Biochemical Characterization of G64W Mutant of Acidic Beta-Crystallin 4

Experimental Eye Research 186 (2019) 107712

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Experimental Eye Research

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Biochemical characterization of G64W mutant of acidic beta-crystallin 4 T Wenqian Lia,b, Qingshan Jic, Zhongjie Weid, Yu-lei Chene, Zhiyong Zhanga, Xueying Yina, Samaneh Khodi Aghmiunia, Muziying Liua, Weirong Chenb, Lei Shic, Quan Chena, Xinzheng Dua, ∗ ∗∗ Li Yua, Min-Jie Caoe, Zhulou Wangd, Shaohui Huangd,g, Tengchuan Jina,f, , Qiwei Wangb, a Division of Molecular Medicine, Hefei National Laboratory for Physical Sciences at Microscale, CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China b Zhongshan Ophthalmic Center, Xian Lie South Road #54, Guangzhou, Guangdong, China c Department of Ophthalmology, The First Aﬃliated Hospital, University of Science and Technology of China, Hefei, Anhui, China d Institute of Biophysics, Chinese Academy of Sciences, Beijing, China e College of Food and Biological Engineering, Jimei University, Xiamen, Fujian, China f CAS Center for Excellence in Molecular Cell Science, Shanghai, China g School of Biological Sciences, University of Chinese Academy of Sciences, Beijing, China

ARTICLE INFO ABSTRACT

Keywords: Crystallins are structural proteins in the lens that last a lifetime with little turnover. Deviant in crystallins can Congenital cataracts cause rare but severe visual impairment, namely, congenital cataracts. It is reported that several mutations in the Acidic β-crystallin 4 acidic β-crystallin 4 (CRYBA4) are related to congenital cataracts. However, the pathogenesis of these mutants is G64W mutant not well understood at molecular level. Here we evaluate the biochemical properties of wild type CRYBA4 Protein folding (CRYBA4WT) and a pathogenic G64W mutant (CRYBA4G64W) including protein folding, polymerization state and Fluorescence correlation spectroscopy protein stability. Furthermore, we explore the diﬀerences in their interactions with α-crystallin A (CRYAA) and Protein interaction basic β-crystallin 1 (CRYBB1) via yeast two-hybrid and pull-down assay in vitro, through which we ﬁnd that G64W mutation leads to protein misfolding, decreases protein stability, blocks its interaction with CRYBB1 but maintains its interaction with CRYAA. Our results deepen our understanding of the pathogenesis of congenital cataracts.

1. Introduction light, oxidation, or the use of steroid (Robman and Taylor, 2005; Bassnett, 2002). The prevalence of cataracts increases sharply for The phenomenon of the lens opacification is called cataracts people over 50 years old (Bassnett, 2002). 8%–25% of the congenital (Moreau and King, 2012b). It can occur in one eye or both eyes. Cat- cataracts are caused by genetic factors and 70% of congenital cataracts aracts can be caused by various reasons such as heredity, aging, local are related to the abnormality of the lens (Haargaard et al., 2004; nutrition supply abnormality, immune abnormality, metabolic ab- Hejtmancik, 2008; Yi et al., 2011). normality, external damage, poisoning and radiation (Ricker et al., Human lens are one of the most important tissues in eyes. It is re- 1994; Chen et al., 2008; Ming et al., 2003; Datta et al., 1997). Among sponsible for transmitting light and focusing the light on the retina. them, aging and heredity are the most important factors. Cataracts can Therefore, the transparency of the lens is prior to the other character- be classified by the age of onset. Cataracts which develop during the istics (Benedek, 1971; Tardieu, 1988; Trokel, 1962). The organized fetal period or within one year after birth are called congenital catar- arrangement of the lens fiber cells is critical for light transmission and acts; those who develop cataracts within ten years of birth are called lens transparency (Kuszak et al., 2004). Crystallins are the major juvenile cataracts; cataracts which develop before the age of 45 are structural protein in the lens. About 90% of water-soluble proteins in called presenile cataracts and cataracts develop after the age of 45 are the lens are crystallins, which contains 40% α-crystallins, 35% β-crys- called age-related cataracts (Shiels and Hejtmancik, 2013). The in- tallins and 25% γ-crystallins (Wistow and Piatigorsky, 1988; Wistow, cidence of age-related cataracts is high due to the absence of protein 2012; Billingsley et al., 2006). The solubility and the structural stability turnover in the lens fiber cells and the external damage such as UV- are essential to crystallins since its concentration in the center of human

∗ Corresponding author. Division of Life Sciences and Medicine, University of Science & Technology of China, Hefei, Anhui, 230007, China. ∗∗ Corresponding author. Zhongshan Ophthalmic Center, Xian Lie South Road #54, Guangzhou, Guangdong, 510060, China. E-mail addresses: [email protected] (T. Jin), [email protected] (Q. Wang). https://doi.org/10.1016/j.exer.2019.107712 Received 29 March 2019; Received in revised form 19 June 2019; Accepted 23 June 2019 Available online 26 June 2019 0014-4835/ © 2019 Elsevier Ltd. All rights reserved. W. Li, et al. Experimental Eye Research 186 (2019) 107712

Fig. 1. Structure and sequence alignment of CRYBA4. (A) The crystal structure of CRYBA4 (PDB ID: 3LWK). (B) Sequence alignment of CRYBA4 in selected species. lens can reach up to 450 mg/ml and last a lifetime with nearly no CRYBA4 gene, namely the c.225G > T, p.G64W mutant, which was protein turnover due to the lack of organelles in the lens fiber cells and reported to cause an autosomal dominant microcornea and congenital arterial or venous blood circulation in the lens (Hejtmancik, 1998; nuclear cataracts in 2010 (Zhou et al., 2010). Gly64 is conserved among Shiels and Hejtmancik, 2013; Bassnett, 2002, 2009; Costello et al., different species through sequence alignment (Fig. 1B). However, the 2013; Morishita et al., 2013; Fagerholm et al., 1981). molecular pathogenesis mechanism is not clear. Here, we evaluated the Both β-crystallins and γ-crystallins belong to the βγ-crystallin family stability, thermodynamic characteristic and protein folding of WT and can fold into two similar domains. Each domain consists of two CRYBA4 and G64W mutant. Furthermore, we studied the effects of motifs that contains about 40 amino acids and consists of four anti- G64W mutant on its protein-protein interactions with CRYBB1 and parallel β-sheets called “Greek-key” (Slingsby and Clout, 1999; CRYAA. These studies provide experimental evidence on the properties Serebryany and King, 2014). Meanwhile, they function through the of the mutant and its molecular pathogenic effects. formation of hetero-oligomers or homo-oligomers. Therefore, the effects of mutations in β-crystallins are highly complex. According to the protein isoelectric point, β-crystallins can be divided into acidic crys- 2. Materials and methods tallins, including acidic β-crystallin 1 (CRYBA1), 2(CRYBA2), 3(CRYBA3) and 4(CRYBA4), and basic crystallins which consist of basic 2.1. Expression and purification of CRYBA4 and CRYBB1 protein β-crystallin 1(CRYBB1), 2(CRYBB2) and 3(CRYBB3) (Slingsby and Bateman, 1990). CRYBA4 is composed of 196 amino acids and accounts The CRYBA4 and CRYBB1 genes were amplified from a human G64W for approximately 5% of acidic β-crystallins in the lens (Lampi et al., cDNA library. The CRYBA4 gene was constructed by site-directed 1997). The crystal structure of CRYBA4 (PDB ID: 3LWK) was solved in mutagenesis as described previously (Zhou et al., 2010; Cormack, WT G64W 2010 as a homodimer (Fig. 1A). CRYBA4 has 47% sequence identity to 2001). CRYBA4 and CRYBA4 gene were cloned into pET28a CRYBB1 and 75% sequence identity to CRYBA3. It can interact with vector with an N-terminal His-tag and into pET30a vector with N- CRYBB1 and CRYBB2 to form hetero-oligomers by exchanging subunits terminal His-tag and maltose binding protein (MBP) tag, respectively. (Serebryany and King, 2014) and participate in maintaining the trans- The recombinant plasmids were transformed into Rosetta™ BL21 (DE3) parency and stability of the lens. Several CRYBA4 mutations have been strain for protein expression. Positive clones were picked into LB reported to cause dominant congenital cataracts, while the mechanism medium containing 50 μg/ml kanamycin and incubated at 37 °C. Ex- is little known (Billingsley et al., 2006; Zhou et al., 2010; Kumar et al., pression of recombinant protein was induced at an OD600 of 1.5 by 2013; Bateman et al., 2003). adding IPTG to a final concentration of 0.4 mM for 4 h at 16 °C. Cells In order to gain molecular insights on CRYBA4 mutation in the were harvested by centrifugation. Bacteria were suspended in a ratio of pathogenesis of cataract, we focused on one of the mutations in one to ten (w/v) with nickel binding buffer (250 mM NaCl, 5 mM imidazole, 20 mM Tris-HCl pH 8.0) plus 0.1 mM PMSF and were lysed by

2 W. Li, et al. Experimental Eye Research 186 (2019) 107712 sonication. The supernatant was loaded onto a 5 ml Hisprep™ IMAC high glucose medium plus 10% fetal bovine serum (FBS), 100 U/ml column (GE Healthcare) and the bound protein was eluted by elution penicillin and 0.1 mg/ml streptomycin and were seeded in 24-well buffer (500 mM NaCl, 130 mM imidazole, 20 mM Tris-HCl pH 8.0). The plates prior to transfection. 36 h post transfection, supernatants of cell WT final protein was further purified by loading onto Superdex™ 200 10/ lysate transfected with pEGFPN1, CRYBA4 -pEGFPN1 and G64W 300 GL column in gel filtration buffer (250 mM NaCl, 20 mM Tris-HCl CRYBA4 -pEGFPN1 respectively were collected and quick-frozen in pH 8.0). When purifying CRYBB1 which was cloned into pET30a vector liquid nitrogen prior to FCS analysis. Supernatant of cell lysate without with N-terminal His-tag and maltose binding protein (MBP) tag, TEV any plasmids transfected was used to establish baseline fluorescence and 5 mM DTT were added into eluted protein for MBP tag removal intensity in our FCS experiments. after nickel elution. The mixture was incubated at 4 °C overnight. After The FCS single particle detection studies were carried out using a dialysis O/N for removing DTT and imidazole, the protein was then CorTector™ SX100 bench-top FCS instrument (LightEdge Technologies, purified again using a 5 ml Histrap™ IMAC column (GE Healthcare). Zhongshan, China). The structure parameter S describes the shape of The final protein was further purified by loading onto Superdex™ 200 the FCS fluorescence detection volume. S was experimentally measured 10/300 GL column in gel filtration buffer (250 mM NaCl, 20 mM Tris- to be 8.2 using 1 nM Rhodamine Green solution, which reflects good HCl pH 8.0). alignment of the fluorescence excitation and detection optical pathways. The excitation 488 nm laser was set at an output level of 18 mW, 2.2. Western blotting with an actual laser power of 96.1 μW measured after the microscope objective. After thawing, the lysate samples were cleared by cen- Lysate of bacterial cell samples expressing CRYBA4WT and trifugation. To achieve single-particle FCS experiment, we set up FCS CRYBA4G64W were boiled in SDS-PAGE sample buffer and centrifuged experiments with 2 s sampling duration and repeated the experiment at 12000 g for 5 min. The supernatant was loaded onto SDS-PAGE gel 1000 times for each cell lysate sample diluted to 10 pM. For single- alongside with a molecular weight marker. Transfer proteins from the particle FCS analysis, each 2 s FCS data with only one particle pulse PAGE gel to a nitrocellulose membrane (100 mA for 2 h) was followed peak was selected and analyzed. The autocorrelation curves were fitted by the incubation of the nitrocellulose membrane in blocking buffer using the analysis software (Correlation Analysis, V2.0) and its size (5% skim milk (w/v), 0.05% Tween 20, 150 mM NaCl, 100 mM Tris- distribution analytical module. HCl pH 7.5) for 1 h at room temperature. The membrane was then incubated with mouse anti-His antibody (Sangon Biotech) and HRP la- 2.6. Circular dichroism beled goat anti-mouse antibody (Sangon Biotech) for 1 h at room temperature sequentially. The positive bands were developed in TMB In order to detect the differences in secondary structure and stability substrates and visualized with a gel imager (Qinxiang). between CRYBA4WT and CRYBA4G64W, circular dichroism (CD) spectra data were recorded using Chirascan Spectrometer (Applied 2.3. Antibiotic resistance measurements Photophysics, Leatherhead, UK). Two far-ultraviolet measurements were collected from 190 nm to 260 nm in 1 nm step with a 4 s averaging CRYBA4WT and CRYBA4G64W genes were cloned into the pMB1-tet- time at 20 °C. The path length and protein concentration were 1 mm pARA-bla-link_long (LFM10, courtesy of Dr. Bardwell) vector (Foit and and 0.3 mg/ml, respectively. The proteins were diluted with phosphate Bardwell, 2013; Foit et al., 2009), which had a linker containing re- buffer saline (PBS) buffer (pH = 7.5). Thermal denaturation data were petitive glycines and serines in TEM1-b-lactamase to harbor the target collected per 5 °C from 20 °C to 90 °C at a rate of 2 °C/min. CD signals at protein in the designated internal loop region (Foit et al., 2009). The 222 nm were used to evaluate the thermostability of CRYBA4WT-MBP plasmids were transformed into NEB 10β competent cells and Spot titer and CRYBA4G64W-MBP fusion proteins. test was used to assess β-lactamase antibiotic resistance. Positive clones were picked into LB and incubated in 37 °C until OD600 reached a value 2.7. Molecular dynamics of 0.5. Then the bacteria were collected by centrifugation and re- WT suspended with 170 mM NaCl until OD600 reached a value of 1.0. In order to gain insights into the folding dynamics of CRYBA4 and − Bacteria were diluted from 100 to 10 6. The diluted bacteria were CRYBA4G64W, we performed molecular dynamics (MD) simulation spotted on LB plate containing 0.5 mg/ml of ampicillin, 50 μg/ml of study. Two independent molecular dynamics simulation were applied tetracycline and 0.2% of arabinose. Plate was incubated at 37 °C for using GROMACS software package. CRYBA4WT and CRYBA4G64W were 18 h and the growth of the cells was used to evaluate the folding of used for molecular dynamics (MD) simulation on a ligand-free form. proteins. The crystal structure of CRYBA4WT was downloaded from PDB (ID: 3LWK) and used as a structural template of CRYBA4G64W. Structural 2.4. Crosslink model of CRYBA4G64W was constructed using Phyre2 (http://www.sbg. bio.ic.ac.uk/phyre2/html/page.cgi?id=index). OPLS-AA force field CRYBA4WT-MBP and CRYBA4G64W-MBP fusion proteins were di- was chosen for MD (Kaminski et al., 2001). The protein models were luted to 10 μM and then incubated with 1 mM disuccinimidyl suberate immerged in cubic box filled with water molecules (SPC/E water (DSS) which was dissolved in DMSO for 1 h at room temperature. Then model). Ions were added to equilibrate the system and then the system 1 M Tris-HCl pH 8.0 was added into the reaction mixture reaching a was conducted under an NPT ensemble. The timescale of simulation final concentration of 50 mM to stop the reaction. The samples were was 100 ns and time step was 2 fs. The temperature of system was 300 K further incubated for 15 min at room temperature and then finally and the pressure was 1 atm. To evaluate the stability of the two pro- analyzed by SDS-PAGE. MBP was used as a control. teins, root mean square deviation (RMSD) was calculated respectively. The quality of simulations was evaluated by VMD via trajectory ana- 2.5. FCS (Fluorescence correlation spectroscopy) lysis. To evaluate the contribution of each atom to the destabilization of protein after simulation with the initial model, root mean square fluc- Fluorescence correlation spectroscopy (FCS) was used to detect the tuation (RMSF) was calculated respectively. The structure alignment of formation of CRYBA4G64W-EGFP aggregation in transfected cell lysates CRYBA4WT and CRYBA4G64W after simulation was performed by Pymol. at single particle level. The pEGFPN1 vector (Clontech), which contains a C-terminal EGFP, was used to make fusion proteins overexpressed in 2.8. Pull-down assay mammalian cells. CRYBA4WT and CRYBA4G64W genes were cloned into the vector for cell transfection. HEK-293T cells were cultured in DMEM In order to investigate the impact of mutant on the interaction with

3 W. Li, et al. Experimental Eye Research 186 (2019) 107712 its partner protein CRYBB1 in vitro, pull-down assay with highly purified protein samples was performed as described (Xu et al., 2017). 5 μM CRYBA4WT-MBP and CRYBA4G64W-MBP fusion proteins were incubated with 5 μM CRYBB1 in IP buffer (10 mM Tris-HCl pH 8.0, 250 mM NaCl, 0.1 mM PMSF) at 4 °C for 4 h. Then the mixture was ® immobilized by Strep-Tactin beads (IBA Life Sciences) at 4 °C for 1 h. After being washed for 3 times with IP buffer, the immuno-precipitated proteins were analyzed by SDS-PAGE and immunoblotted using anti- Strep antibody and anti-His antibody.

2.9. Yeast two-hybrid

Yeast two-hybrid was utilized to detect protein-protein interaction in the yeast cells (Bruckner et al., 2009). Matchmaker GAL4 Two-Hy- brid System 3 (Clontech) was used for yeast two-hybrid assay following Fig. 3. Protein folding properties analysis in E. coli. Antibiotic resistance WT G64W β manufacturer's instruction. CRYBA4WT and CRYBA4G64W genes were measurement of CRYBA4 and CRYBA4 were investigated via TEM1- - cloned into pGAD-T7 as prey plasmids. Meanwhile, CRYAA and lactamase system, ubiquitin (PDB: 1ubq) was used as a control. 1ubq: ubiquitin. CRYBB1 genes were cloned into pGBK-T7 as bait plasmids. For interaction identification, prey constructs and bait constructs were co- protein folds well, the two halves of β-lactamase will be brought to- transformed into S. cerevisiae strain AH109 yeast cells. Cells were plated gether close enough to associate, thereby conferring the expected re- on SD/-Trp/-Leu plates and grown at 30 °C for two days. Then the sistance to β-lactam antibiotics. When the target protein fails in correct positive colonies were picked and spotted on both SD/-Trp/-Leu plates folding, resistance to β-lactam antibiotics will be depredated by the and SD/-Trp/-Leu/-His plates plus 2 μg/ml X-α-Gal and incubated at cell's protein quality control machinery. Therefore, TEM1-β-lactamase 30 °C for three days. Empty vectors were co-transformed as negative will be separated into two halves which results in reduced resistance of controls. host cells to β-lactam antibiotics (Foit et al., 2009; Galarneau et al., 2002). In this study, the target protein was flanked by a linker and fused β 3. Results into TEM1- -lactamase. The antibiotic resistance of bacteria expressing fusion protein was measured by spotting dilutions of cells onto plates 3.1. CRYBA4G64W is prone to form inclusion body when expressed in E. coli containing 0.5 mg/ml ampicillin (Fig. 3). Ubiquitin tripartite fusion protein was used as a control. There were no colonies expressing G64W It is reported that CRYBA4 was unstable when it was expressed in CRYBA4 growing on the plate. On the contrary, colonies expressing CRYBA4WT were found on the fourth line at which the dilution was bacteria or in mammalian cells lacking the help of heteromeric partner, −3 ie CRYBB1 or CRYBB2 (Bateman et al., 2003; Marin-Vinader et al., 10 . This result further supported that the point mutation of G64W 2006). Nevertheless, we obtained pure soluble CRYBA4 with only N- impaired the correct protein folding of CRYBA4 to some extent. terminal His-tag in E. coli express system. Around 48% of the CRY- WT BA4 protein with only N-terminal His-tag was expressed in super- 3.3. G64W increases self-interaction in vitro and in mammalian cell natant after cell lysis. While under the same condition, the vast majority G64W of the CRYBA4 existed in inclusion bodies, with only around 2% in To further investigate the biochemical differences between supernatant as estimated by Western blot (Fig. 2A and B). It is reported CRYBA4WT and CRYBA4G64W, an N-terminal MBP tag was utilized to that partially folded intermediate may be the cause of inclusion bodies promote the solubility of mutant. MBP can promote the solubility and (Serebryany and King, 2014; Evans et al., 2008). This result indicated crystallization of target protein when fused to its C-terminus (Jin et al., ff that the G64W mutant might a ect its folding properties of CRYBA4. 2017; Fang et al., 2018). CRYBA4WT-MBP and CRYBA4G64W-MBP fusion protein were purified using Nickel column and Fast protein liquid 3.2. G64W affects its folding in E. coli chromatography (FPLC) (Fig. 4A). Gel filtration chromatography was carried out to identify oligomerization state of CRYBA4WT-MBP and To explore whether proper protein folding is affected by the mu- CRYBA4G64W-MBP fusion protein (Fig. 4B). The elution volume of tation, the protein folding of CRYBA4WT and CRYBA4G64W were further CRYBA4WT-MBP fusion protein was around 15 ml, which corresponding analyzed via TEM1-β-lactamase system, which is a system for protein to a molecular weight of about 150 kDa (Fig. 4C), suggesting it is a folding screening. TEM1-β-lactamase is an enzyme which provides dimer in solution. Nevertheless, the elution volume of CRYBA4G64W- multi-resistance to β-lactam antibiotics (Foit et al., 2009). When target MBP fusion protein was near 9 ml, which corresponding to a molecular

Fig. 2. CRYBA4G64W is prone to form inclusion body when overexpressed in E. coli. (A) Pellet and supernatant of CRYBA4WT and CRYBA4G64W were analyzed by SDS-PAGE. (B) Western-blotting analysis of the same gel in (A). P: pellet. S: supernatant.

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Fig. 4. G64W promotes self-interaction of CRYBA4. (A) SDS-PAGE analysis of CRYBA4WT-MBP and CRYBA4G64W-MBP fusion protein. (B) Oligomerization state analysis of CRYBA4WT-MBP and CRYBA4G64W-MBP fusion protein by SEC. (C) Molecular standard of Superdex-200 (24 ml) gel filtration column. (D) 5 μM purified CRYBA4WT-MBP and CRYBA4G64W-MBP fusion protein were cross-linked by DSS, respectively. MBP was used as control. (E) Protein self-interaction analysis by yeast two-hybrid. (F) CRYBA4WT -pGAD-T7 and pGBK-T7 or CRYBA4G64W -pGAD-T7 and pGBK-T7 were con-transformed to AH109 to exclude non-specific interaction. (G) The cell lysate of HEK-293T cells transfected with CRYBA4WT-EGFP, CRYBA4G64W-EGFP and EGFP respectively for single-particle FCS analysis. Histograms of diameter distributions of CRYBA4WT-EGFP (blue), CRYBA4G64W-EGFP (red) and EGFP (black) are shown. (H) Probability of small particles (black, diameter ≤26 nm) and large particles (grey, diameter > 26 nm). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

weight of more than 670 kDa (Fig. 4C), indicating the formation of Characteristic diffusion correlation time (τd) of single fluorescent par- large protein aggregates. This result demonstrates that CRYBA4WT-MBP ticles were derived from the fluorescence autocorrelation decay curves G64W forms dimer in vitro and the molecular weight of the CRYBA4 -MBP obtained from single-particle FCS experiments. τd-value reflects the fusion protein was much higher than that of CRYBA4WT-MBP (Fig. 4B). residence time of a freely diffusing fluorescence particle in the FCS WT Crosslink experiment also showed that the CRYBA4 -MBP presented fluorescence detection volume. The larger the τd, the bigger of the mainly as dimers, while the MBP-mutant existed as aggregates single-particle diameter. Our FCS results show that majority of the (Fig. 4D). This result demonstrated that CRYBA4G64W-MBP fusion pro- CRYBA4WT-EGFP and CRYBA4G64W-EGFP fluorescent particles, de- tein is more likely to form aggregates in vitro. The ability of self-inter- tected in cell lysates, have diameters of less than 26 nm, with a higher action was further evaluated by yeast two-hybrid in yeast cells. proportion for the CRYBA4WT-EGFP particles compared to Transformed yeast colonies were picked and spotted on both SD/-Trp/- CRYBA4G64W-EGFP particles. Furthermore, there were more Leu plates and SD/-Trp/-Leu/-His plates supplemented with 2 μg/ml X- CRYBA4G64W-EGFP fluorescent particles with diameters larger than α-Gal respectively (Fig. 4E and F). X-α-Gal which is a chromogenic 40 nm than the WT; very large particles with diameters of 140 nm could substrate used to detect α-galactosidase activity was added into SD/- be detected for the G64W mutant, but neither the WT nor the EGFP Trp/-Leu/-His plates. In yeast, α-galactosidase, encoded by the MEL1 control (Fig. 4G). The diameter of EGFP monomer is about 4 nm ac- gene, will be expressed when GAL4 is activated. The expression of α- cording to its crystal structure. Considering the volume of CRYBA4 and galactosidase hydrolyzes X-α-Gal in the surrounding media and causes the loop between EGFP and CRYBA4, we chose 26 nm as a threshold the yeast colonies turning blue. Thus the stronger the self-interaction is, diameter to differentiate the monomeric and oligomeric fluorescence the higher the expression of MEL1 gene will be, and therefore the particles. For EGFP molecules, 210 single particles were analyzed, re- darker the blue color will be. The color of CRYBA4WT was lighter than sulting in a small particle (i.e., diameter ≤26 nm) probability of CRYBA4G64W, which suggests the strength of the self-interaction of 0.930 ± 0.007 and a large particle (i.e., diameter > 26 nm) prob- CRYBA4WT was weaker than CRYBA4G64W. ability of 0.070 ± 0.007. For CRYBA4WT-EGFP, 237 particles were We used FCS to estimate degrees of aggregation of the G64W mu- analyzed, resulting in a small particle probability of 0.885 ± 0.038 tant compared to its WT counterpart expressed in mammalian cells. and a large particle probability of 0.115 ± 0.038. Significantly, for

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Fig. 5. G64W mutant is less stable than WT CRYBA4. (A) CD spectroscopic analysis of MBP, CRYBA4WT-MBP and CRYBA4G64W-MBP fusion protein. (B) Thermodynamic stability analysis of MBP, CRYBA4WT-MBP and CRYBA4G64W-MBP fusion protein. (C) Structural model of CRYBA4G64W constructed by Phyre2. (D) RMSD ﬂuctuation of the protein backbones during Molecular Dynamics (MD) simulation of CRYBA4WT and CRYBA4G64W. Molecular dynamics was calculated by in the time scale of 100 ns. (E) RMSF of Cα atom of the main chain of CRYBA4WT and CRYBA4G64W. (F) Structure alignment of CRYBA4G64W and CRYBA4WT after simulation.

CRYBA4G64W-EGFP protein, a total of 255 particles were analyzed, re- attributed this result to the presence of MBP tag, which is two times sulting in a probability of small particles of 0.671 ± 0.012 and a large larger and is primarily made up of α-helix. In the meantime, the particle probability of 0.329 ± 0.012. These results indicate that the CRYBB1-MBP fusion protein was used as a control, which showed si- G64W mutant has a tendency of forming larger fluorescent particle milar CD spectrum to the CRYBA4-MBP fusion protein (Fig. 5A). In compared to its WT counterpart (Fig. 4H). thermal stability evaluation by CD spectrum, the fitted Tm value for CRYBA4WT-MBP and CRYBA4G64W-MBP fusion proteins were 57.18 ± 0.78 °C and 54.82 ± 0.43 °C (Fig. 5B), respectively, sug- 3.4. CRYBA4G64W is less stable than WT gesting that the thermodynamic stability of CRYBA4WT-MBP fusion protein was higher than that of CRYBA4G64W-MBP fusion protein. The Explorations on the stability of β-crystallins are difficult since MBP, CRYBA4WT and the mixture of equal molar of MBP and CRY- thermodynamic stability is in some cases inconsistent with thermal BA4WT were also used as controls. The results showed that the Tm of aggregation (Evans et al., 2008). Although we verified that CRY- CRYBA4WT was 70.47 ± 0.44 °C which was higher than MBP fusion BA4G64W protein was prone to form aggregates in vitro, it is still ne- protein and the Tm of the mixture were 56.14 ± 0.56 °C and cessary to compare the thermodynamic stability for CRYBA4WT and 73.12 ± 0.28 °C, respectively. These results showed that, the fusion CRYBA4G64W proteins. Circular dichroism spectroscopy was carried out protein was not simply the mixture of two proteins. These controls were to detect the differences of thermodynamic stability between CRY- performed to assure the results do not reflect denaturation of the entire BA4WT and CRYBA4G64W (Fig. 5A and B). CRYBA4WT, CRYBA4WT-MBP fusion protein rather than single domains. Furthermore, molecular and CRYBA4G64W-MBP fusion proteins were used for the assay due to dynamic (MD) simulation was performed to study the protein dynamics the insolubility of the mutant protein without any tags. The CD result of of CRYBA4WT and CRYBA4G64W. Structural model of CRYBA4G64W was CRYBA4WT showed negative band between 210 and 220 nm, which is in constructed using Phyre2 server (Fig. 5C). Structural stability of the well agree with the high β-sheet content of the CRYBA4 protein systems in the trajectory timescale (100 ns) was evaluated via GRO- (Fig. 5A). However, both in CRYBA4WT-MBP and CRYBA4G64W-MBP MACS and RMSD was obtained (Fig. 5D). The fluctuation of RMSD for fusion proteins showed negative peaks between 210 and 220 nm. We

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G64W mutant was larger than that for CRYBA4WT, which indicated that pET28a. The protein expression of CRYBA4WT, CRYBA4G64W and G64W mutant decreased the stability of CRYBA4. Meanwhile, we cal- CRYAA could be detected by western-blotting. After cell lysis by soni- culated the RMSF of Cα atoms of the main chain of CRYBA4WT and cation, pellet and supernatant were analyzed by western-blotting CRYBA4G64W (Fig. 5E), which represents the contribution of each atom (Fig. 7B). The result showed that in WT group, CRYBA4WT could ex- to the destabilization of protein. The structure alignment after simula- press in supernatant with CRYAA. However, in G64W group, there was tion with the initial model was shown in Fig. 5F to show the con- only CRYAA in supernatant. CRYBA4G64W was still in pellet as inclusion formation change of CRYBA4 after Gly64 was mutated to Trp. The re- body. This result indicated that CRYAA was unable to prevent CRY- sult showed that the RMSF of CRYBA4G64W is higher than CRYBA4WT in BA4G64W from precipitation, which to some extent explains why CRY- most Cα atoms, which means G64W mutation leads to the global de- BA4G64W remains precipitated in the lens regardless the presence of stabilization of protein instead of causing merely local structural fluc- functional CRYAA. tuation. Further structure alignment revealed a significant change in local structure centering at Trp84 (highlighted by red dashed oval). While in the G64W mutant, the loop containing W84 was pushed away. 4. Discussion Furthermore, Gly64 is not the interface of CRYBA4 dimer. These results demonstrate that G64W mutation affects both local and global structure Vision impairment caused by congenital cataracts brings huge ne- of CRYBA4. All the above experimental and computational results de- gative impact on the patient's physical and mental health. About 25% of monstrated that CRYBA4G64W was thermodynamically less stable than congenital cataracts were due to inherited mutations. Although catar- CRYBA4WT. acts is associated with protein aggregation, the molecular mechanism of congenital cataracts is complicated to some extent. Generally speaking, 3.5. G64W blocks its interaction with CRYBB1 missense mutations can be either stable or unstable and are more likely to be unstable. As for the stabilizing mutations, there is a high prob- In addition to folding and dynamic properties, we further studied ability that these corresponding residues can retain the function of the effect of the G64W mutation on its interaction with partner pro- proteins. But for destabilizing mutations, they can cause loss or gain of teins, which may also be involved in pathogenesis of cataracts. It has function and protein misfolding thereby causing aggregation or pre- been reported that CRYBB1 could help to improve the solubility of cipitation which leads to different diseases such as Alzheimer, CRYBA4 in HeLa cells and E. coli when co-expressed with CRYBA4 Parkinson disease and cataracts (Hartl, 2017). CRYBA4, which takes up (Marin-Vinader et al., 2006). Moreover, CRYBB1 is able to interact with 5% of β-crystallins, causes congenital cataracts when Gly64 is mutated CRYBA4 and further form hetero-oligomers (Bateman et al., 2003). to Trp. In this study, we evaluated the consequence of the disease Therefore, yeast two-hybrid system was adopted to investigate the ef- causing G64W mutant of CRYBA4, in the aspect of protein expression fects of G64W mutation on protein-protein interaction. The strength of level, folding properties, thermal stability, molecular dynamics, mole- protein-protein interaction was evaluated by SD/-Trp/-Leu/-His plates cular size, and protein-protein interactions. Our results showed that the supplemented with 2 μg/ml X-α-Gal. On SD/-Trp/-Leu/-His plates, co- formation of precipitation might result from the protein misfolding lonies could grow if CRYBA4WT was present (Fig. 6A). However, there which further lead to the decreasing of protein stability and the abol- was no colonies growing on SD/-Trp/-Leu/-His plate when CRY- ishment of its interaction with CRYBB1 which is necessary for protein BA4G64W was present. This result demonstrated that CRYBA4G64W could stability of CRYBA4. Moreover, the strength of CRYBA4-CRYAA inter- not interact with CRYBB1 in yeast cells. Empty vectors were performed action was maintained. However, the chaperone-like function of as control (Figs. 4E and 6B). Pull-down assay was also used to confirm CRYAA failed to rescue the precipitation of the mutated CRYBA4 in this result using Strep-tag-labeled CRYBB1 and His-tag-labeled MBP- vitro. All these negative effects may lead G64W mutant to precipitation CRYBA4 fusion protein (Fig. 6C). CRYBB1-MBP fusion protein was and therefore cause cataracts. Furthermore, it has been reported that purified by Hisprep™ IMAC column and then MBP was cleaved by TEV I90F, W130E mutant of γD-crystallin and T5P mutant of γC-crystallin and CRYBB1 was isolated from MBP via Hisprep™ IMAC column, pur- can be rescued by α-crystallin (Serebryany et al., 2016; Moreau and ified CRYBB1 protein was obtained through Superdex™ 200 10/300 GL King, 2012a; Liang, 2004). However, there are still several mutants column. Interaction between CRYBA4G64W-MBP and CRYBB1 was whose precipitation cannot be rescued by α-crystallin. For example, weaker than that between CRYBA4WT-MBP and CRYBB1. These results V75D, W42E, I4F mutant of γD-crystallin and I4F mutant of γB-crys- revealed that G64W mutation weakens the interaction between tallin (Serebryany et al., 2016; Moreau and King, 2012a; Liu et al., CRYBA4 and CRYBB1, which may contribute to the formation of pre- 2005; Mishra et al., 2012). Therefore, our studies have found another cipitation in lens fiber cells thereby lead to cataracts. example of an aggregating crystallin mutant that could escape the suppression of α-crystallin. 3.6. G64W maintains its interaction with CRYAA In summary, our findings provide a more comprehensive understanding in the relationship between CRYBA4G64W and cataractogen- α-crystallin A (CRYAA) is considered as chaperone-like protein with esis. the ability of keeping the stability and the solubility of crystallins including CRYBA4. To identify whether the mutation disturbed the strength of CRYBA4-CRYAA interaction, yeast two-hybrid assay was Funding carried out. The strength of protein-protein interaction was evaluated by SD/-Trp/-Leu/-His plates supplemented with 2 μg/ml X-α-Gal. T.J. is supported by the Strategic Priority Research Program of the Colonies of both CRYBA4WT-CRYAA and CRYBA4G64W-CRYAA grew on Chinese Academy of Sciences (XDB29030104), the National Natural SD/-Trp/-Leu/-His plate (Fig. 7A). The color of CRYBA4G64W-CRYAA Science Fund (Grant No.: 31870731 and U1732109), the Fundamental group was darker than CRYBA4WT-CRYAA group. Empty vectors were Research Funds for the Central Universities (WK2070000108). Q.W. is performed as control (Figs. 4E and 6B). This result revealed a higher supported by the National Natural Science Fund for Young Scholars level of interaction strength of CRYBA4G64W and CRYAA compared with (81700812); the Ph.D. Start-up Fund of Natural Science Foundation of CRYBA4WT. It is reported that CRYAA protect mutated γ-crystallin D Guangdong Province [2017A030310214]; and the Guangdong from precipitation (Moreau and King, 2012a). Thus, it is necessary to Provincial Foundation for Medical Scientific Research [A2017016]. Z.Z. assess whether CRYAA could help the folding of CRYBA4G64W and is supported by the National Natural Science Fund (Grant No.: prevent CRYBA4G64W from precipitation. CRYAA-pET22b was co- 21573205). transformed into Rosetta with CRYBA4G64W-pET28a or CRYBA4WT-

7 W. Li, et al. Experimental Eye Research 186 (2019) 107712

Fig. 6. Interaction with CRYBB1 was compromised by G64W mutation. (A) Yeast two-hybrid analysis of interaction strength with CRYBB1. (B) CRYAA-pGAD-T7 and pGBK-T7 or CRYBB1-pGAD-T7 and pGBK-T7 were con-transformed to AH109 to exclude non-speciﬁc interaction. (C) Pull-down assay was used to detect the strength of interaction with CRYBB1, MBP was as control.

Conﬂicts of interest Author contributions

The authors declare that they have no conﬂicts of interest with the Tengchuan Jin and Qiwei Wang designed the study, participated in contents of this article. data analysis and extensively reviewed the manuscript. Weiqian Li performed the experiments, analyzed the data and drafted the manuscript. Other authors participated in the experiments and reviewed the manuscript.

8 W. Li, et al. Experimental Eye Research 186 (2019) 107712

Fig. 7. CRYBA4G64W maintains the interaction with CRYAA. (A) Yeast two-hybrid was adopted to evaluate the strength of interaction with CRYAA. (B) Pellet and supernatant of CRYAA and CRYBA4 co-transformed bacteria were analyzed by western-blotting.

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