Mechanism of the Very Efficient Quenching of Tryptophan Fluorescence in Human γD- and γS-Crystallins: The γ-Crystallin Fold May Have Evolved To Protect Tryptophan Residues from Ultraviolet Photodamage†

Proteins exposed to UV radiation are subject to irreversible photodamage through covalent modification of tryptophans (Trps) and other UV-absorbing amino acids. Crystallins, the major protein components of the vertebrate eye lens that maintain lens transparency, are exposed to ambient UV radiation throughout life. The duplicated β-sheet Greek key domains of β- and γ-crystallins in humans and all other vertebrates each have two conserved buried Trps. Experiments and computation showed that the fluorescence of these Trps in human γD-crystallin is very efficiently quenched in the native state by electrostatically enabled electron transfer to a backbone amide [Chen et al. (2006) Biochemistry 45, 11552−11563]. This dispersal of the excited state energy would be expected to minimize protein damage from covalent scission of the excited Trp ring. We report here both experiments and computation showing that the same fast electron transfer mechanism is operating in a different crystallin, human γS-crystallin. Examination of solved structures of other crystallins reveals that the Trp conformation, as well as favorably oriented bound waters, and the proximity of the backbone carbonyl oxygen of the n − 3 residues before the quenched Trps (residue n), are conserved in most crystallins. These results indicate that fast charge transfer quenching is an evolved property of this protein fold, probably protecting it from UV-induced photodamage. This UV resistance may have contributed to the selection of the Greek key fold as the major lens protein in all vertebrates.National Eye Institute (Grant EY 015834

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

Crystal structures of leucites from synchrotron x-ray powder diffraction data

High resoln. synchrotron x-ray powder diffraction, in conjunction with magic angle spinning 29Si NMR and electron diffraction, were used to det. structures of synthetic analogs based on the 3 dimensional silicate framework of the natural mineral leucite (KAlSi2O6). These structures were refined by the Rietveld method. The structure detns. of dry and hydrothermal synthesized K2MgSi5O12 analogs illustrate the techniques used, and the structural differences between analogs are discussed in terms of cation sizes and synthetic conditions. [on SciFinder(R)

Disulfide isomerization switches tissue factor from coagulation to cell signaling

Cell-surface tissue factor (TF) binds the serine protease factor VIIa to activate coagulation or, alternatively, to trigger signaling through the G protein-coupled, protease-activated receptor 2 (PAR2) relevant to inflammation and angiogenesis. Here we demonstrate that TF·VIIa-mediated coagulation and cell signaling involve distinct cellular pools of TF. The surface-accessible, extracellular Cys(186)–Cys(209) disulfide bond of TF is critical for coagulation, and protein disulfide isomerase (PDI) disables coagulation by targeting this disulfide. A TF mutant (TF C209A) with an unpaired Cys(186) retains TF·VIIa signaling activity, and it has reduced affinity for VIIa, a characteristic of signaling TF on cells with constitutive TF expression. We further show that PDI suppresses TF coagulant activity in a nitric oxide-dependent pathway, linking the regulation of TF thrombogenicity to oxidative stress in the vasculature. Furthermore, a unique monoclonal antibody recognizes only the noncoagulant, cryptic conformation of TF. This antibody inhibits formation of the TF·PAR2 complex and TF·VIIa signaling, but it does not prevent coagulation activation. These experiments delineate an upstream regulatory mechanism that controls TF function, and they provide initial evidence that TF·VIIa signaling can be specifically inhibited with minimal effects on coagulation

Explosive expansion of βγ-Crystallin genes in the ancestral vertebrate

In jawed vertebrates, βγ-crystallins are restricted to the eye lens and thus excellent markers of lens evolution. These βγ-crystallins are four Greek key motifs/two domain proteins, whereas the urochordate βγ-crystallin has a single domain. To trace the origin of the vertebrate βγ-crystallin genes, we searched for homologues in the genomes of a jawless vertebrate (lamprey) and of a cephalochordate (lancelet). The lamprey genome contains orthologs of the gnathostome βB1-, βA2- and γN-crystallin genes and a single domain γN-crystallin-like gene. It contains at least two γ-crystallin genes, but lacks the gnathostome γS-crystallin gene. The genome also encodes a non-lenticular protein containing βγ-crystallin motifs, AIM1, also found in gnathostomes but not detectable in the uro- or cephalochordate genome. The four cephalochordate βγ-crystallin genes found encode two-domain proteins. Unlike the vertebrate βγ-crystallins but like the urochordate βγ-crystallin, three of the predicted proteins contain calcium-binding sites. In the cephalochordate βγ-crystallin genes, the introns are located within motif-encoding region, while in the urochordate and in the vertebrate βγ-crystallin genes the introns are between motif- and/or domain encoding regions. Coincident with the evolution of the vertebrate lens an ancestral urochordate type βγ-crystallin gene rapidly expanded and diverged in the ancestral vertebrate before the cyclostomes/gnathostomes split. The β- and γN-crystallin genes were maintained in subsequent evolution, and, given the selection pressure imposed by accurate vision, must be essential for lens function. The γ-crystallin genes show lineage specific expansion and contraction, presumably in adaptation to the demands on vision resulting from (changes in) lifestyle