Na-acetylation of eye lens proteins: a cryptic phenomenon

Contains fulltext : mmubn000001_02731104x.pdf (publisher's version ) (Open Access)Promotores : H. Bloemendal en G. Tesser199 p

Evolutionary and functional relationships between the basic and acidic β-crystallins

β-Crystallins are complex oligomers composed of many related subunits. In order to understand their interactions we have built molecular models of several bovine β-crystallins, based on their sequence similarity to the well-defined γ-II crystallin structure, using interactive computer graphics techniques. Their common origin with γ-crystallin is displayed in both the retention of four-fold sequence repeats of critical residues involved with stabilizing a folded β-hairpin and the conservation of core-filling hydrophobic side-chains. The β-crystallins have been built as bilobal molecules with each domain composed of two ‘Greek key motifs which associate about an approximate two-fold axis to form β-sheets. The β-crystallin sequences have previously been shown to comprise two families, the basic and acidic subunits, which have extensions of sequence. The three-dimensional models show how the two families appear to stabilize the folded β-hairpin in the N- and C-terminal domains in ways which suggest that they have diverged from a common ancestor in different ways. Acidic β-crystallins, like γ-crystallins, have a regular array of charges on their N-terminal domain which has been interrupted in basic β-crystallins by hydrophobic residues which may be related to the presence of a C-terminal extension. β-Crystallins are more highly charged than γ-crystallins although their charge density is higher in certain regions of the N-terminal domain, particularly in βB1-crystallin. β-crystallins also differ from γ-crystallins in the virtual absence of core-filling sulphydryl groups whereas they have numerous sulphurcontaining side-chains together with tryptophan and histidine rings protruding from the globular domains, particularly in the acidic subunits. The burial of these residues in subunit contacts is consistent with their spectroscopic and electrostatic properties. Protein subunit aggregation commonly occurs through hydrophobic interaction or β-sheet extension. Analysis of the subunit surfaces has identified an N-terminal hydrophobic region common to βB1 and βB2 whereas a C-terminal hydrophobic loop region is common to βB1 and βA1 and may be correlated with their association properties. It is suggested that the polar C-terminal domain of βB2 contributes towards the solubility of higher aggregates by interactions involving β-sheet structure

X-ray diffraction and structure of crystallins

The 3-dimensional organisation of crystallin polypeptides into globular proteins and their interactions into higher order structures are important factors governing optical functions related to refraction, accommodation and transparency. Single crystal X-ray diffraction studies have revealed the tertiary and quaternary structural organisation of β-, γ- and δ-crystallins. Regions of the lens with high refractive index contain high levels of monomeric y-crystallins while the accommodating, hydrated avian lens has largely replaced γ-crystallins with δ-crystallin. The βγ-crystallins form a superfamily of proteins of high symmetry and great diversity in which the basic building block is a 10 kD pseudo-symmetrical 2-Greek key domain. A γ-crystallin comprises two of these β-sheet domains, joined by a linker, and a short C-terminal extension. In β-crystallins the linker has an extended conformation resulting in dimer formation by a mechanism known as domain swapping. Crystallographic analysis of engineered single domains of γ-crystallins, analogous to the ancestral domain, has indicated the importance of the short C-terminal extension in directing domain pairing. γ-crystallins have numerous cysteine residues, some are conserved in the core of the protein molecule and some are variable on the protein surface. The structure of γB-crystallin has been determined at very high resolution using cryo-crystallography allowing the visualisation of the complete protein-protein and protein-water structure at the surface. β-crystallins are seen as tetramers in the crystal structures but their long sequence extensions are harder to visualise in the electron density of the hydrated crystal lattice structure. In one tight packing lattice of βB2 crystallin the N-terminal extension is seen to mediate protein-protein interactions between tetramers to form a 42 helix. The X-ray structure of the taxon-restricted avian δ-crystallin shows that the 50 kD subunit contains 22 helices that form three α-helical domains which dimerise followed by a dimer-dimer interaction to form a tetramer with a 20-helix bundle at the centre. Analysis of the spatial disposition of the sequence conserved regions showed the location of the active site cleft of the superfamily of enzymes related to δ-crystallin and argininosuccinate lyase. A different crystal structure of δ-crystallin solved under more physiological conditions revealed that tetramers assembled as higher order supramolecular helices and that the N-terminal extension may be involved. Combining the observations of higher order helical structures in both the oligomeric β-crystallin and δ-crystallin crystal lattices, we have proposed a highly speculative model for crystallin assembly in the lens fibre cells