title: The importance of the last strand at the C-terminus in βB2-crystallin stability and assembly.
abstract: Congenital cataract is the leading cause of childhood blindness worldwide. Investigations of the effects of inherited mutations on protein structure and function not only help us to understand the molecular mechanisms underlying congenital hereditary cataract, but also facilitate the study of complicated cataract and non-lens abnormities caused by lens-specific genes. In this research, we studied the effects of the V187M, V187E and R188H mutations on βB2-crystallin structure and stability using a combination of biophysical, cellular and molecular dynamic simulation analysis. Both V187 and R188 are located at the last strand of βB2-crystallin Greek-key motif 4. All of the three mutations promoted βB2-crystallin aggregation in vitro and at the cellular level. These three mutations affected βB2-crystallin quite differentially: V187M influenced the hydrophobic core of the C-terminal domain, V187E was a Greek-key motif breaker with the disruption of the backbone H-bonding network, while R188H 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 stability of 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 stabilization of one specific high-order oligomer by mutations might also be deleterious to the stability and folding of the β-crystalllin homomers and heteromers. The long-term structural stability and functional maintenance of β-crystallins are achieved by the precisely regulated oligomeric equilibrium.
authors.affiliation: Eye Center of the 2nd Affiliated Hospital, Medical College of Zhejiang University, Hangzhou 310009, China.
authors.forename: Kai
authors.initials: K
authors.lastname: Zhang
authors.forename: Wei-Jie
authors.initials: WJ
authors.lastname: Zhao
authors.forename: Xiao-Yao
authors.initials: XY
authors.lastname: Leng
authors.forename: Sha
authors.initials: S
authors.lastname: Wang
authors.forename: Ke
authors.initials: K
authors.lastname: Yao
authors.forename: Yong-Bin
authors.initials: YB
authors.lastname: Yan
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D007908:Lens, Crystalline / Q000737:chemistry*; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D017510:Protein Folding; D055503:Protein Multimerization; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012995:Solubility; D038206:beta-Crystallin B Chain / Q000737:chemistry* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D038206:beta-Crystallin B Chain; C113634:beta-crystallin B2
keywords: 1-anilinonaphtalene-8-sulfonate; ADCC; ANS; Autosomal dominant congenital nuclear cataract; BSA; C-terminal domain; CD; CTD; DTT; E(max); GdnHCl; IPTG; Inherited mutation; MD; MW; Molecular dynamic simulation; Protein aggregation; Protein assembly; SDS; SDS-PAGE; SDS-polyacrylamide gel electrophoresis; SEC; T(m); WT; autosomal dominant congenital cataract; bovine serum albumin; circular dichroism; dithiothreitol; guanidine hydrochloride; isopropyl-1-thio-β-d-galactopyranoside; maximum emission wavelength of intrinsic Trp fluorescence; molecular dynamics; molecular weight; size-exclusion chromatography; sodium dodecyl sulfate; the midpoint temperature of protein thermal transition; wild type; βB2-crystallin