The importance of the Abn2 calcium cluster in the endo-1,5-arabinanase activity from Bacillus subtilis.

Arabinanase is a glycosyl hydrolase that is able to cleave the glycosidic bonds of α-1,5-L-arabinan, releasing arabino-oligosaccharides and L-arabinose. The enzyme has two domains, an N-terminal catalytic domain with a characteristic ß-propeller fold and a C-terminal domain whose function is unknown. A calcium ion, located near the catalytic site, serves to stabilize the N-terminal domain, but it has also been proposed to play a key role in the enzyme mechanism. The present work describes the structure of an inactive mutant of the wild-type enzyme (H318Q) and in which the calcium ion has been adventitiously replaced by nickel. These structural studies, together with functional and modelling studies, clearly support the role of the calcium ion in the overall reaction mechanism.

Preliminary X-ray data for an N-terminal fragment of rabbit serum transferrin.

An iron-containing fragment (Mr approximately 39,000) of rabbit serum transferrin has been crystallized from a solution of 25% (w/v) polyethylene glycol 6000, 50 mM-disodium piperazine-N,N'bis(2-ethanesulphonate) adjusted to pH 6.0 at 4 degrees C. The space group is P3(1)21 (or the enantiomorph) with a = b = 66.8(1) A, c = 137.5(3) A and Z = 6. The crystals appear as hexagonal plates, with the unique axis perpendicular to the plate. The crystals, kept at 4 degrees C, are stable in the X-ray beam for at least 130 hours and diffract to better than 1.8 A resolution.

Asp ligand provides the trigger for closure of transferrin molecules. Direct evidence from X-ray scattering studies of site-specific mutants of the N-terminal half-molecule of human transferrin.

Recent X-ray crystallographic and solution X-ray scattering studies have shown that transferrins (serum transferrin, lactoferrin and ovotransferrin) undergo a major conformational change when iron is incorporated into the molecule. Apo-proteins show a structure with open interdomain clefts which close when iron is bound. The closed conformation has been suggested as an important step in the receptor recognition. Here, we report X-ray solution scattering experiments of the mutated N-terminal fragment of human serum transferrin with Asp63-->Ser (Cys). The data provide the first direct experimental evidence for the existence of a trigger mechanism for the closure of the interdomain cleft and that this trigger mechanism is disrupted by mutation of Asp63, the only ligand of iron from domain I.

Close packing of an oligomeric eye lens beta-crystallin induces loss of symmetry and ordering of sequence extensions.

beta-Crystallins are oligomeric eye lens proteins that are related to monomeric gamma-crystallins. The main sequence difference between the two families is the presence of sequence extensions in the beta-crystallins. A major question concerns the role that these extensions play in mediating interactions at the high protein concentrations found in the lens. The predominant beta-crystallin polypeptide, beta B2, can be crystallized in two different space groups, I222 and C222. The I222 crystal structure revealed that the protein packed as a tetramer with perfect 222 symmetry but that the extensions were disordered. The X-ray structure of the C222 lattice of beta B2 has now been refined at 3.3 A, the structure analysed and compared with the I222 lattice. The protein is also a tetramer with 222 symmetry in the C222 lattice but differs in that parts of the N-terminal extensions have been visualized. In the asymmetric unit of the C222 lattice there are four subunits, each comprising a single polypeptide chain, in which certain flexible loops in the N-terminal domains and the N-terminal extensions have various conformations. The tetramers in the C222 lattice are more tightly packed than in the I222 form. Analysis of the tetramer contacts shows that the sites of interaction break the 222 symmetry of the tetramers. The N-terminal extensions play a major role in directing interactions between tetramers. One of the N-terminal extensions interacts with a hydrophobic patch on the N-terminal domain of another tetramer. These crystallographic observations obtained over a physiological concentration range indicate how, in beta-crystallin oligomers, the N-terminal extensions of beta B2 can switch from interacting with water to interacting with protein depending on their relative concentrations. This could be useful in maintaining a gradient of refractive index.

High resolution structure of an oligomeric eye lens beta-crystallin. Loops, arches, linkers and interfaces in beta B2 dimer compared to a monomeric gamma-crystallin.

beta-Crystallins are polydisperse, oligomeric structural proteins that have a major role in forming the high refractive index of the eye lens. Using single crystal X-ray crystallography with molecular replacement, the structure of beta B2 dimer has been solved at 2.1 A resolution. Each subunit comprises an N and C-terminal domain that are very similar and each domain is formed from two similar "Greek key" motifs related by a local dyad. Sequence differences in the internally quadruplicated molecules, analysed in terms of their beta-sheets, hairpins and arches, give rise to structural differences in the motifs. Whereas the related family of gamma-crystallins are monomers, beta-crystallins are always oligomers. In the beta B2 subunit, the domains, each comprising two motifs, are separated by an extended linking peptide. A crystallographic 2-fold axis relates the two subunits of the dimer so that the N-terminal domain of one subunit of beta B2 and the C-terminal domain of the symmetry-related subunit are topologically equivalent to the two covalently connected domains of gamma B-crystallin. The intersubunit domain interface is very similar to the intradomain interface of gamma B, although many sequence differences have resulted in an increase in polar interactions between domains in beta B2. Comparison of the structures of beta B2 and gamma B-crystallins shows that the two families differ largely in the conformation of their connecting peptides. A further extensive lattice contact indicates a tetramer with 222 symmetry. The ways in which insertions and extensions in the beta-crystallin effect oligomer interactions are described. The two kinds of crystallin are analysed for structural features that account for their different stabilities. These studies are a basis for understanding formation of higher aggregates in the lens.

Crystallization and preliminary analysis of an 18,000 Mr fragment of duck ovotransferrin.

Crystals of an 18,000 Mr iron-binding fragment of duck ovotransferrin, corresponding to domain II of the N-terminal lobe, have been obtained. The crystals belong to the trigonal system, P31 (or enantiomer) with a = b = 41.3(1) A, c = 81.2(2) A (1 A = 0.1 nm) and one molecule per asymmetric unit assuming a solvent content of 40% by volume. The crystals are stable at +4 degrees C and diffract to at least 2.3 A resolution.

Metal-induced conformational changes in transferrins.

Recent studies on iron-loaded transferrins have revealed a conformational change upon binding iron due to a domain closure. It has been suggested that the domain closure may be the key for the receptor recognition of the metal loaded transferrin (Grossmann et al., 1992). Small angle X-ray scattering has been used to provide direct structural information on the conformational changes that may take place upon the binding and release of different metals to the transferrins in solution. The data show that In3+ and Cu2+ induce the same domain closure as Fe3+; Al3+ causes a conformational change of somewhat smaller magnitude while Hf4+ does not induce any conformational change. The results are discussed in terms of the molecular recognition of metal loaded transferrin by the receptor.

Packing interactions in the eye-lens. Structural analysis, internal symmetry and lattice interactions of bovine gamma IVa-crystallin.

gamma-Crystallins are a family of low molecular weight proteins found in high concentration in the densely packed regions of high refractive index in vertebrate lenses. Certain members have the characteristic property of a high critical temperature (tc) for phase separation. We report the three-dimensional structure determination of such a protein, bovine lens gamma IVa-crystallin, which has been refined to give an X-ray R-factor of 0.143. Its high tc contrasts with the low tc gamma II-crystallin, whose structure we have already published. The root mean square difference between the alpha-carbon atoms of these two proteins is 0.70 A and gamma IVa has an internal symmetry even higher than that of gamma II. The presence of a protein that exhibits the phenomenon of phase separation at body temperature renders the lens very susceptible to a transformation from transparent to an opaque state due to irregularities in the refractive index. Protein interactions of gamma IVa-crystallin have implications for the mechanism of cataract formation. Modes of self-association behaviour of gamma IVa-crystallin have been inferred from an analysis of the lattice interactions in the crystalline state, where the protein packing density is similar to that of the intact lens. It appears that the point mutation at position 103 from a serine residue in gamma II to a valine in gamma IVa gives rise to a lattice contact formed by two four-stranded beta-sheets in gamma IVa. A group-specific mutation at position 118 from leucine to phenylalanine induces subtle differences in core packing, leading to a reorganization around residue 103. However, the final phase separation determinant may be a complex combination of many side-chain functions.

X-ray solution scattering reveals conformational changes upon iron uptake in lactoferrin, serum and ovo-transferrins.

X-ray solution scattering has been used for studying the structural changes that take place upon uptake and release of iron from serum and chicken ovo-transferrin and human lactoferrin. In the case of chicken ovo-transferrin, data have been obtained for both the intact protein and the isolated N and C-lobes with and without iron. These studies reveal that both lobes undergo a change that is consistent with an opening of the inter-domain cleft when iron is removed from the protein. We suggest that the conformational change of the protein increases the specificity of receptor binding and that the closed configuration of the iron-loaded protein is one, or perhaps the, decisive step in the mechanism for receptor-mediated endocytosis.

Structural comparison of cupredoxin domains: domain recycling to construct proteins with novel functions.

The three-dimensional structures of the copper-containing enzymes ascorbate oxidase, ceruloplasmin, and nitrite reductase, comprised of multiple domains with a cupredoxin fold, are consistent with having evolved from a common ancestor. The presence or absence of copper sites has complicated ascertaining the structural and evolutionary relationship among these and related proteins. Simultaneous structural superposition of the enzyme domains and their known cupredoxin relatives shows clearly that there are at least six cupredoxin classes, and that the evolution of the conserved core of these domains is independent of the presence or absence of copper sites. Relationships among the variable loops in these structures show that the two-domain ancestor of the blue oxidases contained a trinuclear-copper interface but could not have functioned in a monomeric state. Comparison of the sequence of the copper-containing, iron-regulating protein. Ferrous transport (Fet3) from yeast to the structurally defined core and loop residues of the cupredoxins suggests specific residues that could be involved in the ferroxidase activity of Fet3.

New perspectives on the structure and function of transferrins.

Structure-function relationships for transferrins are discussed in the light of recent X-ray crystal structure determinations. A common folding pattern into two lobes, each comprising two domains is adopted; this allows the tight, but reversible binding of iron. Uptake and release of iron involve substantial domain movements which open and close the binding clefts. The iron binding sites are similar and the key role of the CO3(2-) anion bound with each Fe3+ can now be understood; structural differences near the iron binding sites suggest reasons for the different binding properties of serum transferrin and lactoferrin. The glycan moieties do not appear to affect the protein structure or metal binding properties; they are not clearly seen in the X-ray analyses but have been modelled. The accommodation of different metals and anions is illustrated by the crystal structures of Cu2+ and oxalate-substituted lactoferrins; Al3+ binding is of particular interest. New results on transferrin-receptor interactions with transferrin, and melanotransferrin and an invertebrate transferrin (both of which have defective C-terminal binding sites), emphasize possible functional differences between the two lobes. The availability of site-specific mutants of both transferrin and lactoferrin now offers the opportunity to probe the structural determinants of iron binding, iron release, and receptor binding.

Macromolecular crystallography with a third-generation synchrotron source.

The European Synchrotron Radiation Facility (ESRF) at Grenoble, France, is a 6 GeV machine producing hard X-radiation that can be used for pure and applied research in a wide range of disciplines including physics, chemistry, structural biology, materials science, the earth sciences, engineering and medicine. The overall nature of the machine will be described, including the features that give rise to the notation 'third-generation source'. The ESRF is equipped with a number of beamlines which can be used for macromolecular crystallography. Applications include the use of very small crystals, large unit cells, data collection at high resolution, anomalous dispersion measurements for phase determination and time-resolved studies. Key features of these applications will be described.

Hybrid-cluster protein (HCP) from Desulfovibrio vulgaris (Hildenborough) at 1.6 A resolution.

The three-dimensional structure of the hybrid cluster protein from Desulfovibrio vulgaris (Hildenborough) has been determined at 1.6 A resolution using synchrotron X-ray radiation. The protein can be divided into three domains: an N-terminal mainly alpha-helical domain and two similar domains comprising a central beta-sheet flanked by alpha-helices. The protein contains two 4Fe clusters with an edge-to-edge distance of 10.9 A. Four cysteine residues at the N-terminus of the protein are ligands to the iron atoms of a conventional [4Fe-4S] cubane cluster. The second cluster has an unusual asymmetric structure and has been named the hybrid cluster to reflect the variety of protein ligands, namely two mu-sulfido bridges, two mu(2)-oxo bridges, and a further disordered bridging ligand. Anomalous differences in data collected at 1.488 A and close to the iron edge at 1.743 A have been used to confirm the identity of the metal and sulfur atoms. The hybrid cluster is buried in the center of the protein, but is accessible through a large hydrophobic cavity that runs the length of domain 3. Hydrophobic channels have previously been identified as access routes to the active centers in redox enzymes with gaseous substrates. The hybrid cluster is also accessible by a hydrophilic channel. The [4Fe-4S] cubane cluster is close to an indentation on the surface of the protein and can also be approached on the opposite side by a long solvent channel. At the present time, neither the significance of these channels nor, indeed, the function of the hybrid cluster protein is known.

An extended-X-ray-absorption-fine-structure study of freeze-dried and solution ovotransferrin. Evidence for water co-ordination at the metal-binding sites.

Our previous extended-X-ray-absorption-fine-structure (e.x.a.f.s.) study has shown that the probable iron environment in chicken ovotransferrin involves two low-Z ligands (consistent with phenolate linkages) at 0.185(1) nm and four low-Z ligands at 0.204(1) nm [Garratt, Evans, Hasnain & Lindley (1986) Biochem. J. 233, 479-484]. Herein we provide additional information from the e.x.a.f.s. and near-edge structure suggestive of a decrease in the co-ordination number of ovotransferrin-bound iron upon freeze-drying. These effects are reversible, and exposure of the freeze-dried material to a humid atmosphere results in reversion to the solution spectra. Progressive rehydration was monitored by using e.p.r. spectroscopy and was confirmed by recording the high-resolution X-ray-absorption near-edge structure (x.a.n.e.s.). The results suggest the presence of a labile water molecule at the iron-binding sites of ovotransferrin in solution.

An extended-X-ray-absorption-fine-structure investigation of diferric transferrins and their iron-binding fragments.

Iron K-edge extended-X-ray-absorption-fine-structure (e.x.a.f.s.) spectra were recorded for diferric human and rabbit serum transferrins and for diferric chicken ovotransferrin in aqueous solution; for ovotransferrin e.x.a.f.s. spectra from the N-terminal and C-terminal domain fragments were also measured. The overall spectral profiles closely resemble one another, indicating similar iron-binding sites. The simulation of the diferric ovotransferrin spectrum suggests a first co-ordination shell consisting of six low-Z ligands (nitrogen/oxygen), two ligands at a distance of approx. 0.185 nm (1.85 A) and four ligands at approx. 0.204 nm (2.04 A). The two shorter distances may correspond to Fe-O (tyrosine), whereas the longer distance is consistent with Fe-N (histidine) and Fe-O (water). Detailed analysis of the spectra of the N-terminal and C-terminal fragments indicates a difference in the short ligand distance.

An eye lens protein-water structure: 1.2 A resolution structure of gammaB-crystallin at 150 K.

gammabeta-crystallin is a structural protein of the eye lens with a role in the maintenance of an even distribution of protein and water over distances around the wavelength of light, preserving lens transparency. The structure of the 174-residue bovine protein has already been determined at room temperature to 1.47 A resolution. By flash freezing the protein crystals, data have now been collected to a nominal resolution limit of 1.2 A as radiation damage was essentially eliminated. The protein-water model has been refined against this data using the program RESTRAIN converging to an R factor of 18.5% with all data. Atomic positions are clearly indicated in the electron-density maps. Discrete bimodal disorder has been visualized for a few side chains. Out of a total of 498 water molecules present in the crystal asymmetric unit, 394 have been modelled and refined at unit occupancy. The solvent structure is extremely well ordered with an average B value of 23.4 A(2). Partially occupied sites have been identified where disorder in the protein induces concomitant disorder in the local solvent structure. The solvent structure covers 97% of the solvent-exposed surface of the protein in the crystal. 126 water molecules are distributed in second and higher hydration shells. There are networks of hydrogen-bonded solvent extending up to 64 molecules in a network, comprising trimers and tetramers as well as five- and six-membered water-ring structures. The hydration of the protein surface is dominated by arginine and aspartate side chains. Extensive cages of highly ordered solvent molecules are also observed around exposed non-polar groups.

Identification of the prooxidant site of human ceruloplasmin: a model for oxidative damage by copper bound to protein surfaces.

Free transition metal ions oxidize lipids and lipoproteins in vitro; however, recent evidence suggests that free metal ion-independent mechanisms are more likely in vivo. We have shown previously that human ceruloplasmin (Cp), a serum protein containing seven Cu atoms, induces low density lipoprotein oxidation in vitro and that the activity depends on the presence of a single, chelatable Cu atom. We here use biochemical and molecular approaches to determine the site responsible for Cp prooxidant activity. Experiments with the His-specific reagent diethylpyrocarbonate (DEPC) showed that one or more His residues was specifically required. Quantitative [14C]DEPC binding studies indicated the importance of a single His residue because only one was exposed upon removal of the prooxidant Cu. Plasmin digestion of [14C]DEPC-treated Cp (and N-terminal sequence analysis of the fragments) showed that the critical His was in a 17-kDa region containing four His residues in the second major sequence homology domain of Cp. A full length human Cp cDNA was modified by site-directed mutagenesis to give His-to-Ala substitutions at each of the four positions and was transfected into COS-7 cells, and low density lipoprotein oxidation was measured. The prooxidant site was localized to a region containing His426 because CpH426A almost completely lacked prooxidant activity whereas the other mutants expressed normal activity. These observations support the hypothesis that Cu bound at specific sites on protein surfaces can cause oxidative damage to macromolecules in their environment. Cp may serve as a model protein for understanding mechanisms of oxidant damage by copper-containing (or -binding) proteins such as Cu, Zn superoxide dismutase, and amyloid precursor protein.