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Dissimilarity in the contributions of the N-terminal domain hydrophobic core to the structural stability of lens #/#-crystallins Kai Zhang, Wei-Jie Zhao, Ke Yao, and Yong-Bin Yan Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01164 • Publication Date (Web): 30 Apr 2019 Downloaded from http://pubs.acs.org on April 30, 2019
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Biochemistry
Dissimilarity in the contributions of the N-terminal domain hydrophobic core to the structural stability of lens β/γ-crystallins Kai Zhang1,#, Wei-Jie Zhao2,#, Ke Yao1,*, Yong-Bin Yan2,*
1
Eye Center of the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou
310009, China 2
State Key Laboratory of Membrane Biology, School of Life Sciences, Tsinghua University, Beijing
100084, China
#
These authors contributed equally to this work.
*Corresponding authors: Dr. Ke Yao, Eye Center of the 2nd Affiliated Hospital, Medical College of Zhejiang University, Hangzhou,
310009,
China.
Tel:
+86-571-87783897,
Fax:
+86-571-87783908,
E-mail:
[email protected]; or Dr. Yong-Bin Yan, School of Life Sciences, Tsinghua University, Beijing 100084, China. Tel: +86-10-62783477, Fax: +86-10-62772245, E-mail:
[email protected].
Running Title: Hydrophobic core stability of human lens β/γ-crystallins Abbreviations: ANS, 1-anilinonaphtalene-8-sulfonate; BSA, bovine serum albumin; CD, circular dichroism; CTD, C-terminal domain; DTT, dithiothreitol; Emax, emission maximum wavelength of intrinsic fluorescence; GdnHCl, guanidine hydrochloride; MD, molecular dynamics; NTD, N-terminal domain; IPTG, isopropyl-1-thio-β-D-galactopyranoside; SDS, sodium dodecylsulfate; SEC, size-exclusion chromatography; WT, wild type
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Abstract Vertebrate lens β/γ-crystallins share a conserved tertiary structure composing four Greek-key motifs divided into two globular domains. Numerous inherited mutations in β/γ-crystallins have been
linked to cataractogenesis. In this research, the folding mechanism underlying cataracts caused by the I21N mutation in βB2 was investigated by comparing the effect of mutagenesis on the structural features and stability of four β/γ-crystallins, βB1, βB2, γC and γD. Our results showed that the four β/γ-crystallins differ greatly in solubility and stability against various stresses. The I21N mutation greatly impaired βB2 solubility and native structure as well as its stability against denaturation induced by guanidine hydrochloride, heat treatment and UV irradiation. However, the deleterious effects were much weaker for mutations at the corresponding sites in βB1, γC and γD. Molecular dynamic simulations indicated that the introduction of a nonnative hydrogen bond contributed to twisting Greek-key motif I outwards, which might direct the misfolding of I21N mutant of βB2. Meanwhile, partial hydration of the domain hydrophobic interior induced by the mutation destabilized βB1, γC and γD. Our findings highlight the importance of nonnative hydrogen bond formation and hydrophobic core hydration in crystallin misfolding caused by inherited mutations.
Keywords: β/γ-crystallin; congenital cataract-causing mutation; hydration of protein interior; nonnative hydrogen bond; protein aggregation; protein folding
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Biochemistry
Introduction Cataracts account for about half of human blindness cases worldwide. Cataractogenesis is complicated process with many risk factors, while aging and UV irradiation are two common factors for age-related cataracts and inherited mutations are frequently observed in congenital cataracts
(1-3).
Although cataracts can be clinically catalogued into a number of phenotypes, a general feature of various cataracts at the protein level is the formation of large light-scattering particles from various misfolded crystallins, which are the predominant structural proteins in vertebrate lens
(4-6).
Vertebrate
crystallins is comprised of three families: -crystallins (A and B), β-crystallins (βA1/A3, βA2, βA3, βB1, βB2 and βB3) and γ-crystallins (γA, γB, γC, γD, γE, γF and γS). Among them, multimeric -crystallins are small heat shock proteins possessing molecular chaperones functions
(7).
In the lens,
γ-crystallins exist as monomers, while the acidic and basic β-crystallins form homomers or heteromers with a size distribution ranging from dimer to octamer
(5).
The dynamic oligomeric equilibrium in -
and β-crystallins have been proposed to be important to their function and structural stability
(8-11).
Although it is unclear yet why the lens cells are evolved to possess more than a dozen types of crystallins, it is certain that crystallin structural stabilities are vital to the lifelong maintenance of lens functions. Although β/γ-crystallins differ in their oligomeric states, they share a common two-domain fold and each domain is comprised of two Greek-key motifs. The N-terminal domain (NTD) and C-terminal domain (CTD) are structurally conserved compact globular domains with a compact dehydrated hydrophobic core (12-17). Modifications of intra- or inter-subunit domain interactions have complicated effects on β/γ-crystallin stability and aggregatory behaviors under stressed conditions (11, 18-21). NTD is generally less stable than CTD and a folding intermediate with intact CTD but less structured NTD can be detected during the folding of various β/γ-crystallins
(6, 22-28).
Disruption of Greek-key motif
integrity is thought to be an important mechanism underlying congenital cataracts caused by some of β/γ-crystallin mutations
(27, 29-33).
Most cataract-causing mutations in β/γ-crystallins are highly
conserved, especially for those structural residues contributing to domain structure integrity. Previously I21N mutation in βB2 has been identified in human patients with autosomal dominant 3 ACS Paragon Plus Environment
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congential nuclear cataracts in a Chinese family
(34).
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I21 is located at the first β-strand of Greek-key
motif I in NTD and its side-chain contributes to the hydrophobic core in NTD (Figure 1A). The location of I21 is just prior to the Y/FXXXXY/FXG signature sequence, which identifies the β/γ-crystallin type of Greek-key fold
(35, 36).
Intriguingly, I21 is fully conserved in βB2 across various
species, but is substituted by Val, Leu or Phe in other types of β/γ-crystallins (Figure 1B). Notably, substitution of the structurally identical residue L6 in γD or F9 in γS by Ser has been associated with cataracts in mice (33, 37). To study the molecular basis of cataracts induced by I21N mutation in βB2 and the contributions of hydrophobic core in various β/γ-crystallins, in this research we compared the structural features and stabilities of four mutants, βB1-V63N, βB2-I21N, γC-F6N and γD-L6N. Interestingly, the four types of β/γ-crystallins had quite dissimilar sensitivities to chemical denaturant, heat treatment and UV damage. The mutations resulted in different effects on the structures and stabilities of the four crystallins, while the I21N mutation in βB2 was the most deleterious by destructuring the NTD of βB2. MD simulations suggested that the introduced Asn formed a nonnative hydrogen bond with T42, which was not observed in the other β/γ-crystallin mutants. The mutations at the corresponding sites in the other three crystallins led to partial hydration of the hydrophobic core. These findings provide novel insights into the structural basis underlying the deleterious effects of cataract-causing mutations.
Figure 1. Structure of βB2-crystallin N-terminal domain and sequence alignment of β/γ-crystallins. (A) Crystal structure of βB2-crystallin N-terminal domain (PDB ID: 1YTQ). Residues contributing to the formation of NTD hydrophobic core are shown by stick model. I21 is highlighted by the space-filling model. (B) Sequence alignment of amino acid residues around the mutation site 4 ACS Paragon Plus Environment
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Biochemistry
among various β/γ-crystallins. The sequences used for alignment are: βA1 (P05813), βA2 (P53672), βA4 (P53673), βB1 (P53674), βB2 (P43320), βB3 (P26998), γA (P11844), γB (P07316), γC (P07315), γD (P07320) and γS (P22914).
Materials and Methods Materials Sodium dodecyl sulfate (SDS), ultrapure guanidine hydrochloride (GdnHCl), imidazole, 1-anilinonaphtalene-8-sulfonate (ANS) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich. Dithiothreitol (DTT) and isopropyl-1-thio-β-D-galactopyranoside (IPTG) were obtained from Promega. Taq DNA polymerase, various restriction endonucleases and Taq DNA ligase were from Takara. The pET28a plasmid was from Invitrogen. E. coli DH5 and E. coli Rosetta (DE3) were from Novagen. Site-directed mutagenesis The human CRYBB1, CRYBB2, CRYGC and CRYGD genes were cloned as described previously (38-41). Site-directed mutagenesis was achieved by using the following forward (F) and reverse (R) primers: βB1-V63N-F:
5’-ACAGGCTGGTGAACTTCGAACTGGAA-3’;
βB1-V63N-R:
5’-TCCAGTTCGAAGTTCACCAGCCTGT-3’;
βB2-I21N-F:
5’-CAAGATCATCAACTTTGAGCAG-3’;
βB2-I21N-R:
5’-CTGCTCAAAGTTGATGATCTTG-3’;
γC-F6N-F:
5’-AAGGATCCATGGGGAAGATCACCAACTATGA-3’;
γC-F6N-R:
5’-GGAAGCTTTTAATACAAATCCACCACTCTCC-3’;
γD-L6N-F:
5’-AAGGATCCATGGGGAAGATCACCAACTACGA-3’
and
γD-L6N-R:
5’-
GAAGCTTTCAGGAGAAATCTATGACTCT-3’. The recombinant plasmids were obtained by inserting the wild type (WT) and mutated genes into pET28a and used for used for overexpression in the E. coli cells. Protein expression and purification The His-tagged recombinant proteins were overexpressed in E. coli Rosetta (DE3) using similar protocols as those described previously with modifications of the incubation temperatures and IPTG concentrations
(39-41).
In brief, the optimal incubating temperature and IPTG concentration were 5 ACS Paragon Plus Environment
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screened for each protein ranged from 16 to 37 C and 0.1 to 1 mM, respectively. The final incubating temperatures, IPTG concentrations and incubation time were 16 C, 0.1 mM and 24 h for β-crystallins, and 37 C, 0.1 mM and 4 h for γ-crystallins, respectively. The His-tagged recombinant proteins were extracted from the soluble fractions of cell lysates by a 5 mL Ni-column, followed by a further step of purification using a Superdex 200HR 10/300GL column equipped on an ÄKTA purifier. All protein solutions were prepared using buffer A (20 mM sodium phosphate, 150 mM NaCl, 1 mM DTT and 1 mM EDTA, pH 7.2). The purity was evaluated by SDS-PAGE using a 12% acrylamide separating gel under reducing conditions. The purified final products were identified to exist in a homogenous oligomeric status analyzed by size-exclusion chromatography (SEC). Protein concentration was determined by the standard Bradford method (42). SEC analysis SEC analysis was performed using 150 μL protein solutions with a concentration of 0.8 mg/mL by a Superdex 200HR 10/300GL column at 4 C. Before injected into the SEC column, all protein solutions were equilibrated at 4 C for 2 h and the column was pre-equilibrated using buffer A for at least one column volume (24 mL). The injection volume was 100 μL and the flow rate was 0.4 mL/min. Solubility The solubility of the proteins in buffer A was determined using a MILLIPORE micro-concentrator (30).
During concentrating, aliquots were taken frequently and diluted by buffer A to determine protein
concentration. The solubility was determined by the maximum concentration after long-term concentrating. The presented data were shown by averageS.D. from three independent repetitions. Spectroscopy Spectroscopic experiments were performed at 25C using the same protocols as those described previously (30, 38). In brief, near- and far-UV circular dichroism (CD) spectra were measured on a Jasco J-715 spectrophotometer (Jasco Corp.). The cell pathlength was 1 mm and 1 cm, while the protein concentration was 0.2 mg/mL and 0.5 mg/mL for far- and near-UV CD measurements, respectively. Intrinsic and extrinsic fluorescence of proteins were recorded on an F-2500 fluorescence spectrophotometer (Hitachi Ltd.). The cuvette pathlength was 1 cm and the protein concentration was 0.2 mg/mL. The excitation wavelength was 295 nm and 380 nm for intrinsic and extrinsic 6 ACS Paragon Plus Environment
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Biochemistry
fluorescence measurements, respectively. The extrinsic fluorescence was measured using ANS as the probe of hydrophobic exposure. Turbidity experiments were performed on an Ultraspec 4300 pro UV/Visible spectrophotometer (Amersham Pharmacia Biotech) using a wavelength of 400 nm. Protein stability analysis Protein stabilities against heat treatment, UV irradiation and GdnHCl denaturation were performed according to the protocols described elsewhere
(30, 38).
In brief, the protein concentration was 0.2
mg/mL in buffer A for denaturation induced by heat and GdnHCl, while 1 mg/mL for UV treatment. Protein thermal denaturation was performed by increasing temperatures of protein solutions from 20 to 86 C. UV treatment was conducted by irradiating the protein solutions by UV light (254 nm) on ice. Turbidity of the UV-treated samples was measured every 30 min. Buffer A was used as the blank. GdnHCl-induced denaturation was performed by the proteins by 0-6 M GdnHCl in buffer A overnight at ambient temperature. Changes in protein structures were probed by CD, fluorescence, light scattering and turbidity data. Molecular dynamic simulations and structural anlaysis Molecular dynamic (MD) simulation studies were the same as that described previously (20, 21, 43). Since the mutation sites were in the NTD hydrophobic core of β/γ-crystallins, only the structures of NTD were used for MD simulations to facilitate the study of mutational effects on the structural changes of NTD within limited simulating time. The crystal structures of βB1 (PDB ID: 1OKI) (13), βB2 (PDB ID: 1YTQ) (44), γC (PDB ID: 2V2U) (17) and γD (PDB ID: 1HK0) (14) were used as the template structures. The
starting
structures
of
the
mutants
were
created
by
(http://www.pymol.org/). The simulations were performed by VMD
mutagenesis (45).
using
PyMol
Proteins in a water box
containing 150 mM NaCl was equilibrated for 10 ns at 450 K and 1 atm using NAMD 2.9
(46).
After
equilibration, the simulations were performed up to 20 ns with a time-interval of 2 fs. Simulation parameters were calculated by VMD. Visualization, analysis and rendering of the structures were achieved by PyMol (http://www.pymol.org/).
Results Effects of the mutations on β/γ-crystallin solubility and structural features Various vertebrate crystallins are known to have extremely high solubility under physiological 7 ACS Paragon Plus Environment
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conditions
(5).
Page 8 of 28
Intriguingly, the solubility of βB1, βB2, γC and γD varied significantly under our
experimental conditions ranging from 764 to 2765 mg/mL (Figure 2). Our results indicated that solubility of β/γ-crystallins was independent on their oligomeric status since βB2 and γC had higher solubility than βB1 and γD. It is unclear yet for the biophysical basis of the dramatic difference in solubility among these four types of β/γ-crystallins. No correlation could be identified between solubility determined experimentally and the sizes of the minimal solvating water shell of the four proteins obtained using their crystal structures (data not shown). Furthermore, the mutations replacing the hydrophobic core residue by the polar residue Asn also exhibited dissimilar impacts on βB1, βB2, γC and γD solubility. The mutations significantly reduced β-crystallin solubility but had little impact on γ-crystallins. The most deleterious effect was seen for the I21N mutation in βB2, which reduce βB2 solubility from 2765 mg/mL to 0.950.02 mg/mL. The poor solubility of the I21N mutant might be the main cause of congenital nuclear cataract in patients with the βB2-I21N mutation (34).
Figure 2. Solubility of β/γ-crystallins and their mutants. *** represents p Glu point mutants of human gamma-D crystallin provides a model for hereditary or UV-induced cataract, Protein Sci. 25, 1115-1128.
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Xu, J., Zhao, W. J., Chen, X. J., Yao, K., and Yan, Y. B. (2018) Introduction of an extra tryptophan fluorophore by cataract-associating mutations destabilizes betaB2-crystallin and promotes aggregation, Biochem. Biophys. Res. Commun. 504, 851-856.
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Biochemistry
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Biochem. Biophys. Res. Commun. DOI: 10.1016/j.bbrc.2018.10.175.
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