The importance of the last strand at the C-terminus in βB2-crystallin stability and assembly

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Biochimica et Biophysica Acta xxx (2013) xxx–xxx

BBADIS-63806; No. of pages: 12; 4C: 2, 6, 7, 8, 9, 10, 11

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

j ourna l homepage: www.e lsev ie r .com/ locate /bbad is

The importance of the last strand at the C-terminus in βB2-crystallinstability and assembly

OFKai Zhang a,1, Wei-Jie Zhao b,1, Xiao-Yao Leng b,1, Sha Wang b, Ke Yao a,⁎, Yong-Bin Yan b,⁎⁎

a Eye Center of the 2nd Affiliated Hospital, Medical College of Zhejiang University, Hangzhou 310009, Chinab State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing 100084, China

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Abbreviations: ADCC, autosomal dominant coanilinonaphtalene-8-sulfonate; BSA, bovine serum albumiC-terminal domain; DTT, dithiothreitol; Emax, maximum eTrp fluorescence; GdnHCl, guanidine hydrochloridegalactopyranoside; MD, molecular dynamics; MW, molecuchromatography; SDS, sodium dodecyl sulfate; SDS-PAGE,phoresis; Tm, the midpoint temperature of protein thermal⁎ Corresponding author. Tel.: +86 571 87783897; fax:⁎⁎ Correspondence to: Y.-B. Yan, School of Life Science100084, China. Tel.: +86 10 6278 3477; fax: +86 10 627

E-mail addresses: [email protected] (K. Yao), ybyan@ts1 These authors contributed equally to this work.

0925-4439/$ – see front matter © 2013 Published by Elsehttp://dx.doi.org/10.1016/j.bbadis.2013.10.001

Please cite this article as: K. Zhang, et al., ThBiophys. Acta (2013), http://dx.doi.org/10.10

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Article history:Received 19 August 2013Received in revised form 29 September 2013Accepted 1 October 2013Available online xxxx

Keywords:βB2-crystallinInherited mutationAutosomal dominant congenital nuclearcataractMolecular dynamic simulationProtein aggregationProtein assembly

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RCongenital cataract is the leading cause of childhood blindness worldwide. Investigations of the effects ofinheritedmutations on protein structure and function not only help us to understand themolecularmechanismsunderlying congenital hereditary cataract, but also facilitate the study of complicated cataract and non-lensabnormities caused by lens-specific genes. In this research, we studied the effects of the V187M, V187E andR188Hmutations onβB2-crystallin structure and stability using a combination of biophysical, cellular andmolec-ular dynamic simulation analysis. Both V187 and R188 are located at the last strand of βB2-crystallin Greek-keymotif 4. All of the three mutations promoted βB2-crystallin in vitro and at the cellular level. These three muta-tions affected βB2-crystallin quite differentially: V187M influenced the hydrophobic core of the C-terminal do-main, V187E was a Greek-key motif breaker with the disruption of the backbone H-bonding network, whileR188H perturbed the dynamic oligomeric equilibrium by dissociating the dimer and stabilizing the tetramer.Our results highlighted the importance of the last strand in the structural integrity, folding, assembly and stabilityof β-crystallins. More importantly, we proposed that the perturbation of the dynamic equilibrium between β-crystallin oligomers was an important mechanism of congenital hereditary cataract. The selective stabilizationof one specific high-order oligomer by mutations might also be deleterious to the stability and folding ofthe β-crystalllin homomers and heteromers. The long-term structural stability and functional maintenance ofβ-crystallins are achieved by the precisely regulated oligomeric equilibrium.

© 2013 Published by Elsevier B.V.

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Congenital cataract is the leading cause of childhood blindnessworldwide [1]. Cataract is the opacification of the lens, which is trans-parent in normal lens to transmit and focus visible light onto the retina.Many factors have been linked to the onset of congenital cataract [2],while 8.3–25% of the cases are caused by genetic alterations [3]. Atpresent, at least 26 foci have been linked to congenital cataract, andhalf of the associated genes are crystallins [3]. Although surgical treat-ment is a well-established method for cataract [4], investigations ofthe molecular mechanism underlying cataract remain an importanttask for both prenatal diagnosis and drug design. Particularly, the

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ngenital cataract; ANS, 1-n; CD, circular dichroism; CTD,mission wavelength of intrinsic; IPTG, isopropyl-1-thio-β-D-lar weight; SEC, size-exclusionSDS-polyacrylamide gel electro-transition; WT, wild type+86 571 87783908.s, Tsinghua University, Beijing7 1597.inghua.edu.cn (Y.-B. Yan).

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e importance of the last stran16/j.bbadis.2013.10.001

molecular mechanisms of most genetic variations have not been exten-sively studied yet,which limits our understanding of the occurrence anddevelopment of the diseases. Furthermore, the so called lens-specificgenes such as crystallins are also widely expressed in the other organsand tissues. Mutations in some of the crystallin genes have been associ-ated with non-cataract abnormities such as neurodegenerative disease,cardiomyopathy, tumorigenesis, and retinal and vascular diseases [5–7].Thus advances in the structures and functions of lens-specific proteinswill facilitate the investigations of the molecular mechanisms underly-ing these non-lens disorders.

Crystallins are the predominant structural proteins of vertebratelens and constitute about 90% of water-soluble proteins of the lens [5].According to the elution positions on the size-exclusion chromatogra-phy (SEC) profile, crystallins in vertebrate lens can be divided intothree classes: α-, β- and γ-crystallins (reviewed in [8]). α-Cystallinsare molecular chaperones with characteristic structural features ofsmall heat shock proteins, and can assemble into large homomers orheteromers containing an average of 24 subunits. β/γ-Crystallins arestructural proteins with the same fold in tertiary structure composingfour Greek-key motifs divided into two domains (Fig. 1). β-Crystallinsexist as homomers or heteromers with 2–8 subunits, while γ-crystallins aremonomers. The short-order arrangements amongvariouscrystallins, cytoskeletal structures and lens membranes are believed to

d at the C-terminus in βB2-crystallin stability and assembly, Biochim.

http://dx.doi.org/10.1016/j.bbadis.2013.10.001

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Fig. 1. (A) Crystal structure of humanβB2-crystallin (PDB ID: 1YTQ). The tetrameric struc-ture was created and rendered by PyMol (http://www.pymol.org/). The four subunits areshown in different colors. The positions of V187 (magenta) and R188 (cyan) are highlight-ed by the space-filling model. (B) Sequence alignment of the C-terminus of human β/γ-crystallins. The residues corresponding to V187 and R188 in βB2-crystallin are coloredin magenta and cyan, respectively.

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be crucial to the functional requirements of the lens such as transparen-cy, refractive index and long-term survival [8–10]. Consequently,crystallins are the major components in the water-insoluble fractionsof the cataract lens, and numerous mutations in crystallins have beenlinked to congenital or juvenile cataract [1,3,10]. The extensive studiesduring the past decade have revealed that these mutations may leadto protein aggregation via one or more of the following mechanisms:modifications of the native structure, decrease in solubility, formationand accumulation of an aggregation-prone intermediate, impairmentof the normal functions of crystallins, disruption of the protein interac-tion network, overload of the intracellular protein quality controlsystem, alterations in the subcellular location or increase of the sensitiv-ities to various stresses such as chemical denaturants, heat, UV irradia-tion and proteolysis (reviewed in [3,5,11,12] and [13–19]). It seemsthat the onset of cataract is a very complex process, and various muta-tions may affect the lens via quite different mechanism(s).

The effect of mutations on the structure and function of themultimeric β-crystallins could be much more complicated than thaton the monomeric γ-crystallins although they share the same fold intheir tertiary structures. In theory, the mutations may affect one ormore of the following aspects: the structure and/or folding of the sub-unit, the subunit interactions in the homomers, the assembly of theheteromers and the dynamic equilibrium between various oligomers.For example, theΔG91mutation results in a totally insoluble expressionin Escherichia coli [20] and decreases subunit interactions of βA1/βA3-crystallin [21]. A spontaneous insertion mutation in CRYBA1/A3 impairsphagosome degradation in the Nucl rat [18]. The A2V mutation in βB2-

Please cite this article as: K. Zhang, et al., The importance of the last stranBiophys. Acta (2013), http://dx.doi.org/10.1016/j.bbadis.2013.10.001

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crystallin decreases the ability of tetramerization and promotes aggre-gation during kinetic refolding [22]. The Q155X mutation alters the na-tive structure and decreases the stability of βB2-crystallin [23]. TheS129R mutation in βB1-crystallin is a stability-beneficial mutation, butit dissociates the dimer, reduces the protection effect on βA3-crystallin aggregation [24] and increases the sensitivity to proteolysis[19]. The above observations suggest that it remains very important tostudy the complex mechanisms underlying cataract caused by β-crystallin mutations.

In this research, we studied the effects of the mutations V187M,V187E and R188H on βB2-crystallin structure and stability. V187Mhas been associated with autosomal dominant congenital cataract(ADCC) with nuclear phenotype [25], while R188H was identified in afamily with ADCC having an anterior axial embryonal nuclear pheno-type [26]. The V187E mutation has been identified in the Aey2 mousemutant with progressive cataract [27]. Both of V187 and R188 are locat-ed at the last strand of the fourth Greek-keymotif of βB2-crystallin, andare conserved in human β/γ-crystallins (Fig. 1). The distinct chemicalnature and positions of the side chains of valine and arginine suggestthat V187 and R188may have different roles in βB2-crystallin structureand stability although all mutations at these two residues are linked tocongenital nuclear cataract. Herein we found that V187 plays a crucialrole in the structural integrity and hydrophobic core stability, whileR188 plays a regulatory role in the dynamic oligomeric equilibrium.These results highlighted the importance of the last strand at the C-terminus in βB2-crystallin structure, assembly and stability. Moreimportantly, our results suggested that the dynamic oligomeric equilib-rium was well encoded in the primary sequences of β-crystallins, andany perturbation of the equilibrium could be deleterious to β-crystallin structure, stability and folding.

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2. Materials and methods

2.1. Materials

Sodium dodecyl sulfate (SDS), ultra-pure guanidine hydrochloride(GdnHCl), dithiothreitol (DTT), isopropyl-1-thio-β-D-galactopyranoside(IPTG), 1-anilinonaphtalene-8-sulfonate (ANS), yeast alcohol dehydro-genase, chicken ovalbumin, horsemyoglobin and bovine serum albumin(BSA) were Sigma products. All the other chemicals were local productsof analytical grade.

2.2. Site-directed mutagenesis

The pET28a plasmid containing the wild type (WT) human βB2-crystallin gene was obtained as that described previously [22]. A six-His tag sequence was fused to the N-terminus of the open readingframe of the gene to facilitate further purification by Ni2+-column.Site-directed mutagenesis was performed by overlap extension usingpolymerase chain reaction (PCR). The common PCR primers were:Forward (For), 5′-AAGGATCCATGGCCTCAGATCACCAG-3′ and Reverse(Rev), 5′-GGAAGCTTCTAGTTGGAGGGGTGGAA-3′. The primers forconstructing the V187M, V187E and R188H mutants were: For-V187M, 5′-AGGTGCAGTCCATGCGCCGTAT; Rev-V187M, 5′-ATACGGCGCATGGACTGCACCT-3′; For-V187E, 5′-AGGTGCAGTCCGAGCGCCGTAT-3′; Rev-V187E, 5′-ATACGGCGCTCGGACTGCACCT-3′; For-R188H, 5′-TGCAGTCCGTGCACCGTATCC-3′ and Rev-R188H, 5′-GGATACGGTGCACGGACTGCA-3′, respectively. The amplified fragments were digestedby BamHI and HindIII, and inserted into the expression vector pET28a.The recombinant pET28a plasmids containing the WT or mutatedgenes were transformed into E. coli DH5α cells, and the sequenceswere confirmed by DNA sequencing. Then the plasmids were trans-formed into E. coli Rosetta (DE3) for the production of the recombinantproteins.


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2.3. Protein expression and purification

The E. coli Rosetta (DE3) cells with the transformation of theWT andmutated CRYBB2 geneswere grown in the Luria–Bertani medium for 4hat 37 °C. Then 0.1mM IPTG was added to induce the overexpression ofthe recombinant proteins. After induction, the E. coli cells containingthe genes of the WT βB2-crystallin and V187M were grown in theLuria–Bertani medium for 4 h at 37 °C, while those having the R188Hgenes were incubated for 15 h at 20 °C. The conditions for the overex-pression of the V187E mutant were screened by ranging the tempera-ture from 10 °C to 37 °C and the IPTG concentration from 0.01 to0.1mM. After incubation, the cells were harvested by centrifugation at8000 g, washed by 0.9% sodium chloride and sonicated by ultrasonic.Then the soluble fractions were separated by centrifugation at12,800g at 4°C. TheHis-tagged recombinant proteins in the supernatantwere collected by a Ni-NTA affinity column, and were further purifiedby a Hiload 16/60 Superdex 200 prep-grade column equipped onÄKTA purifier. The His-tag was not removed and the His-tagged pro-teins had identical properties with the proteins with the cleavage ofthe tag (data not shown). The purified proteins were concentratedusing the 10KMWCOMillipore concentrators (Millipore Corp., Billerica,MA, USA) and stored at −80 °C. The protein concentration was deter-mined by the Easy Protein Quantitative Kit (Trans Gen Biotech Corp.,Beijing China) following the Bradford method using BSA as a standard[28]. The protein solutions were prepared in buffer A containing20mM sodium phosphate, 150mM NaCl, 1mM EDTA, and 1mM DTT,at pH7.2.

2.4. SDS- and native-PAGE

The SDS-polyacrylamide gel electrophoresis (PAGE) was performedunder the reduced conditions with a separating gel concentration of12.5%. The native-PAGE analysis was conducted using similar gel condi-tions except that the proteins were not denatured by heating and SDS.The protein concentration was 0.2 mg/ml for both the SDS- andnative-PAGE analysis.

2.5. Spectroscopy

All spectroscopic experimentswere carried out at 25°C usingproteinsamples prepared in buffer A. The circular dichroism (CD) spectra wererecorded on a Jasco J-715 spectropolarimeter (Jasco Corp., Tokyo,Japan). The protein concentration was 0.2mg/ml and 1mg/ml for thefar- and near-UV CD measurements, respectively. A 1 mm or 10 mmpathlength cell was used for the far- or near-UV CD measurements, re-spectively. The protein samples for far-UV CD spectroscopy were pre-pared in the low-salt buffer by a 40-fold dilution of buffer A withddH2O. The fluorescence spectra were recorded on an F-2500 fluores-cence spectrophotometer (Hitachi Ltd., Tokyo, Japan) with a slit widthof 5 nm for both excitation and emission. The protein concentrationwas 0.2 mg/ml for the fluorescence measurements. The excitationwavelength of the intrinsic Trp fluorescence was 280 or 295 nm.When excited at 295nm, the fluorescence is dominated by the Trp fluo-rescence due to the weak absorbance of Tyr at this wavelength, whilethe excitation at 280nm produces fluorescence from both the Trp andTyr fluorophores. Parameter A, which sensitively reflects the positionand shape of the Trp fluorescence spectra [29], was calculated by divid-ing the fluorescence intensity at 320 nm by that at 365 nm. The ANSfluorescence was determined with an excitation wavelength of380nm. The samples for ANS fluorescence experiments were preparedby mixing the protein and ANS stock solutions to reach a final molarratio of 75:1 (ANS:protein) and equilibrated in the dark for 30min at25 °C. The turbidity of the samples was monitored by the absorbanceat 400 nm (A400) using an Ultraspec 4300 pro UV/Visible spectropho-tometer (Amersham Pharmacia Biotech, Uppsala, Sweden). The


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resonance Rayleigh light scattering was measured with an excitationwavelength at 295nm as described previously [30].

2.6. Size-exclusion chromatography

The size-exclusion chromatography (SEC) analysis was carriedout using a Superdex 200HR 10/30 column on an ÄKTA fast proteinliquid chromatography (Amersham Pharmacia Biotech, Sweden)as described previously [22,31]. In brief, the column was pre-equilibrated with buffer A for about 10 column volumes of mobilephase, and then 100 μl protein solutions were injected into the col-umn and run at a flow rate of 0.35ml/min. The oligomeric equilibri-um of the homomeric proteins was analyzed by ranging the proteinconcentration from 0.2 to 4mg/ml. The samples were prepared by di-luting the 8mg/ml stock solutions in buffer A and equilibrated for 2hat 4 °C. The SEC analysis of the βA3/βB2 heteromers was performedby injecting the samples equilibrated for 0 h or 12 h at 37 °C after afast manual mixing of 0.5 mg/ml homomeric protein solutions. Thecolumn was calibrated with molecular weight (MW) markers yeastalcohol dehydrogenase (MW 140 kDa), BSA (MW 66 kDa), chickenovalbumin (MW 43 kDa) and myoglobin (MW 17 kDa). The peakarea was determined by fitting the SEC profiles with one or twoLorentz peak(s) using Origin 8 (OriginLab Corp.).

2.7. Protein thermal denaturation

Protein thermal denaturation was carried out by heating 0.2mg/mlprotein solutions continuously from 20 °C to 86 °C. The thermal transi-tion curves were obtained by measuring the intrinsic Trp fluorescenceand turbidity every 2 °C after a 2min equilibration at a given tempera-ture. The temperature was controlled by a water-bath.

2.8. Protein denaturation induced by GdnHCl

The equilibrium unfolding of proteins induced by GdnHCl was per-formed by incubating the WT and mutated βB2-crystallins in buffer Acontaining various concentrations of GdnHCl ranging from 0 to 6 Movernight at 25 °C. The final protein concentration was 0.2mg/ml. Theunfolded samples were then used for turbidity, light scattering, CD, in-trinsic and ANS fluorescence measurements to monitor the structuralchanges and appearance of aggregates during unfolding.

2.9. Protein aggregation experiments

Thermal aggregation kinetics was measured by heating the proteinsolutions at a given temperature and recording the turbidity dataevery 2 s. Protein concentrations were varied from 0.1 mg/ml to0.5 mg/ml. The time-course aggregation during kinetic refolding wasobtained by measuring the turbidity every 2 s at 25 °C immediatelyafter the initiation of kinetic refolding by a 1:40 fast manual dilutionof the fully GdnHCl-denatured proteins into buffer A. The GdnHCl-denatured proteins were prepared by incubating the proteins in bufferA containing 4MGdnHCl overnight at room temperature. The final pro-tein concentration was 0.2mg/ml and the final GdnHCl concentrationwas 0.1 M. The aggregation measurements were quenched after10min reaction and the protein samples were centrifuged at 12,800 gat 4°C for 10min. Then the proteins in the supernatant and precipitationwere analyzed by 12.5% SDS-PAGE.

2.10. Protein stability against UV irradiation and long-term incubation

Protein stability against UV irradiation was performed using thesame conditions as those described previously [32]. In brief, the sampleswith a protein concentration of 1mg/ml in buffer A were irradiated by254nmUV light. The solutions were placed on ice to eliminate the pos-sible heating effect of the light. Time-course aggregation induced byUV-



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Fig. 2. Effect of the mutations on βB2-crystallin solubility. (A) Overexpression and purifi-cation of the recombinant proteins from E. coli. Left panel: V187Emainly existed in the in-clusion bodies and was unable to be purified from the soluble fractions whenoverexpressed in E. coli. The band of V187E is indicated by the open box. The overexpres-sion of V187E was induced by 0.1mM IPTG at 20 °C for 15h. Right panel: identification ofthe purifiedWTβB2-crystallin, V187Mand R188H by SDS-PAGE.M represents the band oftheMWmarkers. The MWs of themarkers are labeled on the left side of the lanewith theunit of kDa. The protein concentration was 0.2mg/ml for SDS-PAGE analysis. (B) Effect ofV187M and R188H mutations on βB2-crystallin solubility. The solubility limits of the WTand mutated proteins were determined by microconcentration in buffer A. The solubilityof each protein was calculated from three independent experiments. *: P b 0.005 (WT vs.mutant).


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irradiation was monitored by recording the turbidity every 30min. Asfor the long-term protein stability at the physiological temperature,the protein solutions with a concentration of 3mg/ml were incubatedin the water-bath at 37 °C for 48 h, and time-course aggregation wasmeasured at 37 °C.

2.11. Solubility measurements

The protein samples were centrifuged continuously using micro-miniature 10K MWCOMillipore concentrators (Millipore Corp., Billeri-ca, MA, USA) at 12,800g and 4°C. The concentration of the proteins wasdetermined frequently till the concentration reached its maximum. Allsolubility experiments were repeated three times.

2.12. Molecular dynamic simulation

Details regarding molecular dynamic (MD) simulation analysis werethe sameas those described previously [19]. In brief, the crystal structureof the WT human βB2-crystallin (PDB ID: 1YTQ) [33] was used as thetemplate structure to create the starting structure of the mutants byPyMol (The PyMOL Molecular Graphics System, Version 0.99rc6,Schrödinger, LLC, http://www.pymol.org/). The simulation systemswere generated by adding a water box and 150mM NaCl by VMD [34].The equilibrium simulation was performed using NAMD2.8 [35] at 2 fstime-steps for 10ns at room temperature (300K) and constant pressure(1atm). The free energies of monomer–monomer and dimer–dimer in-teractions as well as the hydrogen-bonding networks were calculatedusing the VMDplugins NAMD energy and hydrogen bonds, respectively.The analysis and rendering of the structures were performed using thesoftware PyMOL.

2.13. Cell culture and immunofluorescence

The genes of theWT andmutated CRYBB2were ligated to the vectorpEGFP-C3 for overexpression in the HeLa cells. The HeLa cells were cul-tured in six-well plates with DMEM high glucose (30mM) supplement-edwith 10% fetal bovine serum (FBS) and 5% CO2 at 37°C. The cellswereseeded on the coverslides treatedwith 2.5MNaOH and 60% ethanol oneday before transfection. The plasmids containing genes of the WT andmutated CRYBB2 were transfected into the cells using VigoFect (Vigor-ous) following the manufacturer's instructions. After transfection for4 h, the medium was refreshed by DMEM high glucose to remove thetransfection reagent and residual DNAs. After 30h incubation, the cellswere fixed with 4% paraformaldehyde for 40 min, washed with PBStwice and penetrated with 0.4% Triton-100 for 30 min. Then the cellswere dyed with 1 μM Hoechst 33342 for 3 min in the dark, followedby 10 min washing with PBS for three repetitions. Finally, thecoverslides were mounted and analyzed on a Zeiss LSM710 laser scan-ning microscope.

3. Results

3.1. Effect of the mutations on βB2-crystallin solubility

When overexpressed in the E. coli Rosetta cells, the three mutationshad quite different effects on the yields of soluble proteins. The V187Mmutant behaved similar to the WT βB2-crystallin with most of the re-combinant proteins existed in the soluble fractionswhen overexpressedat 37°C. The R188Hmutantmainly existed in the inclusion bodieswhenoverexpressed at 37°C, but could successfully fold into soluble forms ata low temperature of 20°C. However, themajority of the V187Emutantwas found in the insoluble fractions for all overexpressing conditionsscreened (Fig. 2A), implying that the V187E mutation completelyblocked βB2-crystallin to fold into the functional form in the E. colicells. In this case, only the recombinant V187M and R188H mutantswere purified for further studies at the protein level, while the effect


of the V187Emutation onβB2-crystallin structurewas evaluated by cel-lular studies and MD simulations.

The solubility limits of theWT andmutatedβB2-crystallinswere de-termined by microconcentration in buffer A. Under our conditions, thesolubility limit of the WT βB2-crystallin was ~295 mg/ml (Fig. 2B),which is within the range of crystallin concentrations in the lens [8].The two mutations, V187M and R188H, reduced the solubility limit ofβB2-crystallin to 263 mg/ml and 253 mg/ml, respectively. However,the N250 mg/ml solubility suggested that the mutants are extremelysoluble and that the 10%–15% decrease in solubility might not play adominant role in cataract formation.

3.2. Effect of the mutations on βB2-crystallin structure by biophysicalexperiments

Spectroscopic methods were used to investigate the effects of theV187M and R188H mutations on βB2-crystallin secondary and tertiarystructures. As shown in Fig. 3A, the almost identical far-UV CD spectraof the three proteins indicated that the mutations had no influence onβB2-crystallin secondary structures. The effect of mutations on the ter-tiary structureswas evaluated by near-UVCDand intrinsic fluorescence.The results in Fig. 3B–D indicated that onlyminor changeswere inducedby the twomutations. The shape of the near-UV CD spectra was not sig-nificantly altered and the intensities of the negative peaks were slightlydecreased by themutations. Meanwhile, themaximum emission wave-lengths of intrinsic Trp fluorescence (Emax) were ~329 nm for all three


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Fig. 3. Effect of the V187M and R188Hmutations on the secondary and tertiary structures of βB2-crystallin by spectroscopic methods. (A) Far-UV CD spectra. (B) Near-UV CD spectra. (C)Intrinsic Trp fluorescence excited at 295 nm. (D) Intrinsic Trp and Tyr fluorescence excited at 280 nm. (E) Extrinsic ANS fluorescence excited at 380 nm. The proteins were prepared inbuffer A with a concentration of 0.2mg/ml except for that a concentration of 1mg/ml was used for the near-UV CDmeasurements. The positions of the maximum emission wavelengthsof intrinsic fluorescence are indicated by the dotted lines in panels (C) and (D).


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served for the Trp & Tyr fluorescence. As for the hydrophobic exposure,about 4-fold increase in the ANS fluorescence was induced by theR188H mutation, while no significant change was observed for theV187M mutation. These spectroscopic results indicated that the twomutations had very limited impacts on the secondary structures andthe microenvironments of the Trp and Tyr fluorophores. The most sig-nificant change was that the R188H mutation dramatically increasedthe ANS-accessible hydrophobic sites of βB2-crystallin.

The effect of the mutations on the oligomeric equilibrium of βB2-crystallin was investigated by the SEC method. As shown in Fig. 4, theWT βB2-crystallin eluted as a single peak with an apparent MW ofaround 45 kDa, which indicated that the protein existed in a dimer–monomer equilibrium in diluted solutions [22,36,37]. With the increaseof protein concentration, the elution volume of βB2-crystallin shifted toa smaller elution volume, corresponding to the stabilization of thedimer. The behavior of the mutant V187M was identical to the WTβB2-crystallin as revealed by the same elution volumes at variousprotein concentrations. This indicated that the V187M did not affectthe association properties of homomeric βB2-crystallin. Unlike the WTprotein and V187M, R188H contained two well separated peaks in theSEC profiles, which was denoted as Peak 1 and Peak 2, from small tolarge volumes, respectively. Peak 2 had an apparent MW close to themonomeric form,while that of Peak 1was between the dimeric and tet-rameric forms. Thus Peak 2 could be assigned as a state in dimer–mono-mer equilibriumwith the preference of monomer, while Peak 1 existedin a state of tetramer–dimer equilibrium. As protein concentration in-creased, the elution volumes of both Peak1 and Peak2 shifted to smaller


values, indicating the stabilization of the tetramer and dimer, respec-tively. Meanwhile, the content of Peak 2 increased exponentially from~5% to over 30% (inset of Fig. 4A). These observations suggested thatthe R188H mutation destabilized the dimeric structure, but stabilizedthe monomeric and tetrameric structures. The existence of the variousoligomeric states of R118H in solutions was also confirmed by the ap-pearance of multiple bands in the native-PAGE gel (data not shown).

3.3. Effect of the mutations on βB2-crystallin stability and folding

Protein stability was evaluated by classical methods of denaturationinduced by heat, long-term incubation, UV-irradiation and chemical de-naturants. As shown in Fig. 5A, themidpoint temperature of the thermaltransition (Tm) was 65.7±0.1 °C for the WT βB2-crystallin when mon-itored by Trp fluorescence, consistent with the previous result [22]. TheTmofV187Mwas 63.6±0.2°C,whichwas 2°C lower than that of theWTprotein. A dramatic destabilization effect was observed for the R188Hmutation, which led to a 16.5 °C decrease in the Tm. During thermal de-naturation, theWTprotein began to aggregate at ~72°C (Fig. 5B),wherethe thermal unfolding was completed when monitored by Trp fluores-cence (Fig. 5A). The aggregation of the mutants was more serious andappeared at much lower temperatures, 66 °C and 52 °C for V187M andR188H, respectively. Thermal aggregation kinetic at 59°C (Fig. 5C) indi-cated that no aggregation occurred for the WT βB2-crystallin when theprotein concentration ranged from 0.1 to 0.5mg/ml, while the aggrega-tion of two mutants was concentration-dependent. The critical concen-tration of protein thermal aggregation was between 0.1 and 0.2mg/mlfor both mutants, while R188H was more aggregation-prone as



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Fig. 4. SEC analysis of the oligomeric equilibrium of βB2-crystallin. (A) Representative SECprofiles of the proteins with protein concentrations of 0.5mg/ml and 2mg/ml in buffer A.The two elution peaks of R188H are denoted as Peak 1 and Peak 2. The proteinconcentration-dependenceof thepeak area of Peak1was calculated byfitting the SEC pro-files of R188H by two Lorentz peaks. The inset shows the percentages of the relative peakarea of Peak 1 at various protein concentrations. The elution volumes of the standard pro-teins used for calibration are shown on the top of the figure. The dotted line shows the po-sition of the WT protein with a concentration of 0.5 mg/ml. (B) Protein concentration-dependence of the elution volumes of the peaks in the SEC profiles.


UNCreflected by the pronounced shorter lag time and larger aggregation

rate.To evaluate the long-term stability, the proteins with a concentra-

tion of 3 mg/ml were incubated at around physiological temperature(37 °C) for 48 h. As shown in Fig. 5D, both the WT protein and V187Mmaintained transparent after 48 h incubation, while the turbidity ofR188H increased continuously till the maximum was reached after24h incubation.Moreover, the three proteins had dissimilar sensitivitiestoUV irradiation (Fig. 5E). That is, theWTproteinwas themost resistantto aggregation induced by UV irradiation, followed by V187M, andR188H was the most sensitive one. Similar effects of the mutationscould also be observed for the protein stability against chemical dena-turant GdnHCl (Fig. 6). Both mutations destabilized βB2-crystallin bylowering the midpoint concentration of GdnHCl-induced unfolding(Fig. 6A), and the R188H mutation was more deleterious. The V187Mmutation did not affect the hydrophobic exposure during GdnHCl-induced unfolding (Fig. 6B). However, the R188Hmutation significantlyincreased the hydrophobic exposure at lowGdnHCl concentrations. The


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significant increase in ANSfluorescence could be caused by the accumu-lation of the intermediate with large hydrophobic areas or the appear-ance of aggregates with high ANS-binding ability [38]. However, nochanges in the turbidity (absorbance at 400 nm) or light scattering at295 nm were observed for all of the three proteins, indicating that theproteins did not form aggregates under our denaturing conditions(data not shown). Thus the increase in ANS fluorescence induced bythe R188Hmutationwasmore likely to be the stabilization of an interme-diate with large hydrophobic area although we could not exclude thepossibility of forming transient small aggregates by R188H under lowGdnHCl concentrations. Consistent with the equilibrium unfolding data,only minor fractions of the WT and V187M undergo the misfolding/aggregation pathway during kinetic refolding as reflected by turbidityand SDS-PAGE analysis (Fig. 6C). TheR188Hmutation promoted themol-ecules to be more easily trapped into the misfolding/aggregation path-way as revealed by the altered aggregation kinetics and larger amountsof molecules in the insoluble fraction of the refolded sample.

The above biophysical data are well consistent with the overexpres-sion results in E. coli (Fig. 2). To gain insight into the effect of the muta-tions on the status of βB2-crystallin in human cells, the genes of theWTand mutated CRYBB2 were fused by EGFP-tag and transfected into theHeLa cells. As shown in Fig. 7, control HeLa cells transfected withEGFP-C3 exhibited fluorescence broadly distributed throughout boththe cytoplasm and nucleus. Cells overexpressing the WT βB2-crystallinhad a similar distribution pattern with the control cells. An alterationof the distribution pattern was observed for the cells harboring the mu-tated βB2-crystallins, which forms several granule-like structures in thecytoplasm. R188Hwas also found to have a dense distribution in the nu-cleolus, which is quite different from the others. A quantitative analysisof the ratio of cells with granule-like aggregates indicated that V187Eand R188H had similar ability of forming intracellular aggregates(P=0.44),while that of V187Mwasweaker than the two othermutants(Pb0.05).

3.4. Effect of the βB2-crystallin mutations on the assembly and stability ofβA3/βB2-crystallin heteromer

Heteromers can be formed between basic and acidic β-crystallins[36,37], and the stable basic β-crystallins can protect the unstable acidicones to fight against stress-induced denaturation and aggregation[39,40]. Consistent with those previous observations [22,36,37], theWT βB2-crystallin could form stable heteromers with βA3-crystallinas revealed by the shift of the elution peak of the protein mixtures to alower volume after 12h incubation at 37 °C (Fig. 8A). The βA3/V187Mpeak eluted at a position with a smaller apparent MW than the WTone. This suggests that the V187Mmutationmodified the protein inter-actions between βA3- and βB2-crystallins although it did not affectβB2-crystallin homo-oligomerization (Fig. 4). R188H could also formheteromers with βA3-crystallin. Similar to the bi-state behavior ofR188H homomers at high protein concentrations, the equilibratedβA3/R188Hwith a concentration of 0.5mg/ml also contained two stablestates. The fitting of the βA3/R188H peak indicated that one componenteluted at a volume similar to the dimeric βA3/βB2, while the other ap-peared at a volume corresponding to a tetramer. The time-coursestudywas conducted by native-PAGE analysis of samples with a proteinconcentration of 0.2mg/ml (Fig. 8B). Consistentwith the SEC results, theV187M mutation did not affect the association of βB2-crystallin withβA3. Different from the one single band for the WT βB2-crystallin andV187M, R188H contained several bands in the native-PAGE gel, whichconfirmed that R188H perturbed the oligomerization equilibrium ofβB2-crystallin. Unexpectedly, the association between R188H andβA3-crystallin was much faster than the WT protein and V187M, sug-gesting that the interference of the homo-oligomerization might facili-tate hetero-oligomerization.

The stability of theβ-crystallin heteromerswas investigated by ther-mal aggregation kinetics measured at 59 °C and 65 °C (Fig. 8C).



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Fig. 5. Effect of the V187M and R188Hmutations on βB2-crystallin stabilities against heating, long-term incubation and UV irradiation. (A) Protein thermal denaturationmonitored by themaximum emission wavelength of intrinsic Trp fluorescence (Emax). (B) Protein thermal aggregation monitored by turbidity (absorbance at 400 nm). (C) Thermal aggregation kineticsmonitored by turbidity every 2 s when incubating the proteins at 59 °C. No aggregation was observed for the WT protein with all of the three given concentrations as well as V187Mand R188Hwith a concentration of 0.1mg/ml. (D) Long-term stability revealed by incubating 3mg/ml proteins at 37 °C continuously, and turbidity data weremeasured at given intervalsat 37°C. The inset shows the photographs of theproteins incubated for 24h at 37°C. (E) Sensitivity to UV-irradiation of the proteinswith a concentration of 1mg/ml. The tubeswere placedon the ice to eliminate the heating effect. The left panel shows the aggregation kinetics, while the right panel shows the photographs of the proteins after irradiated for 1.5 h and 24 h,respectively. The tubes containing buffer A were used as the controls.


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Consistent with the previous results [19,24,40,41], βA3-crystallin isprone to aggregate during heat treatment. The formation of βA3/βB2heteromer significantly stabilized βA3-crystallin against thermal aggre-gation by elongating the lag time and reducing the aggregation rate.V187M could protect βA3-crystallin, but the efficiency was muchlower than that of the WT βB2-crystallin. The R188H mutationcompletely eliminated the protection effect of βB2-crystallin on βA3-crystallin aggregation. It is worth noting that 0.1mg/ml R188H did notaggregate at 59 °C. Thus the dramatic increase of the aggregation rateand decrease of the lag time revealed that the aggregation of 0.2mg/mlβA3/R188H was much heavier than the sum of 0.1 mg/ml βA3-crystallin and R188H homomers. This suggested that the R188Hmutation might introduce non-native packing between βA3- andβB1-crystallins, which produced unstable heteromers with modifiedquaternary structures.

3.5. Structural basis of mutation-induced structural perturbations by MDsimulations

The above biophysical and cellular studies indicated that the threemutations had quite dissimilar effects on βB2-crystallin structure, olig-omerization, stability and folding in the cells. To explore the underlyingstructural basis, MD simulations were performed for the WT proteinand the V187M, V187E and R188H mutants using the crystal structureof human βB2-crystallin [33] as the template structure. The protein


molecules were solvated in a water box containing 150mM NaCl, andthe simulations were run under 300 K for 10 ns. During simulations,only the movements of flexible loops were observed for both of the di-meric and tetrameric structures of the WT βB2-crystallin, and the corestructures were well maintained (data not shown).

As shown in Fig. 9A, V187Mmaintained all of the regular secondarystructure elements of the WT βB2-crystallin, and the variations in thedimeric structure were caused by themovements of the flexible regionswhen simulated at room temperature. When the C-terminal domain(CTD) was used for alignment, the two Greek key motifs were almostsuperimposed, suggesting that the V187M mutation did not affect thecore structure of the protein. Considering that the side chain of V187is part of the hydrophobic core of CTD, a close inspection indicatedthat the positions of the residues involved in the stabilization of theCTD hydrophobic core were not significantly affected by the V187Mmutation (Fig. 9B). The dominant change was that the packing of thehydrophobic side chains in the hydrophobic core was slightly loosedand expanded by the enlarged side chain of the substitutedMet at posi-tion 187.

Compared with V187M, the V187E mutation produced much moredramatic changes in the secondary structure components of βB2-crystallin (Fig. 9C). Particularly, the fourth Greek-key motif, whereV187 is located, was almost completely destroyed by the mutation.The H-bonding network that defines the fourth Greek-key motif wassignificantly altered by the mutation (Fig. 9D). The backbone hydrogen



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bonds between V187/R188 and the adjacent residues were fullydisrupted, which led to the disorder of the originally well-defined β-strands in the WT protein. In addition to the deleterious effect on thebackbone H-bonding network, the carboxyl group of E187 formed anew H-bond with the backbone NH of V152, which participates into

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Fig. 7. Effects of the V187M, V187E and R188H mutations on the aggregation ofoverexpressed βB2-crystallin in HeLa cells. (A) Cellular distributions of the overexpressedproteins visualized by the GFP tag fused to the N-terminus of the proteins. The nuclei arestained in blue using Hoechst 33342. (B) Percentages of cells containing at least one pro-tein aggregate. At least 100 cells were picked from 10 random viewing fields. *:P b 0.00001 (WT vs. mutant).

Fig. 6. Protein denaturation induced by GdnHCl. (A) Transition curves of protein equilibri-um unfolding monitored by Emax of Trp fluorescence. (B) Hydrophobic exposure duringGdnHCl-induced unfolding monitored by the intensity at 470 nm of ANS fluorescence.The Emax and ANS data did not change at GdnHCl concentrations above 1.5M. (C) Proteinaggregation kinetics during kinetic refolding initiated by a fast 1:40manual dilution of thedenatured proteins into buffer A. The inset shows the SDS-PAGE analysis of the superna-tant (S) and precipitation (P) fractions of the reacted solutions. M is the marker, and theMWs are 25, 35, 40, 55, 70, 100, 130 and 170 kDa, from the bottom to top, respectively.βA3-crystallin, which cannot self-refold into the soluble native state during kineticrefolding [40], was used as a positive control.

Please cite this article as: K. Zhang, et al., The importance of the last strand at the C-terminus in βB2-crystallin stability and assembly, Biochim.Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbadis.2013.10.001


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Fig. 8. Effects of the V187M and R188H mutations on the formation and stability of βA3/βB2-crystallin heteromer. (A) SEC analysis of the samples incubated for 0 h and 12 h at37 °C after mixing equal molar of βA3- and βB2-crystallins. The peak in the SEC profileof βA3/R188H was fitted by two Lorentz peaks. For clarity, the SEC profiles of β-crystallin homomers are not shown, and the position of the peaks are indicated on thetop axis. Theprotein concentrationwas 0.5mg/ml. (B) Time course study of theheteromerformation analyzed by native-PAGEwith a protein concentration of 0.2mg/ml. The red ar-rows indicate the R188H tetramer and the black arrows indicate the dimer andmonomer,respectively. βA3/R188H reached its equilibriumwithin 2 h incubation at 37 °C. (C) Ther-mal aggregation kinetics of βA3/βB2-crystallin heteromers at 59 °C (solid lines) and 65 °C(dashed lines). The protein concentration was 0.2mg/ml for the heteromers.


the H-bonding network in theWT protein by formingH-bondswith thebackbone C_O of R188 (Fig. 9E). Thus the competition of the substitut-ed E187 resulted in the breakdown of the native backbone H-bonding


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network. The formation of non-native H-bonds distorted the fourthGreek-key motif of βB2-crystallin. It is worth noting that the MD simu-lations were performed from a starting structure based on the crystalstructure of the WT protein. The failure to complete the folding of thefourth Greek-keymotif might bemore deleterious to the correct foldingof the V187E mutant in vivo, which is supported by the overexpressionresults in the E. coli cells and HeLa cells.

Biophysical studies indicated that the effect of the R188H mutationon βB2-crystallin structure and stability was much more complicatedthan those of V187M and V187E. The dimeric structure of βB2-crystallin was not significantly affected by the R188H mutation(Fig. 10A). In the crystal structures of human and bovine βB2-crystallins [33,42], R188 plays an important role in the stabilization ofthe conserved domain pairing interface by forming inter-subunit ionpairs with E75 (Fig. 10B). The substitution of this fully conserved Argby His did not produce charge repulsions between the two domainssince His is also positively charged under neutral pH. However, the ionpairs between R188 and E75 were disrupted by the mutation, whichfurther allowed the water molecules to penetrate in and dissociate thedimeric βB2-crystallin thereafter. Consequently, the monomer–mono-mer binding energy was significantly reduced (Fig. 10E). In the tetra-meric βB2-crystallin structure, R188 is also close to the dimer–dimerinterface although it is not directly involved in the stabilization of thedimer–dimer interactions (Figs. 1 and 10). MD simulations for the WTβB2-crystallin and R188H tetramers indicated that an additional ionpairing network around the mutation site was induced across thedimer–dimer interface by the R188H mutation (Fig. 10D). The averagenumbers of H-bonds/ion pairs in the dimer–dimer interface were 6.51for the WT protein and 9.04 for R188H. The newly introduced ionpairs pulled the dimer–dimer interface closer and strengthened the in-teractions between the dimers (Fig. 10E). The time-course study duringsimulations clearly showed that there was large divergence betweenthe WT and mutated proteins for the dimer–dimer binding energy(Fig. 10F).

4. Discussion

Bothβ- andγ-crystallins are defined by fourGreek-keymotifs divid-ed into two domains [8]. The integrity of the conserved Greek-keymotifs plays a vital role in the structure, stability and function of β/γ-crystallins as well as the onset of congenital cataracts caused by muta-tions [5,8,11,12,43]. The bulk of the N50 mutations in β/γ-crystallins islocated at the Greek-key motifs [43]. Experimental work of γD-crystalllin mutations has also led to the proposal that the distortion ofeven one Greek-key motif will promote the aggregation of β/γ-crystallins and the onset of nuclear cataract thereby [43]. The impor-tance of the Greek-key motifs in β-crystallins has been reflected bythe study of the deletion mutation ΔG61 in βA3-crystallin [20] and thenonsense mutation Q155X in βB2-crystallin [23]. It is worth notingthat the deletion or truncation mutations will lead to a serious foldingproblem for the mutant. In this research, the role of the Greek-key mo-tifs in β-crystallin structure and assembly was investigated using threemissense mutations V187M, V187E and R188H. Our results indicatedthat the two residues, V187 and R188, located at the last strand ofGreek-key motif 4, had quite distinct roles in βB2-crystalllin structureand assembly.

V187 is located in themiddle of the last strand of Greek-key motif 4,and the side chain participates in the formation of the CTD hydrophobiccore (Figs. 1 and 9). Sequence alignments indicated that V187 is con-served in βB2-crystallins across species (data not shown), but can besubstituted by the other hydrophobic amino acids such as Leu, Ile orPhe in human β/γ-crystallin family (Fig. 1B). This suggests that a hydro-phobic residue is necessary for residue 187 inβB2-crystallin, but there isno critical requirement for the size or hydrophobicity of the side chain.Consequently, we found that the V187Mmutation, the substitution by apolar residue, hardly affected the structure, oligomerization and long-



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Fig. 9. Effects of the V187M and V187Emutations on βB2-crystallin structure byMD simulations. (A) Overall structures (top) and the structures of CTD (bottom) of theWT βB2-crystallin(green) and V187M (magenta). (B) Structure of the CTD hydrophobic core. (C) A comparison of the overall structure (top) and the structure of CTD (bottom) between the WT βB2-crystallin and V187E. The WT protein and V187E are rendered in green and yellow, respectively. (D) Disruption of the backbone H-bonding network of βB2-crystallin Greek-key motif4 by the V187E mutation. The positions of codon 187 are indicated by red arrows. (E) The negatively charged side chain of E187 competed with the backbone carboxyl group of R188to form a new H-bond with V152.


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term stability of βB2-crystallin. The main effect of the V187Mmutationon βB2-crystallin was the slightly loosened packing of the CTD hydro-phobic core (Fig. 9B), which further produced a minor/moderate de-crease in the stability of βB2-crystallin or βA3/βB2-crystallin againstheat, chemical denaturant and UV irradiation. When the conserved hy-drophobic residue V187 was replaced by a negatively charged residueGlu, Greek-key motif 4 of βB2-crystallin was destroyed with the break-down of the backbone H-bonding network defining the motif and theintroduction of non-native H-bonds/ion pairs distorting the well-organized backbone structures (Fig. 9C–E). The inability to maintainthe native Greek-key motif 4 impeded the accomplishment of the fold-ing of βB2-crystallin from the nascent polypeptides, and aggregateswere formed from the misfolded proteins in both the E. coli and HeLacells.

Unlike V187, R188 is fully conserved in the β/γ-crystallin family(Fig. 1B). This positively charged residue plays a somewhat similarstabilization role between the N- and C-terminal domains, eitherintra- or inter-subunit, in β- and γ-crystallin structures. In mono-meric γ-crystallins, the equivalent residue R168 is one residue ofthe C-terminal charge cluster and participates the inter-domainion-pairing network to stabilize the interface between the N- andC-terminal domains [44]. In multimeric β-crystallins, R188 plays avital role in the stabilization of the monomer–monomer interface[33,42]. As for βB2-crystallin, the side chain of R188 in CTD of onesubunit forms ion pairs with E75 from the N-terminal domain ofthe other subunit in the dimer (Fig. 10B). The substitution of R188by His blocked the inter-subunit ion pairs and destabilized themonomer–monomer interactions in the dimer thereby. At low pro-tein concentrations, the R188H mutant preferred to exist as a mono-mer (Fig. 4), which is fully consistent with the knowledge obtainedfrom the crystal structure. At high protein concentrations, the dy-namic equilibrium of βB2-crystallin oligomers shifted to the tetra-meric form, a dimer of dimer [8,33,42]. The R188H mutationpromoted the formation of the tetrameric form by the introductionof a new ion-pairing network between the Arg residues adjacent toH188 and D127–D128 from one subunit in the other dimer.


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The relationship between β-crystallin oligomeric state and stabilitymay be muchmore complex than a simple linear relationship. Previousstudies have shown that the removal of the N-terminal extension,which is important for both homo- and hetero-oligomerization, doesnot affect βB1-crystallin stability [45,46]. The dimer disrupting muta-tion S129R is proposed to be a stability-beneficial mutation to decreaseβB1-crystallin aggregation against heat treatment [19]. The A2V muta-tion, which has no impact on dimerization but retard tetramerization,slightly decreases protein stability and increases the propensity of ag-gregation of βB2-crystallin [22]. Herein we found that the perturbationof the dynamic equilibrium by the R188H mutation dramatically re-duced βB2-crystallin stability and promoted protein aggregation whensubjected to stresses. It seems that the stabilization of the tetramerbut not the dissociation of thedimerwas the dark face of theR188Hmu-tation due to the following observations. First, R188H was more proneto denature and aggregate than theWT protein under all in vitro stressconditions when the protein concentration was above 0.2 mg/ml.Secondly, the aggregation of R188H exhibited a strong protein-concentration dependency, and fewer aggregates were observed atlower protein concentrations (Fig. 5). Thirdly, at a low protein concen-tration of 0.1 mg/ml where R188H mainly existed as a monomer(Fig. 4), R188H did not aggregate during heat treatment. Finally, theβA3/R188H heteromer was much more unstable than the WT proteineven though βA3/R188H can associate into larger oligomers. The resultsherein as well as those in literature [19,22,33,36,42,46] suggested thatthe dynamic nature of oligomerization was important to the stabilityand solubility of β-crystallins. The shift of the dynamic equilibriummight be a naturalmechanism to stabilize the proteins and fight againstoff-pathway aggregation when subjected to stresses.

It has been well established that β-crystallins can form bothhomomers and heteromers (reviewed in [5,8]). However, the structuralbasis of heteromer formation remains unclear due to the lack of high-resolution structure. It is interesting to find that the V187M mutation,which had no influence onβB2-crystallin homo-oligomerization, slight-ly affect the hetero-oligomerization as revealed by a smaller apparentMW in the SEC profile. The R188H mutation, which dramatically



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Fig. 10. Structural analysis of the perturbation of βB2-crystallin oligomeric equilibrium by the R188H mutation. (A) Dimeric structures of the WT βB2-crystallin (green) and the mutantR188H (cyan). (B) Disruption of the inter-subunit ion pairs between R188 and E75 by the R188Hmutation. (C) Tetrameric structures of theWT βB2-crystallin (green) and R188H (cyan).(D) Formation of an additional ion-pairing network induced by the R188Hmutation. (E) Monomer–monomer and dimer–dimer binding energies in the dimeric and tetrameric proteins.(F) Time course study of the changes of the binding energy for dimer–dimer association in the tetrameric proteins during MD simulations.


UNCOimpaired the dynamic equilibrium of homo-oligomerization, facilitated

heteromer formation by accelerating the association rate and increasethe size distribution of heteromers (Fig. 8). These observations sug-gested that the last strand of βB2-crystallin might play a regulatorybut not essential role in heteromer formation. Similar to homomers, itseems that the stability of heteromers also depended on the dynamicassociation/dissociation process. The aggregation of the βA3/R188Hheteromers was more serious than the sum of equivalent molar ofhomomers, suggesting that there were considerable structural adjust-ments during heteromer formation.

From the view at the protein level, cataract is closely associatedwiththe deposition of the soluble structural proteins in the lens, and thelarge aggregates scatter the light directly and/or are toxic to the cellsto perturb the normal structure and function of the lens [11,12,47]. Un-like the thoroughly studied γ-crystalllin mutations, the molecularmechanisms remain to be elusive for most of the β-crystalllin muta-tions, which may partially be caused by the complicated folding path-ways of the multimeric proteins [48] and the dynamic nature of thehomomeric/heteromeric β-crystallin oligomers. In this research, wefound that the mutations of two adjacent residues in the last strand ofβB2-crystallin Greek-key motif 4 might lead to congenital nuclear


cataract via quite dissimilar mechanisms: V187M loosened the CTD hy-drophobic core, V187E was a Greek-key motif breaker, while R188Hperturbed the oligomeric equilibrium. Our results reinforced the impor-tance of the Greek-key motifs in the structural integrity, folding andstability of β/γ-crystallins. More importantly, we showed that the per-turbation of the dynamic equilibrium of β-crystallin oligomers mightbe an important mechanism in cataract formation caused by inheritedmutations. The selective stabilization of a specific high-order oligomerby mutationsmay also be deleterious. The long-term stability and func-tion of β-crystallins are achieved by a precise balance/dynamics inhomo- and hetero-oligomerization determined by the informationencoded in the primary sequences.

Acknowledgements

This study was supported by funds from the Ministry of Science andTechnology of China (2010CB912402 and 2012BAI08B01), National Nat-ural Science Foundation of China (81371001 and 81130018), ZhejiangKey Innovation Team Project of China (2009R50039), Zhejiang Key Lab-oratory Fund of China (2011E10006) and Project of National Clinical KeyDiscipline of Chinese Ministry of Health. Sha Wang was supported by a



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fellowship from Tsinghua-Peking Center for life Sciences, TsinghuaUniversity.

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The importance of the last strand at the C-terminus in βB2-crystallin stability and assembly

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