FEBS Letters 586 (2012) 330–336

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Cataract-linked cD-crystallin mutants have weak affinity to lens chaperonesa-crystallins

Sanjay Mishra, Richard A. Stein, Hassane S. Mchaourab ⇑Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232, USA

a r t i c l e i n f o

Article history:Received 6 December 2011Revised 12 January 2012Accepted 13 January 2012Available online 28 January 2012

Edited by Gianni Cesareni

Keywords:cD-crystallinDenaturant unfoldingBimane fluorescenceaB-crystallin phosphorylationa-Crystallinb-CrystallinChaperoneSmall heat-shock proteinCataract

0014-5793/$36.00 � 2012 Federation of European Biodoi:10.1016/j.febslet.2012.01.019

Abbreviations: aB-D3, aB-crystallin S19D/S45Dhydrochloride; sHSP, small heat-shock proteins; T4L,⇑ Corresponding author.

E-mail address: hassane.mchaourab@vanderbilt.ed

a b s t r a c t

To test the hypothesis that a-crystallin chaperone activity plays a central role in maintenance of lenstransparency, we investigated its interactions with c-crystallin mutants that cause congenital cata-ract in mouse models. Although the two substitutions, I4F and V76D, stabilize a partially unfoldedcD-crystallin intermediate, their affinities to a-crystallin are marginal even at relatively high con-centrations. Detectable binding required further reduction of cD-crystallin stability which wasachieved by combining the two mutations. Our results demonstrate that mutants and possiblyage-damaged c-crystallin can escape quality control by lens chaperones rationalizing the observa-tion that they nucleate protein aggregation and lead to cataract.

Structured summary of protein interactions:gammaD Crystallin and alphaB Crystallin bind by molecular sieving (View interaction)gammaD Crystallin and alphaB Crystallin bind by fluorescence technology (View interaction)gammaD Crystallin and alphaA Crystallin bind by fluorescence technology (View interaction)gammaD Crystallin and alphaA Crystallin bind by molecular sieving (View interaction)

� 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction

A unique cellular and molecular architecture enables the ocularlens to transmit and focus light on the retina. The lens consists oflayered terminally differentiated cells, the fiber cells, which losetheir organelles early in development and are unable to synthesizeor turn-over proteins [1]. Transparency and refractivity entail short-range order packing of highly water soluble proteins, a-, b- andc-crystallins, which account for more than 90% of the lens proteinweight [2]. A highly tuned balance of forces prevents long rangeorder or crystallization at the high crystallin concentrations foundin fiber cells [3,4].

Under assault by environmental factors, the optimized proteinpacking is eventually perturbed leading to protein aggregationand light scattering. The ‘‘wear and tear’’ of lens aging is recordedat the molecular level through accumulated modifications of thecrystallins [5,6] such as truncations [7,8], glycation [9,10], oxida-tions [11] and deamidations [12]. In the absence of protein turn-

chemical Societies. Published by E

/S59D; GdnHCl, guanidineT4 lysozyme; WT, wild type

u (H.S. Mchaourab).

over, loss of crystallins stability and/or solubility alters the balanceof forces between proteins thereby compromising lens transpar-ency and refractivity. Age-related cataract is an opacification ofthe lens which can result from protein unfolding and subsequentaggregation [1] and/or changes in the surface properties of mole-cules leading to loss of solubility [13–15]. One of the mechanismshypothesized to delay aggregation in the lens is the activity of res-ident small heat-shock proteins (sHSP) [16] aA- and aB-crystallin.In his seminal work, Horwitz proposed [17,18] that by inhibitinglens proteins aggregation, a-crystallins delay the onset of cataract.The protein unfolding hypothesis for age-related cataract positsthat progressive modifications of b- and c-crystallins reducetheir free energies of unfolding and promote their binding toa-crystallin. The disappearance of soluble a-crystallin from thecenter of normal human lenses by age 40 [19] is hypothesized toset the stage for aggregation of destabilized proteins. Aging is char-acterized by a progressive shift in the molecular weight distribu-tions of fiber cells proteins [20]. Similar to late-onset aggregationdiseases, the gradual accumulation of modified proteins in the lensoverwhelms the chaperone machinery.

Despite their abundance, b- and c-crystallins do not have adefined role in lens development. They are considered structural pro-teins whose packing and interactions are optimized for transparencyand refractivity [1]. Both b- and c-crystallins are members of the

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S. Mishra et al. / FEBS Letters 586 (2012) 330–336 331

same protein superfamily [21]. The folds of the dimeric b- and mono-meric c-crystallins consist of four greek key motifs organized in twodomains [22,23]. The main sequence difference between the twofamilies is N-terminal extensions in the b-crystallins, with the basicisoforms having additional C-terminal extensions. Seven c-crystal-lins are expressed in the human lens; the most abundant, cD-crystal-lin, accounts for 11% of the total protein content in the fiber cells.

A number of mutations in c-crystallins have been associatedwith congenital cataracts in humans and mouse models. These in-clude ‘‘solubility mutants’’ where residue substitutions change thesurface property of the molecule [24,25]. Another class of mutantsis associated with changes in stability, i.e. shifts in the unfoldingequilibrium [26,27]. Studies of two such mutants in mouse modelssuggest multiple mechanistic origins of cataract [26,27]. The sub-stitution of I4F in cB-crystallin results in large inter-fiber spacesand large aggregates in the inner fiber cells [26]. Although the mu-tant appears to associate with a-crystallin in mouse lens extract,temperatures higher than 37 �C were required to form the complexin vitro. cD-crystallin mutant V76D forms intranuclear aggregatesthat disrupt denucleation of fiber cells [27].

The existence of cataract-causing, destabilized mutants of lensproteins poses a conundrum for the chaperone hypothesis ofa-crystallin function. aA-crystallin constitutes 30% of total lensproteins and its binding capacity could reach one substrateper subunit of equivalent molecular mass [16]. Thus, either thec-crystallin mutants are not efficiently bound by a-crystallins orc-crystallin may have a yet undefined cellular role in lens develop-ment that is inhibited by its destabilization and subsequent inter-action with a-crystallin. To distinguish between these possibilities,we investigated the interaction between both a-crystallin subunitsand destabilized mutants of cD-crystallin. The experimental designfollows our established approach that emphasizes a direct quanti-tation of binding in the absence of aggregation [28]. For this pur-pose, cD-crystallin mutants associated with congenital cataractin mice were constructed and their unfolding equilibrium charac-terized. Binding to a-crystallins was detected by changes in thefluorescence intensity and anisotropy of a bimane probe site-spe-cifically attached to cD-crystallin. We find remarkably low affini-ties in vitro suggesting that these mutants may escape qualitycontrol by a-crystallin chaperone. These findings are interpretedin the context of the folding equilibrium of c-crystallin and built-in repulsive interactions between crystallins critical for lens refrac-tivity and transparency.

2. Materials and methods

2.1. Expression and purification of crystallins

Human cD-crystallin was a generous gift from Dr. C. Slingsby,Birbeck College. The I4F, V76D, and I4F/V76D mutants were gener-ated by QuikChange procedure (Stratagene). Mutagenesis was con-firmed by DNA sequencing.

The proteins were expressed and purified as described by Junget al. [29] except that cD-crystallin expression was at 25 �C for10 h and the final gel filtration purification was carried out in Buf-fer A (9 mM MOPS, 6 mM Tris, 50 mM sodium chloride, 0.1 mMEDTA, 0.02% (w/v) sodium azide; pH 7.2) supplemented with 20%glycerol (v/v). Bimane was added after the second cation exchangecolumn concurrently with pH adjustment to 7. The reactions wereincubated for 2 h at room temperature with 10� monobromobi-mane. The excess label was then removed by the gel filtration step.Stoichiometric labeling at cysteine, C111, was confirmed by MS/MSof intact protein as well as by MS/MS of tryptic protein fragments(data not shown). Predicted molar extinction coefficient of43235 M�1 cm�1 was used to determine cD concentrations. aA-,aB- and the triply phosphorylated analog (S19D/S45D/S59D) of

aB-crystallin, referred to as aB-D3, were purified as previouslydescribed [30–32]. Protein concentrations for aA- and aB-D3were determined from extinction coefficients [33]: 16507 and19005 M�1 cm�1, respectively.

2.2. Circular Dichroism (CD) spectroscopy

Spectra were collected on a Jasco J-810 Spectropolarimeter in20 mM phosphate buffer, pH 7.1. Near-UV spectra (330–250 nm)were obtained using a 0.5 mg/ml protein solution at room temper-ature. Far-UV spectra (260–185 nm) were collected at room tem-perature using a 0.1 mg/ml protein solution.

2.3. Binding of cD-crystallin to a-crystallin

25 lM cD-crystallin was mixed with a-crystallins at the appro-priate molar ratio then incubated at 37 �C for 2 h. Bimane emissionspectra were collected from 420 to 500 nm following excitation at380 nm (Photon Technology International (PTI) L-format spectro-photometer). The fluorescence anisotropy of cD-crystallin wasmeasured on a T-format spectrophotometer (PTI). The fluorescenceintensities parallel and perpendicular to the direction of polarizedlight were analyzed to determine steady state anisotropy values.The samples were excited at 380 nm, and the fluorescence signalwas monitored at 465 nm. Binding isotherms were generated byplotting bimane emission or anisotropy at 465 nm as a functionof the [a-crystallin/cD-crystallin] ratio.

2.4. Analytical size exclusion chromatography (SEC)

Binding between bimane-labeled cD-crystallins anda-crystallins was analyzed by gel filtration on a Superose 6 10/300 GL Column (GE Healthcare Life Sciences) equilibrated in BufferA. Samples were incubated at 37 �C for 2 h prior to injection. Pro-tein elution was monitored by tandem absorbance (280 nm) andfluorescence (excitation 380 nm; emission 475 nm).

2.5. Thermodynamic analysis of cD-crystallin mutants

Denaturant-unfolding curves were constructed by monitoringtryptophan fluorescence of cD as a function of guanidine hydro-chloride concentration. Bimane-labeled cD-crystallin constructs(5 lM final concentration) were mixed with 1–5 M guanidinehydrochloride (8 M stock solution in Buffer A supplemented with1 mM DTT, pH 7.2) and were incubated at 25 �C or at 37 �C for10 h. The denaturation curves did not change upon further incuba-tion for up to 24 h. The curves were fit to a three-state unfoldingmodel [34] by non-linear least-squares methods using the soft-ware Origin (Origin Lab).

3. Results

3.1. Methodology

The topological location of the cD-crystallin mutants, whichcause congenital cataract in mice, is shown in Fig. 1. I4 and V76are located in the N-terminal domain of the molecule in a buriedenvironment. I4F substitutes a conserved isoleucine in the firstb-strand of the greek key motif by a phenylalanine. Because themutation preserves the hydrophobic nature of the side chain, its ef-fects on stability must be mediated by steric repacking. In contrast,the substitution of valine 76, which is located in a loop betweenb-strands 5 and 6, by an aspartate introduces a charged residuein the packed protein interior. Although Wang et al. [27] noted thatthe aspartate’s carboxylate group could hydrogen bond to nearbyarginine residues, this does not appear sufficient to neutralize its

Fig. 1. Ribbon representation of cD-crystallin structure (pdbID 1HK0). The N-terminal domain is colored in blue; sites of cataract-causing mutations arehighlighted in red and cysteine 111 in green.

332 S. Mishra et al. / FEBS Letters 586 (2012) 330–336

destabilizing effect. Finally, we have combined both mutationswith the goal of further destabilizing the protein. Our model ofa-crystallin chaperone activity (Scheme 1) [16] links its apparentaffinity to the fractions of: (1) activated a-crystallin, (sHSP)a and[35,36] (2) partially or globally unfolded substrate (I and U in Eq.1). Thus, in comparing multiple mutants with different free ener-gies of unfolding, their relative affinities to a-crystallin areexpected to parallel reduction in the stability of the native state N.

To investigate cD-crystallin binding to a-crystallin, we selec-tively attached a bimane label at cysteine 111 (Fig. 1). Bimanelabeling marginally reduces the stability of the WT without signif-icant changes in the shape and apparent cooperativity of theunfolding curve (Supplementary Fig. 1).

3.2. Structure and unfolding equilibrium of cD-crystallin mutants

Fig. 2A shows that, at room temperature, I4F and V76Dsubstitutions do not change the overall secondary structure ofcD-crystallin. The minimum at 218 nm suggests that cD-crystallinmutants maintain their b-sheet content although it is somewhatdiminished for I4F/V76D. In contrast, the near-UV CD spectrum(Fig. 2B) reports significant alterations in the tertiary structure spe-cifically in the environment of aromatic amino acids. These aremore accentuated for the V76D and the double mutant I4F/V76Dconsistent with the expected disruptive effects of a charged residuein the protein hydrophobic core.

That these mutations destabilize cD-crystallin is evident indenaturant-unfolding curves (Fig. 3). At 25 �C, left shifts relativeto the WT are observed in the low denaturant region leading to bi-phasic curves. The presence of an unfolding intermediate, hereafterreferred to as I, is manifested by a plateau in the 0.5–2.5 M GdnHClrange. Mutations in c-crystallin N-terminal domain, includingV76D, have been shown to stabilize an intermediate with anunfolded N-terminal domain [37]. The midpoint of the transitionfrom the native state, N, to I shifts to lower denaturant concentra-tions in the mutants relative to the WT with the largest shiftobserved for the double mutant. The second transition in theunfolding curves obtained at higher denaturant concentrations re-flects destabilization of the C-terminal domain leading to a globallyunfolded state, U. Thus, Fig. 3A demonstrates that I4F and V76Ddestabilize the native state (N) relative to an intermediate withan unfolded N-terminus. The combination of the two mutationswhich are in close proximity in the three dimensional structure

Scheme 1.

(Fig. 1) further destabilizes N. Table 1 reports the free energy,DG0, associated with each transition and the dependence of DG0

on denaturant concentration, m. Because the WT unfolding curvedoes not have two explicit transitions, the fit errors in DG0 are con-siderable. In contrast, the parameters for the mutants are morenarrowly defined revealing that the V76D substitution is moredestabilizing than the I4F substitution.

As expected, stabilities of WT and mutants c-crystallin are fur-ther reduced at the physiological temperature of 37 �C (Table 1).The midpoints for both transitions, from N to I and I to U, shift tolower denaturant concentrations. At this temperature, the nativestate of the double mutant is marginally stable. The equilibriumconstant for the first transition, [I]/[N], is 0.014 suggesting that Iis relatively populated even in the absence of denaturant (by com-parison the fraction of U is on the order of 10�7).

3.3. a-Crystallins interactions with cD-crystallin mutants

Neither aA-crystallin nor aB-crystallin chaperone binds WTcD-crystallin at detectable level; even at high concentrations andmolar ratios (data not shown). Furthermore, only marginal bindingby aA-crystallin was detected for each of the single mutants(Fig. 4A). Thus, the I4F and V76D mutations which destabilize thenative state relative to the intermediate do not cross the energeticthreshold for stable binding to the a-crystallin subunits.

Further destabilization of cD-crystallin native state by the com-bination of I4F and V76D is sufficient to promote stable binding toaA-crystallin. As previously described [28], complex formationbetween a-crystallin and bimane-labeled substrates changes theintensity and/or wavelength of the bimane group. Fig. 4A comparesthe binding levels of the three mutants. Although the fluorescenceof I4F and V76D changes upon addition of a-crystallin, the data isscattered and does not trace the shape of a binding isotherm indi-cating weak binding despite the high concentrations of cD-crystal-lin single mutants (50 lM compared to 25 lM for the doublemutant) and large molar excess of aA-crystallin. In contrast, largeand consistent changes in bimane intensity are observed for thedouble mutant as a function of increasing aA-crystallin molar ex-cess. The binding isotherm can be fit assuming that a-crystallinbinds only through its high affinity mode [28], i.e. with a ratio of4 aA subunits to each c-crystallin subunit. The complexes betweena-crystallin and cD-crystallin formed within two hours of incuba-tion at 37 �C and binding did not increase with longer incubationtimes (Supplementary Fig. 2).

While the double mutant did not effectively bind aB-crystallin,it formed stable complexes with its phosphorylation mimic(Fig. 4B). Phosphorylation of aB-crystallin at serines 15, 45 and 59[38] enhances binding by shifting the equilibrium of Eq. 2 (Scheme1) towards an activated chaperone form [30]. Substitution of theseresidues by aspartates to generate aB-D3 mimics phosphorylation’seffects on aB-crystallin oligomer equilibrium and chaperone activ-ity. Binding of cD-crystallin mutants to aB-D3 produced a marginalchange in intensity. Therefore, complex formation was detectedthrough changes in bimane anisotropy. Molar excess of aB-D3 leadsto a progressively larger anisotropy of bimane-labeled cD-crystallindue to the expected larger size of the complex between the two pro-teins. Similar to aA-crystallin, binding of the double mutant wasmore robust than the single mutants (data not shown) althoughthe dissociation constant was smaller than that obtained withaA-crystallin indicating higher affinity.

3.4. Analysis of the a-crystallin/c-crystallin complex by size exclusionchromatography

The binding trends described above were verified by size exclu-sion chromatography. Binding of monomeric cD-crystallin to the

Fig. 2. Room temperature CD analysis of cD-crystallin mutants. (A) Far-UV spectra showing a pronounced minimum at 218 nm. (B) Near-UV spectra reporting changes in thetertiary fold as a consequence of the mutations.

Fig. 3. Denaturant-unfolding curves of cD-crystallin mutants at (A) 25 �C and (B) 37 �C. The substitutions lead to the appearance of two explicit transitions compared with theWT monophasic curve. The first transition corresponds to the population of an intermediate with an unfolded N-terminal domain. The second transition corresponds tounfolding of the C-terminal domain. The solid lines represent non-linear least-squares fit yielding the parameters reported in Table 1.

Table 1Equilibrium unfolding/refolding parameters for cD-crystallin wild-type and mutants at pH 7.2.

cD-crystallin Temperature (�C) N � I I � U

DG0N?I (kcal/mol) m (kcal/mol/M) C½ (M) DG0

I?U (kcal/mol) m (kcal/mol/M) C½ (M)

WT 25 6.23 ± 17.9 2.08 ± 3.90 2.74 10.4 ± 24.1 3.12 ± 6.77 3.2437 10.7 ± 18.7 5.46 ± 9.46 2.03 10.3 ± 3.88 4.08 ± 1.60 2.51

I4F 25 8.50 ± 2.18 5.26 ± 1.39 1.58 7.67 ± 1.30 2.48 ± 0.40 3.0037 5.99 ± 0.78 4.52 ± 0.62 1.35 7.56 ± 0.62 2.83 ± 0.23 2.65

V76D 25 5.48 ± 0.66 4.71 ± 0.58 1.18 7.85 ± 0.77 2.59 ± 0.25 2.9637 4.36 ± 0.92 5.38 ± 1.08 0.79 8.14 ± 0.84 3.07 ± 0.31 2.55

I4F/V76D 25 3.96 ± 0.62 5.99 ± 0.83 0.62 9.68 ± 1.04 3.24 ± 0.35 2.7937 2.67 ± 0.54 5.07 ± 0.74 0.50 7.37 ± 0.36 2.81 ± 0.13 2.59

DG0, m, and C½ are the free energy, the denaturant dependence of DG0, and the concentration of urea at the transition midpoint, respectively, for the two transitions of cD-crystallin unfolding. DG0, m are reported with the associated fit error.

S. Mishra et al. / FEBS Letters 586 (2012) 330–336 333

oligomeric a-crystallin shifts the bimane fluorescence to shorterretention times due to the larger mass of the complex [39]. Forhigh affinity binding, the retention times are close to those ofa-crystallin while for low affinity binding, the chaperone/substratecomplex runs at the void volume of a superose 6 column [16].

Fig. 5A shows that incubation of aA-crystallin with WT cD-crys-tallin or the single mutants I4F (data not shown) and V76D doesnot result in detectable binding. In contrast, incubation with thedouble I4F/V76D leads to the concomitant reduction in the c-crys-tallin peak intensity (both absorbance and fluorescence) and

appearance of a fluorescence peak migrating with a retention timesimilar to the chaperone. This indicates the formation of a stablecomplex between the two proteins. At large molar excess ofaA-crystallin, the binding is manifested by a distinct change inthe shape of its UV absorbance peak.

Fig. 5B compares the binding of cD-crystallin mutants toaB-crystallin and its phosphorylation mimic aB-D3. A 50-foldexcess of aB-crystallin is required to detect fluorescence of thecomplex migrating at retention times similar to the aB-crystallinpeak. In agreement with the conclusion of Fig. 4, SEC analysis

Fig. 4. Binding isotherms of cD-crystallin mutant to (A) aA-crystallin and (B) aB-crystallin and its phosphorylation mimic aB-D3. The concentration of the single mutantswas 50 lM while the double mutant was 25 lM. Samples were incubated at 37 �C for two hours at the appropriate ratio as described in the methods section. The solid linesfor the double mutant I4F/V76D in (A) and (B) are non-linear least-squares fit assuming high affinity binding where 4 a-crystallin subunits bind one c-crystallin. Thedissociation constants are 43 ± 7 and 27 ± 6 lM for aA-crystallin and aB-D3, respectively. For aB-crystallin, the dissociation constant was larger than 300 lM.

334 S. Mishra et al. / FEBS Letters 586 (2012) 330–336

indicates that aB-D3 has the highest affinity to the cD-crystallinmutants showing detectable binding even for the two single mu-tants (Fig. 5B). The aB-D3/cD complex is detectable by absorbanceat 280 nm through a distinct peak running as a shoulder to theaB-D3 peak indicating an apparent larger size for the complex asexpected (Arrows in Fig. 5B and C).

4. Discussion

Although previous studies investigated a-crystallin suppressionof c-crystallin thermally-induced aggregation, the data presentedhere is the first direct quantitation of binding between the twoproteins under conditions where c-crystallin native state is moreenergetically favorable than its intermediate or unfolded states.We find that destabilization of cD-crystallin induces binding toa-crystallin. In addition, the stability of the a-crystallin/c-crystal-lin complex evidenced by size exclusion chromatographyconfirms that the interaction between the two proteins is drivenby a-crystallin chaperone activity. However, despite the perturba-tion of the native structure, binding levels for the single mutants

Fig. 5. Analysis of cD-crystallin mutant binding to a-crystallin by size exclusion chromat(A) Samples of aA-crystallin and cD-crystallin were incubated at the indicated molar ramanifested by a fluorescence peak with retention time similar to that of aA-crystallin. (Bexcess of the former is required for detectable binding of the double mutant while aB-Dpeak. (C) The complex between aB-D3 and cD-crystallin is detected as a distinct peak,

I4F and V76D are low suggesting that destabilization by thesemutations is not sufficient to trigger binding. This is unexpectedconsidering the disruption of the tertiary structure by V76D, man-ifested by the near-UV CD spectrum, and the marginal stability ofits native state at 37 �C (Table 1).

To frame these results into a more general context, it is instruc-tive to compare the binding characteristics of the cD-crystallinmutants to the only other protein to which a-crystallin apparentaffinity was extensively characterized, T4 Lysozyme (T4L). Similarto cD-crystallin, T4L is a highly stable protein with DGunf of15 kcal/mol [31]. Mutations that reduce stability were found totrigger binding to sHSP despite limited structural perturbation ofthe native state [31]. Apparent dissociation constants of T4L bind-ing to a-crystallin are at least an order of magnitude smaller thanthose measured for the most destabilized cD-crystallin mutantinvestigated here, I4F/V76D [28,40]. Weak affinities to a-crystallinwere also observed for bB1-crystallin mutants and attributed tothe population of a dimeric intermediate with low affinity toa-crystallin [41]. Together, these results suggest that interactionbetween lens proteins and a-crystallin in the ‘‘chaperone mode’’

ography (SEC) detected by in-line absorbance at 280 nm and fluorescence at 475 nm.tios and injected on a Superose 6 column. The binding of cD-crystallin I4F/V76D is) Comparative SEC analysis of binding to aB-crystallin and aB-D3. A 50-fold molar3 binds the single mutants. The y axes were scaled to show details of the complex

indicated by the arrow, migrating at a larger mass than aB-D3 alone.

Fig. 6. Fraction of N, I and U as a function of changes in DG0N?I for (A)

DG0I?U = 10 kcal/mol and (B) DG0

I?U = -2 kcal/mol. For (B) shifts in the DG0N?I

lead to a large increase in the fraction of U. These simulations apply to three-stateunfolding equilibria.

S. Mishra et al. / FEBS Letters 586 (2012) 330–336 335

departs from the mechanistic principles deduced from studies ofmodel substrates.

The cD-crystallin mutants studied here shift the unfolding equi-librium towards I. Table 1 shows that to a first approximationDG0

N?I decreases while DG0I?U remains relatively constant. To

illustrate the effects of changes in DG0N?I on the fractional popu-

lation of N, I and U, we carried out simulations based on a three-state equilibrium (Fig. 6). Unlike a two-state folding equilibrium,the increase in the population U upon destabilization of N is scaledby the equilibrium constant of the second transition, KI?U, whichfor c-crystallin is much smaller than 1. Thus, the fractional popu-lation of U remains negligible (Fig. 6A). Fig. 6B demonstrates thatsignificant population of U requires a reduction in the value ofDG0

I?U which for cD-crystallin describes the unfolding of theC-terminal domain.

The coupled model of sHSP chaperone activity (Scheme 1) pre-dicts that shift in the substrate folding equilibrium triggers bindingif it increases the population of a substrate state recognized by thesHSP. Thus, the relatively large dissociation constants suggest thatthe cD-crystallin intermediate, I, has low affinity to a-crystallin.We conclude that similar to T4L [40], a-crystallin high affinitybinding requires global unfolding of cD-crystallin.

The central finding of this paper is that a subclass of destabiliz-ing, cataract-linked mutants of c-crystallin can escape detection,binding and sequestration by a-crystallin. While a-crystallin bind-ing to destabilized proteins has been extensively studied usingmodel substrates, the results reported here suggest that conclusions

based on these studies are not directly extendable to lens proteins.Each mutation or modification has to be investigated in the contextof the proper unfolding equilibrium and a quantitative analysis ofbinding carried out. Another critical and confounding factor not ad-dressed here is molecular crowding [42] which has been invariablyneglected regardless of substrate identity or the method of its desta-bilization. Crowding in lens fiber cells is more peculiar involving thethree molecules whose interaction is to be studied. The results pre-sented here suggest that a detailed understanding of these molecu-lar interactions may not be readily derived from in vitro studies.Thus analysis of these interactions would require adequate cellularand/or organism models that better capture the intricacies of theprotein environment in lens fiber cells. Finally, our findings mayhave direct relevance to age-related cataract. It is possible thatage-related modifications of c-crystallins, that likewise selectivelystabilize I, fail to interact with a-crystallin allowing them to nucle-ate aggregation.

Acknowledgments

The authors thank Edward Monson for technical assistance andHanane Koteiche for critically reading the manuscript. This workwas supported by Grants EY12018 and P30EY008126 from theNational Institutes of Health.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.febslet.2012.01.019.

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