fibrils in the
`cardiovascular system in the late-onset disease senile systemic
`amyloidosis, affecting as much as 25% of the population over the
`age of 80 (15). In addition, there are ⬎80 TTR variants
`associated with early onset amyloid diseases,
`including the
`V30M variant, which preferentially deposits in the peripheral
`nervous system (16–18).
`TTR is a 55-kDa homotetrameric protein, comprised of
`127-residue ␤-sheet rich subunits, which is present in the cerebral
`spinal fluid and serum. In blood, TTR serves as the secondary
`thyroxine carrier protein (by using ⬇10% of its capacity) and
`transports retinol via binding ⱕ1 equivalent of holo retinol-
`binding protein (RBP) (19). It appears that all of the TTR
`deposited as amyloid is derived from plasma. Rate-limiting
`tetramer dissociation of TTR into monomers is necessary but not
`sufficient for TTR fibril formation, as tertiary structural changes
`within the monomer are also required (20–23).
`We created a TTR variant (WT M-TTR), which proved to be
`monomeric, normally folded, and nonamyloidogenic under phys-
`iological conditions, through the introduction of a mutation in
`each of the quaternary structural interfaces (F87M兾L110M)
`(20). A denaturing stress (such as acidic pH) induces a confor-
`
`mational change in WT M-TTR facilitating its self-assembly into
`amyloid fibrils at a rate ⬎100 times faster than the tetrameric
`WT protein because of the rate-limiting dissociation of the WT
`tetramer required for fibril formation (20).
`It is estimated that ⬇4% of African Americans (1.3 million
`people) are heterozygous for the V122I allele (12). Although
`the age of onset (typically ⬎60 years of age) is similar for senile
`systemic amyloidosis (WT) and FAC (V122I) patients, the
`latter are much more likely to suffer cardiac failure (especially
`in the case of V122I homozygotes) (12, 13, 24). Here we
`compare the V122I homotetramer to the WT TTR homotet-
`ramer using a biophysical approach in an attempt to define the
`mechanism of the amyloidogenicity associated with the pa-
`thology of FAC. The monomeric WT and V122I M-TTR
`variants were also compared focusing on the influence of the
`V122I cardiac mutation on tertiary structural stability and
`amyloidogenicity. Here we show that the V122I FAC variant
`destabilizes the TTR tetramer and lowers the kinetic barrier
`associated with tetramer dissociation, resulting in a greater
`extent and faster rate of folded monomer formation, a struc-
`ture that rapidly undergoes partial denaturation and self-
`assembles into amyloid fibrils (in vitro).
`
`Materials and Methods
`Protein Expression and Purification. Recombinant WT, V122I,
`F87M兾L110M (WT M-TTR), and V122I兾F87M兾L110M (V122I
`M-TTR) TTR proteins were expressed and purified as described
`previously (25). All proteins were further purified by using gel
`filtration chromatography on a Superdex-75 column (Amersham
`Pharmacia). Protein concentrations were determined by mea-
`suring UV absorbance at 280 nm, by using an extinction coef-
`ficient of 7.76 ⫻ 104 M⫺1䡠cm⫺1. The pH 7 buffer used contains
`50 mM sodium phosphate, 100 mM KCl, and 1 mM EDTA.
`Fibril Formation Assay. TTR (0.4 mg兾ml) in 10 mM phosphate
`buffer with 100 mM KCl (pH 7) was diluted 1:1 with 200 mM
`buffer (100 mM KCl and 1 mM EDTA) to jump to the desired
`pH (sodium citrate for pH 3.2, sodium acetate for pH 3.6–5.2,
`and phosphate buffer for pH ⬎5.2). The solutions subjected to
`a denaturation stress were incubated at 37°C for 72 h, after which
`the suspensions were vortexed and optical density measured at
`
`This paper was submitted directly (Track II) to the PNAS office.
`
`Abbreviations: FAC, familial amyloidotic cardiomyopathy; TTR, transthyretin; WT M-TTR,
`F87M兾L110M variant; ThT, thioflavin T; V122I M-TTR, V122I兾F87M兾L110M variant; WT, wild
`type; RBP, retinol-binding protein.
`
`See commentary on page 14757.
`‡To whom reprint requests should be addressed. E-mail: jkelly@scripps.edu.
`
`The publication costs of this article were defrayed in part by page charge payment. This
`article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
`§1734 solely to indicate this fact.
`
`www.pnas.org兾cgi兾doi兾10.1073兾pnas.261419998
`
`PNAS 兩 December 18, 2001 兩 vol. 98 兩 no. 26 兩 14943–14948
`
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`400 nm. For time course experiments, a 0.3- to 0.4-mg兾ml TTR
`sample was quickly diluted 1:1 with pH 4.4 buffer while moni-
`toring turbidity at 400 nm (37°C) by using a UV spectrometer
`equipped with a Peltier temperature control unit.
`Fibril formation was also assessed by thioflavin T (ThT)
`binding, where a 25-␮l aliquot of sample (vortexed to achieve
`homogeneity) was mixed with 173 ␮l of 50 mM Tris buffer (100
`mM KCl, pH 8.0) and 2 ␮l of ThT stock solution (1 mM) in 10
`mM phosphate (pH 7.4). The mixed sample was then excited at
`440 nm, and emission at 482 nm was recorded.
`
`Gel Assay-Monitored Quaternary Structure Changes. Samples pre-
`pared identically to those (containing 0.2 mg兾ml of TTR) used
`for the fibril formation assay were incubated at 25°C (instead of
`37°C) for 40 h to evaluate the extent of pH-induced tetramer
`dissociation. A 1,200 ␮l sample at each pH was then mixed with
`24 ␮l of a 25 mg兾ml zwitterionic detergent (Z 3–14) stock
`solution, which was immediately combined with 350 ␮l of a 1 M
`sodium phosphate buffer (pH 7) solution containing 0.5 mg兾ml
`of Z 3–14 to neutralize the pH for gel
`loading. [We have
`previously established that the concentration of Z 3–14 used
`does not allow reconstitution of TTR monomers to tetramers or
`dissociate the tetramer present in the mixture (26).] Ten micro-
`liters of the mixture was mixed with the same volume of 4%
`nonreducing SDS gel-loading buffer and, without boiling, the
`samples were loaded on a 12% SDS acrylamide gel. The gel was
`stained with Coomassie staining reagent (Pierce) and, after
`drying, the lanes were analyzed by densitometry and quantified
`using the program SCION IMAGE.
`
`Trp Fluorescence Monitored Tertiary Structural Changes. Urea de-
`naturation studies were carried out by diluting a TTR stock
`solution (0.4 mg兾ml) with varying concentrations of chaotrope
`at pH 7, obtaining a final protein concentration of 0.02 mg兾ml.
`Samples were incubated at room temperature for 24 and 96 h
`before fluorescence measurements were made. Refolding sam-
`ples were prepared by diluting 0.4 mg兾ml of TTR denatured in
`8 M urea (incubated at 4°C for 96 h) into 50 mM phosphate
`buffer (100 mM KCl, pH 7), obtaining a final protein concen-
`tration of 0.02 mg兾ml. Refolded samples were incubated at room
`temperature for 24 h before fluorescence measurements were
`made. Concentrations of the urea stock solutions were deter-
`mined by refractive index measurements (27). Tryptophan flu-
`orescence was used to monitor TTR tertiary structural changes.
`Samples (25°C) were excited at 295 nm and the emission
`measured from 310 to 410 nm on an Aviv Model ATF105
`spectrofluorometer (Aviv Associates, Lakewood, NJ). Fraction
`unfolded was calculated by using the F355兾F335 value at each
`protein concentration, knowing the folded minimum (F355兾F335
`⫽ 0.81) and the denatured maximum (F355兾F335 ⫽ 1.35) fluo-
`rescence ratios, assuming a linear dependence.
`
`Rate of Tetramer Dissociation Measured by Linking Tertiary Structural
`Changes. The evaluation of tetramer dissociation rates was
`carried out by removing 200-␮l aliquots from a denaturing
`TTR (0.02 mg兾ml) sample (10 ml) in 4.5 M urea [50 mM
`phosphate buffer (100 mM KCl, pH 7, 25°C)]. The Trp
`fluorescence emission ratio (F355兾F335) as a function of time
`(25°C) was measured.
`
`Results
`The V122I Homotetramer Dissociates to Folded Monomers 3-Fold
`Faster than WT Homotetramers. Mounting evidence suggests that the
`intact TTR tetramer is not directly amenable to denaturation by
`urea (20, 28). Instead, the tetramer has to dissociate to folded
`monomers before it can be denatured by urea. The slow dissociation
`rate of the native tetramer explains the slow approach to equilib-
`rium observed during urea denaturation (Fig. 1A), which is slower
`
`(A) Urea induced denaturation of V122I (circles) and WT (triangles)
`Fig. 1.
`TTR, after incubation for 24 (open symbols) and 96 h (closed symbols), as
`detected by Trp fluorescence. Lines through the V122I (solid) and WT (dashed)
`data are smoothing curves to guide the eye. The lower value of WT plateau (at
`⬎3 M urea, 96 h) indicates this protein has not completely reached equilib-
`rium. (B) Urea denaturation curves of monomeric V122I M-TTR (F) and WT
`M-TTR (Œ) (20) (0.02 mg兾ml 24 h incubation). That tetrameric V122I TTR (E) (96
`h incubation) denaturation occurs via the monomer is demonstrated by the
`indistinguishable denaturation curves (F and E, within error). The solid line
`through the V122I M-TTR data are fitted to a two-state model yielding a ⌬GH2O
`value of 4.7 ⫾ 0.2 kcal䡠mol⫺1 and m value of 1.4 ⫾ 0.1 kcal䡠mol⫺1䡠M⫺1. (C) The
`rate of urea- (4.5 M) induced V122I (E) and WT (F) TTR tetramer dissociation
`detected by very fast linked tertiary structural changes. Solid lines are fitted to
`a first order single exponential function.
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`BIOPHYSICS
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`(A) Fibril formation of V122I TTR (0.2 mg兾ml) monitored by turbidity
`Fig. 2.
`(dark gray) and ThT binding (light gray) as a function of pH (37°C, 72 h). Fibril
`formation from WT TTR is shown by white bars, monitored by turbidity as a
`function of pH (37°C, 72 h). The relative amount of V122I fibril formation
`observed for the pH 4.2 sample was assigned to be unity (monitored by either
`ThT fluorescence or turbidity). The WT fibril yield is less than V122I at all pHs.
`(B) The rate of fibril formation at pH 4.2 (37°C) for V122I (F) and WT (E) TTR
`(0.2 mg兾ml) ascertained by turbidity at 400 nm. (C) Kinetics of fibril formation
`from monomeric V122I M-TTR (F) and WT M-TTR (E) at 37°C (0.15 mg兾ml, pH
`4.4). The lines through the data points are smoothing curves to guide the eye.
`
`for WT than V122I TTR homotetramers. The tryptophan fluores-
`cence senses tertiary structural changes, not quaternary structural
`changes, on the basis of the fact that the tetramer and recently
`introduced folded monomeric version of TTR (WT M-TTR) have
`indistinguishable fluorescence spectra (20).
`The identical midpoints (Cm) of the urea denaturation
`curves exhibited by WT and V122I TTR (0.02 mg兾ml; 25°C)
`reflect identical tertiary structural stabilities (Fig. 1 A). This
`interpretation is verified by comparing the denaturation curves
`of WT and V122I M-TTR monomers, which are identical in
`terms of Cm and amplitude (Fig. 1B) (20). Moreover, the Cms
`exhibited by WT and V122I TTR (Fig. 1 A) are identical to the
`Cms displayed by the monomeric versions of these two se-
`quences (Fig. 1B), consistent with the fact that tertiary struc-
`tural changes are being monitored. The differences in ampli-
`tude as a function of time for the data displayed in Fig. 1 A
`reflect the different rates of tetramer dissociation monitored
`by linked tertiary structural changes of WT and V122I TTR.
`A slow approach to equilibrium is not observed for the M-TTR
`constructs (Fig. 1B), because the monomers denature on the
`millisecond timescale (20). The rate of dissociation of the WT
`and V122I homotetramers can be monitored by coupling the
`slow (hour timescale) quaternary structural changes (not
`detectable by fluorescence) to the fast (millisecond timescale)
`tertiary structural changes monitored by Trp fluorescence,
`provided the urea concentration used is in the posttransition
`region for tertiary structural changes (4.5 M urea). Under
`these conditions, the monomeric subunits resulting from tet-
`ramer dissociation unfold in a few milliseconds and remain
`unfolded, allowing the rate of tetramer dissociation to be
`measured (Fig. 1C). The WT tetramer exhibits a dissociation
`half-life of ⬇37.7 h, which is 3-fold longer than that exhibited
`by V122I TTR (t1/2 ⬇11.5 h). These half-lives are referred to
`as approximate, because there appears to be a minor slower
`phase (⬍10%) that interferes with a perfect fit of the data to
`a single exponential function, possibly derived from an anion
`stabilized tetramer (28). Nevertheless, it is clear the V122I
`cardiac mutation increases the velocity of the rate-determining
`step for amyloidosis 3-fold relative to WT TTR at 4.5 M urea.
`The dependence of the rates of WT and V122I tetramer
`dissociation on urea concentration is nearly identical, as
`revealed by identical slopes in a ln kdissociation vs. urea concen-
`tration plot (P. Hammarstro¨m, X.J., and J.W.K., unpublished
`data).
`
`The V122I Homotetramer Dissociates to the Amyloidogenic Interme-
`diate Faster than WT TTR Under a Mild Acid Denaturation Stress. WT
`TTR is converted into amyloid by pH-mediated tetramer disso-
`ciation linked to tertiary structural changes, resulting in the
`formation of a so-called monomeric amyloidogenic intermediate
`that self-assembles into amyloid fibrils (20–23). The V122I FAC
`variant forms fibrils over a pH range very similar to WT TTR
`(Fig. 2A). The yield of pH-induced fibril formation from the
`V122I homotetramer is higher than that of WT TTR at all pH
`values tested (pH 3.2–6.8; Fig. 2 A). The ionic strength in these
`experiments is high enough (⬎0.2 M) that significant changes in
`amyloidogenicity are not expected because of ionic strength
`increases over the pH range of 3.6–5.2 (acetate buffer, ionic
`strength increase ⬇0.09 M). At the optimal denaturation stress
`for maximal V122I fibril formation (pH 4.2), V122I forms ⱖ20%
`more fibrils than WT, as judged by turbidity and 1.7-fold more,
`as judged by ThT binding (see Fig. 5, which is published as
`supporting information on the PNAS web site, www.pnas.org).
`Moreover, the extent of V122I fibril formation is ⱖ20% that
`afforded by WT TTR (pH 4.2) at all time points after the initial
`period up to and beyond the half-life of TTR in plasma (8–18 h;
`Fig. 2B; ref. 29). The FAC variant formed amyloid fibrils ⬇2-fold
`faster than WT TTR at pH 4.2, as discerned by turbidity data
`
`Jiang et al.
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`PNAS 兩 December 18, 2001 兩 vol. 98 兩 no. 26 兩 14945
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`(A) SDS兾PAGE analysis of TTR quaternary structure as a function of pH. (Upper) V122I TTR; (Lower) WT TTR. (B) Mol fraction of monomer as a function
`Fig. 3.
`of pH calculated by using densitometry from the gels shown in A. A smoothing curve applied to the V122I (F) and WT (E) data guides the eye. (C) Tetramer stability
`as a function of TTR concentration for V122I (F) and WT (E) in 4 M urea. Samples were incubated for 96 h at 25°C before analysis.
`
`(Fig. 2B). The 2-fold faster rate of V122I TTR fibril formation
`from the homotetramer relative to WT implies that the V122I
`tetramer is less stable than the WT tetramer (see below). That
`tetramer dissociation is rate limiting for TTR amyloidosis was
`demonstrated by comparing the time courses of amyloid fibril
`formation from monomeric WT and V122I M-TTR (Fig. 2C),
`which are identical and 50-fold faster than amyloid formation
`from the V122I homotetramer (Fig. 2B). The pH-dependent
`yield of fibrils from monomeric and tetrameric V122I TTR was
`very similar at long incubation times (see Fig. 6, which is
`published as supporting information on the PNAS web site),
`consistent with the idea that in both cases a misfolded amyloi-
`dogenic monomer is the direct precursor to amyloid.
`
`The V122I Tetramer Is Destabilized Relative to WT. The hypothesis
`that the V122I mutation alters tetramer stability was probed by
`comparative pH-dependent tetramer–monomer equilibrium
`measurements and concentration-dependent equilibrium con-
`stant measurements at a fixed chaotrope concentration. We
`followed the acid-induced quaternary structure changes as a
`function of pH by using an SDS兾PAGE method previously
`validated by analytical ultracentrifugation (23, 26). After TTR
`was incubated at the desired pH, the zwitterionic detergent
`Z3–14 was added before neutralizing the protein solution
`to prevent refolding while preserving the TTR quaternary
`structure (Fig. 3A). The FAC variant dissociates to the mo-
`nomeric amyloidogenic intermediate over nearly the same
`pH range as the WT protein (Fig. 3B; pHm 4.4), the major
`difference being that V122I TTR (0.2 mg兾ml) is about
`10% monomeric at neutral pH, whereas dissociated WT
`
`monomers are nearly undetectable (Fig. 3 A and B). The extent
`of V122I monomer formation at a given pH is increased by
`10–20% relative to WT TTR (Fig. 3B). These data imply that
`the native V122I tetramer is destabilized by ⬍1 kcal兾mol
`
`[⌬⌬G ⫽ ⫺RT ln(KeqWT兾Keq
`V1221] relative to the WT tetramer
`under these conditions.
`It is possible that in vivo the presence of one equivalent of RBP
`complexed to TTR might eliminate the differences in the
`tetramer-folded monomer equilibrium observed in Fig. 3 A and
`B because of tetramer binding (300 nM Kd for WT) and
`stabilization (30). However, the gel assay still identifies ⬇5–10%
`monomeric V122I at neutral pH (see Fig. 7, which is published
`as supporting information on the PNAS web site) in the presence
`of holo RBP, consistent with rapid on兾off rates of RBP binding,
`validating the physiologic relevance of this study.
`The mol fraction of TTR tetramer observed in solution under
`denaturing conditions (4 M urea; 75% denatured at 0.02 mg兾ml,
`96 h, 25°C) should be concentration dependent. The dissociation
`constant (Kd) for a tetrameric protein ⫽ 256䡠C3䡠(1 ⫺ ␣)4兾␣,
`where ␣ is the percentage of tetrameric protein at a given
`denaturant concentration, and C is the protein concentration. A
`comparison of the protein concentration dependence of the
`dissociation constants characterizing the V122I and WT TTR
`tetramer-unfolded monomer equilibrium is a direct reflection of
`the stability of the tetramers. This experiment is not complicated
`by monomer stability differences, because V122I and WT M-
`TTR have identical stabilities (Figs. 1B and 2C). The WT protein
`displayed a dramatic increase in the mol fraction of tetramer
`observed over the concentration range of 0.1–0.7 mg兾ml (96-h
`incubation, 25°C; Fig. 3C), whereas the fraction of tetramer
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`On the basis of estimates of the free energy difference
`between WT and V122I TTR (⬇0.5 kcal兾mol), one would expect
`less than a 5-fold increase in the rate of dissociation for the V122I
`tetramer, consistent with the 3-fold increase observed. The
`3-fold increase in tetramer dissociation rate translates into a
`2-fold increase in the rate of V122I TTR fibril formation. The
`V122I mutation has no detectable effect on the stability of the
`TTR tertiary structure nor does it alter the amyloid fibril
`formation rate from the engineered monomeric version of TTR
`(identical in both respects to WT M-TTR). The V122I mutation
`selectively acts by lowering the stability of the tetramer (Fig. 3)
`and consequently lowering the kinetic barrier for tetramer
`dissociation compared with WT TTR (Figs. 1C and 2B). The
`5–10% folded monomeric TTR in equilibrium with the V122I
`tetramer is a significant risk factor for FAC, because the folded
`monomer forms amyloid on the time scale of minutes (Fig. 2C)
`(20), whereas the WT tetramer has to dissociate first, which
`occurs on a tens of hours time scale (Fig. 2B). In contrast,
`monomeric WT TTR is hard to detect at neutral pH (Fig. 3A).
`The physiological relevance of the lowered ability of the V122I
`tetramer to be stabilized by anions relative to WT is unclear,
`although this could play a role in FAC by making a greater
`fraction of the tetramer amenable to dissociation and amyloid
`formation.
`Of the ⬎80 disease-associated TTR variants known, less
`than 10 have been studied by biophysical methods thus far (14,
`23, 26, 33). From emerging and published data, the majority of
`the pathogenic mutations destabilize the tetramer and lower
`the kinetic barrier for tetramer dissociation required for
`amyloidosis (26). The two mutations characterized to date that
`suppress the onset of amyloid disease have the opposite
`influence on kinetics and thermodynamics (26, 32, 33). We
`predict that the uncharacterized variants will also fall into
`these categories. What is not yet clear because of lack of data
`is whether the disease-associated mutations will generally alter
`tertiary structural stability in addition to quaternary structural
`stability (14). It is interesting that the V122I FAC mutation
`does not alter the tertiary structural stability relative to WT
`TTR. Ongoing studies should clarify this issue. Although there
`are other examples of mutations that appear to alter quater-
`nary structure stability without altering tertiary structure
`stability, rarely have the isolated tertiary structures been
`studied carefully (34–37). In addition to familial TTR amyloid
`diseases, familial amyotrophic lateral sclerosis and several
`cancers appear to be associated with the quaternary structural
`destabilization (misfolding) of superoxide dismutase and P53
`tumor suppressor, respectively (38–42).
`In retrospect, the V122I selective destabilization of the
`tetramer is not surprising, as this mutation is in the ␤-sheet
`mediated quaternary structural interface. Ile-122 is located on
`the periphery of the H strand, which makes an antiparallel
`interaction with strand H⬘ of another monomer,
`␤-sheet
`stabilizing the dimer interface. The side chain of Ile-122 packs
`against the side chains of Phe-87⬘ and Tyr-114⬘ of the neigh-
`boring subunit. The packing between the Ile-122 and Tyr-114⬘
`side chains is slightly altered relative to the Val-122兾Tyr-114⬘
`interaction. The subtle movement of the Tyr-114⬘ side chain in
`the V122I homotetramer alters its interactions with the AB
`loop of a second dimer at the face-to-face dimer–dimer
`interface, rationalizing the observed tetramer destabilization
`(43). A previously described mutation in the AB loop (V20I)
`similarly destabilizes the TTR tetramer by altering the inter-
`action of this loop with Tyr-114⬘ (the AB–AB⬘ loop interaction
`is also changed), destabilizing the face-to-face dimer interface,
`a perturbation resulting in cardiomyopathy (14).
`There is strong genetic, medical, and biochemical evidence for
`the hypothesis that amyloid fibrils are the causative agent of TTR
`amyloid diseases, including FAC (32, 44–46). The V122I muta-
`
`Inhibition of WT (E) and V122I (F) fibril formation as a function of Cl⫺
`Fig. 4.
`ion concentration. At 0.6 M Cl⫺, 50% of WT fibrilization is inhibited, whereas
`V122I amyloidogenecity is reduced only by 10%.
`
`formed by V122I TTR over the same concentration range is
`dramatically reduced. An estimation of the difference in free
`energy between the WT and V122I tetramers (pH 7) at physi-
`ological protein concentration (0.25 mg兾ml) is ⬇0.5 kcal兾mol in
`4 M urea. Although we are close to equilibrium in the experi-
`ments shown in Fig. 3C, it could be that the slower dissociation
`rate of WT TTR has a contribution.
`We have recently shown that anion binding to the TTR
`tetramer stabilizes this quaternary structure (slowing tetramer
`dissociation dramatically) inhibiting amyloid fibril formation
`(28). Although it is not yet known whether there may be a
`physiologically relevant anion, we studied whether the WT and
`V122I tetramers were equally susceptible to anion stabilization
`by Cl⫺ ion. The WT protein is much more susceptible to
`concentration-dependent anion stabilization and inhibition of
`amyloid fibril formation than is the V122I cardiac variant (Fig.
`4), a feature that may contribute to the amyloidogenicity of
`V122I TTR.
`
`Discussion
`TTR is a very interesting protein in that tetramer dissociation
`[measured by subunit exchange (31) or the data in Fig. 1C] is
`quite slow, exhibiting a half life of hours to days. Pathogenic
`mutations like V122I that destabilize the TTR quaternary
`structure increase its rate of tetramer dissociation and amyloid-
`osis (Fig. 1C), whereas a mutation like T119M, which protects
`compound heterozygotes from disease by interallelic trans-
`suppression stabilizes the TTR tetramer dramatically, increasing
`the barrier for tetramer dissociation, almost completely inhib-
`iting dissociation (32). These observations imply that the tran-
`sition state for tetramer dissociation does not resemble the
`tetrameric ground state in structure or energy, because the
`mutations do not affect the transition state nearly as much as the
`ground state. Hence changes in stability translate into similar
`changes in activation barriers. Because the kinetic barrier(s)
`are already high for WT TTR, stabilizing mutations make
`them even higher, whereas destabilizing mutations lower the
`kinetic barrier(s). Thus both kinetics and thermodynamics
`have to be considered when explaining the influence of a given
`TTR mutation on amyloidogenicity.
`
`Jiang et al.
`
`PNAS 兩 December 18, 2001 兩 vol. 98 兩 no. 26 兩 14947
`
`Page 5
`
`

`

`tion causing familial amyloidotic cardiomyopathy shifts the
`tetramer-folded monomer equilibrium toward monomer (lowers
`tetramer stability) and lowers the kinetic barrier for tetramer
`dissociation, which increases the extent and rate of amyloid fibril
`formation relative to WT TTR. The increase in the velocity of
`rate-limiting tetramer dissociation required for amyloid fibril
`formation, coupled with the presence of folded monomer under
`physiological conditions, may explain why the V122I cardiac
`disease penetrance approaches 100%, whereas senile systemic
`amyloidosis, involving WT TTR amyloid deposition in the heart
`
`(similar age of onset), affects less than 25% of the population
`above age 80.
`
`We thank Kristina Berecic for help in the initial stages of this project,
`Joleen White (The Scripps Research Institute) for providing the RBP,
`and Dr. Per Hammarstro¨m for helpful discussions. We are grateful for
`primary financial support from the National Institutes of Health [R01
`DK46335 (J.K.) and R01 AG15916 (J.B.)] and secondary support from
`The Skaggs Institute of Chemical Biology and The Lita Annenberg
`Hazen Foundation.
`
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