Potential implications of the glycosylation patterns in collagen α1(I) and α2(I) chains for fibril assembly and growth.

O-Glycosylation of hydroxylysine (Hyl) in collagen occurs at an early stage of biosynthesis before the triple-helix has formed. This simple post-translational modification (PTM) of lysine by either a galactosyl or glucosylgalactosyl moiety is highly conserved in collagens and depends on the species, type of tissue and the collagen amino acid sequence. The structural/functional reason why only specific lysines are modified is poorly understood, and has led to increased efforts to map the sites of PTMs on collagen sequences from different species and to ascertain their potential role in vivo. To investigate this, we purified collagen type I (Col1) from the skins of four animals, then used mass spectrometry and proteomic techniques to identify lysines that were oxidised, galactosylated, glucosylgalactosylated, or glycated in its mature sequence. We found 18 out of the 38 lysines in collagen type Iα1, (Col1A1) and 7 of the 30 lysines in collagen type Iα2 (Col1A2) were glycosylated. Six of these modifications had not been reported before, and included a lysine involved in crosslinking collagen molecules. A Fourier transform analysis of the positions of the glycosylated hydroxylysines showed they display a regular axial distribution with the same d-period observed in collagen fibrils. The significance of this finding in terms of the assembly of collagen molecules into fibrils and of potential restrictions on the growth of the collagen fibrils is discussed.

Assessment of pelt quality in leather making using a novel non-invasive sensing approach.

Excessive removal of structural material from skin during leather processing results in unattractive crease formation in leather. It is difficult to detect this in pelts at an early processing stage as it only becomes really apparent once the skin is made into leather. There would be great advantages in detecting the problem at the pickled pelt stage (skins treated with sodium sulphide and lime, bated with enzymes, and then preserved in NaCl and sulphuric acid) so that adjustments to the processing could be made to mitigate the effect. A novel bio-sensor for inspection of pickled lamb pelts has been fabricated and developed. The sensor has the planar Interdigital structure. The experimental results show that the sensor has a great potential to predict the quality of leather in a non-invasive and non-destructive way.

Use of a proteomics approach to identify favourable conditions for production of good quality lambskin leather.

It is necessary to understand the changes that occur during the initial processing of lamb skins, because these will affect the final quality of the leather. The types of collagen, their macro and micro structures, the presence of proteins other than collagens, and the quantity and the type of proteoglycans, all have a profound effect on the quality of leather. Proteins isolated from untreated or raw sheep skin and from pickled skin (skins treated with sodium sulfide and lime followed by bating with enzymes, then preserved in sodium chloride and sulfuric acid) were significantly different when analysed by use of 2D gel electrophoresis and mass spectrometry. Agarose gel electrophoresis with a very sensitive sequential staining procedure has been used to identify the glycosaminoglycans present in raw and treated skin and their impact on quality of leather. Results showed that effective removal of proteoglycans acting as inter-fibrillar adhesives of collagen fibrils seemed to improve leather quality. Removal of these molecules not only opens up the fibre structure of the skin but may also be important in wool removal. The presence of elastin, which imparts elastic properties to skin, is of significant importance to tanners. The amino acids desmosine and isodesmosine, found exclusively in elastin, were quantitatively analysed to assess the role of elastin in leather quality.

Structure of human apolactoferrin at 2.0 A resolution. Refinement and analysis of ligand-induced conformational change.

The three-dimensional structure of a form of human apolactoferrin, in which one lobe (the N-lobe) has an open conformation and the other lobe (the C-lobe) is closed, has been refined at 2.0 A resolution. The refinement, by restrained least-squares methods, used synchrotron radiation X-ray diffraction data combined with a lower resolution diffractometer data set. The final refined model (5346 protein atoms from residues 1-691, two Cl- ions and 363 water molecules) gives a crystallographic R factor of 0.201 (Rfree = 0. 286) for all 51305 reflections in the resolution range 10.0-2.0 A. The conformational change in the N-lobe, which opens up the binding cleft, involves a 54 degrees rotation of the N2 domain relative to the N1 domain. This also results in a small reorientation of the two lobes relative to one another with a further approximately 730 A2 of surface area being buried as the N2 domain contacts the C-lobe and the inter-lobe helix. These new contacts also involve the C-terminal helix and provide a mechanism through which the conformational and iron-binding status of the N-lobe can be signalled to the C-lobe. Surface-area calculations indicate a fine balance between open and closed forms of lactoferrin, which both have essentially the same solvent-accessible surface. Chloride ions are bound in the anion-binding sites of both lobes, emphasizing the functional significance of these sites. The closed configuration of the C-lobe, attributed in part to weak stabilization by crystal packing interactions, has important implications for lactoferrin dynamics. It shows that a stable closed structure, essentially identical to that of the iron-bound form, can be formed in the absence of iron binding.

Altered domain closure and iron binding in transferrins: the crystal structure of the Asp60Ser mutant of the amino-terminal half-molecule of human lactoferrin.

The crystal structure of a site-specific mutant of the N-terminal half-molecule of human lactoferrin, Lf(N), in which the iron ligand Asp60 has been mutated to Ser, has been determined at 2.05 A resolution in order to determine the effects of the mutation on iron binding and domain closure. Yellow monoclinic crystals of the D60S mutant, in its iron-bound form, were prepared, and have unit cell dimensions a = 110.2 A, b = 57.0 A, c = 55.2 A, beta = 97.6 degrees, space group C2, with one molecule of 333 residues in the asymmetric unit. The structure was determined by molecular replacement, using the wild-type Lf(N) as search model, and was refined by restrained least-squares methods. The final model, comprising 2451 protein atoms (from residues 2 to 315) one Fe3+ and one CO2-(3), and 107 water molecules, gives an R-factor of 0.175 for all data in the resolution range 20.0 to 2.05 A. The model conforms well with standard geometry, having root-mean-square deviations of 0.014 A and 1.2 degrees from standard bond lengths and angles. The structure of the D60S mutant deviates in two important respects from the parent Lf(N) molecule. At the mutation site the Ser side-chain neither binds to the iron atom nor makes any interdomain contact as the substituted Asp does; instead a water molecule fills the iron coordination site and participates in interdomain hydrogen bonding. The domain closure is also changed, with the D60S mutant having a more closed conformation. Consideration of crystal packing suggests that the altered domain closure is a genuine molecular property but both the iron coordination and interdomain contacts are consistent with weakened iron binding in the mutant. The implications for iron binding in transferrins generally are discussed.

The three-dimensional structure of PNGase F, a glycosylasparaginase from Flavobacterium meningosepticum.

BACKGROUND: Peptide:N-glycosidase F (PNGase F) is an enzyme that catalyzes the complete removal of N-linked oligosaccharide chains from glycoproteins. Often called an endoglycosidase, it is more correctly termed an amidase or glycosylasparaginase as cleavage is at the asparagine-sugar amide linkage. The enzyme is widely used in structure-function studies of glycoproteins. RESULTS: We have determined the crystal structure of PNGase F at 1.8 A resolution. The protein is folded into two domains, each with an eight-stranded antiparallel beta jelly roll configuration similar to many viral capsid proteins and also found, in expanded form, in lectins and several glucanases. Two potential active site regions have been identified, both in the interdomain region and shaped by prominent loops from one domain. Exposed aromatic residues are a feature of one site. CONCLUSIONS: The finding that PNGase F is based on two jelly roll domains suggests parallels with lectins and other carbohydrate-binding proteins. These proteins either bind sugars on the concave face of the beta-sandwich structure (aided by loops) or amongst the loops themselves. Further analysis of the function and identification of the catalytic site should lead to an understanding of both the specificity of PNGase F and possibly also the recognition processes that identify glycosylation sites on proteins.

Purification and crystallization of the endoglycosidase PNGase F, a peptide:N-glycosidase from Flavobacterium meningosepticum.

The endoglycosidase peptide: N-glycosidase, secreted by the Gram-negative bacterium Flavobacterium meningosepticum (PNGase F), has been isolated, purified to homogeneity and crystallized from polyethylene glycol solutions using vapour diffusion and seeding techniques. The crystals are orthorhombic, space group P2(1)2(1)2, with unit cell dimensions a = 85.07 A, b = 85.14 A, c = 48.50 A, and are suitable for high resolution X-ray structure analysis.

Enzymatic deglycosylation as a tool for crystallization of mammalian binding proteins.

Enzymatic deglycosylation has been used in attempts to crystallize several glycoproteins with the aim of overcoming the problems resulting from heterogeneity and flexibility of the attached glycan chains. An endoglycosidase preparation from Flavobacterium meningosepticum, comprising the enzymes endo F and PNGase-F, was used in experiments on the mammalian binding proteins lactoferrin and haemopexin. Significant differences were found in the susceptibility of different proteins to deglycosylation. For human lactoferrin (Lf) and its recombinant N-terminal half-molecule (Lf(N)), deglycosylation was rapid and complete, and was essential for obtaining high-quality crystals of both apo-Lf and Lf(N); for bovine Lf, however, complete deglycosylation did not occur. Similarly, for rabbit haemopexin the carbohydrate chain on the C-terminal domain was easily removed, but the three chains on the N-terminal domain proved more resistant and their removal led to some fragmentation of the protein. Nevertheless, this approach provided the only means of crystallizing the C-terminal domain and is likely to be useful for other glycoproteins.

Three-dimensional structure of lactoferrin in various functional states.

The three-dimensional structures of various forms of lactoferrin, determined by high resolution crystallographic studies, have been compared in order to determine the relationship between structure and biological function. These comparisons include human apo and diferric lactoferrins, metal and anion substituted lactoferrins, the N-terminal half molecule of human lactoferrin, and bovine diferric lactoferrin. The structures themselves define the nature and location of the iron binding sites and allow anti-bacterial and putative receptor-binding regions to be mapped on to the molecular surface. The structural comparisons show that small internal adjustments can allow the accommodation of different metals and anions without altering the overall molecular structure, whereas large-scale conformational changes are associated with metal binding and release, and smaller, but significant, movements accompany species variations. The results also focus on differences in flexibility between the two lobes, and on the importance of interactions in the inter-lobe region in modulating iron release from the N-lobe and in possibly enabling binding at one site to be signalled to the other.

Domain closure in lactoferrin. Two hinges produce a see-saw motion between alternative close-packed interfaces.

Lactoferrin is an iron transport protein. Upon binding iron, the two domains in the N-terminal half of the molecule move together. Previous work has shown that this domain closure involves two hinges. Using the newly refined structure of the open form, the structural mechanism underlying this motion is analyzed here in detail. Upon closure the domains rotate 54 degrees essentially as rigid bodies. The axis of rotation passes through the two beta-strands linking the domains. These strands contain hinges in the sense that three large torsion angle changes are responsible for the bulk of the motion while smaller torsion angle changes in neighboring residues are responsible for the remainder of the motion. The rotation axes of these three torsion angle changes are nearly parallel to the axis of the overall 54 degrees rotation, so the local motion in the hinges can be directly related to the overall motion. A crucial feature of the hinge residues is that they have very few packing constraints on their main-chain atoms. The domains make different packing contacts with each other in the open and closed forms. These contacts form two interdomain interfaces arranged on either side of the hinges. Pivoting about the hinges produces a see-saw motion between the two interfaces. That is, when the domains close down, residues in the interface on one side of the hinges become buried and close-packed and residues on the other side become exposed. The situation is reversed when the domains open up. Lactoferrin provides a particularly clear example of the general features of hinged domain motion. It is compared to other instances of hinged domain closure and contrasted with instances of shear domain closure, where the overall motion is a summation of many small sliding motions between close-packed segments of polypeptide.

Crystallization of the C-terminal domain of rabbit serum hemopexin.

The C-terminal domain of rabbit serum hemopexin, comprising residues 215 to 435, has been crystallized following removal of the attached carbohydrate using the endoglycosidase Endo F. The crystals, grown by vapour diffusion from solutions containing polyethylene glycol 1500, are orthorhombic, with cell dimensions a = 41.0 A, b = 64.2 A, c = 85.2 A, space group P2(1)2(1)2(1), and one molecule in the asymmetric unit. The crystals diffract to 2.4 A resolution and are suitable for X-ray structure analysis.

Preliminary crystallographic studies of the amino terminal half of human lactoferrin in its iron-saturated and iron-free forms.

The amino terminal half of human lactoferrin (LfN) produced from transfected baby hamster kidney cells has been crystallized in its iron-saturated and iron-free forms. The crystals of glycosylated LfN and deglycosylated LfN are monoclinic, space group C2, with cell dimensions a = 133.0 A, b = 58.3 A, c = 58.3 A, alpha = 90.0 degrees, beta = 114.7 degrees, gamma = 90.0 degrees, and one molecule per asymmetric unit. Crystals of apo LfN have also been prepared using deglycosylated protein. These crystals are tetragonal, space group P4(1)2(1)2 (or P4(3)2(1)2), with cell dimensions of a = b = 58.4 A and c = 217.2 A and one molecule per asymmetric unit. Both the iron-saturated and the iron-free crystals are suitable for high resolution X-ray analysis.

Molecular replacement solution of the structure of apolactoferrin, a protein displaying large-scale conformational change.

The crystal structure of an orthorhombic form of human apolactoferrin (ApoLf) has been determined from 2.8 A diffractometer data by molecular replacement methods. A variety of search models derived from the diferric lactoferrin structure (Fe2Lf) were used to obtain a consistent solution to the rotation function. An R-factor search gave the correct translational solution and the model was refined by rigid-body least-squares refinement (program CORELS). Only three of the four domains were located correctly by this procedure, however; the fourth was finally placed correctly by rotating it manually onto three strands of electron density which were recognized as part of its central beta-sheet. The final model, refined by restrained least-squares methods to an R factor of 0.214 for data in the resolution range 10.0 to 2.8 A, shows a large domain movement in the N-terminal half of the molecule (a 54 degree rotation of domain N2) and smaller domain movements elsewhere, when compared with Fe2Lf. A feature of the crystal structure is that although the ApoLf and Fe2Lf unit cells appear very similar, their crystal packing and molecular structures are quite different.

Structure, function and flexibility of human lactoferrin.

X-ray structure analyses of four different forms of human lactoferrin (diferric, dicupric, an oxalate-substituted dicupric, and apo-lactoferrin), and of bovine diferric lactoferrin, have revealed various ways in which the protein structure adapts to different structural and functional states. Comparison of diferric and dicupric lactoferrins has shown that different metals can, through slight variations in the metal position, have different stereochemistries and anion coordination without any significant change in the protein structure. Substitution of oxalate for carbonate, as seen in the structure of a hybrid dicupric complex with oxalate in one site and carbonate in the other, shows that larger anions can be accommodated by small side-chain movements in the binding site. The multidomain nature of lactoferrin also allows rigid body movements. Comparison of human and bovine lactoferrins, and of these with rabbit serum transferrin, shows that the relative orientations of the two lobes in each molecule can vary; these variations may contribute to differences in their binding properties. The structure of apo-lactoferrin demonstrates the importance of large-scale domain movements for metal binding and release and suggests that in solution an equilibrium exists between open and closed forms, with the open form being the active binding species. These structural forms are shown to be similar to those seen for bacterial periplasmic binding proteins, and lead to a common model for the various steps in the binding process.

Apolactoferrin structure demonstrates ligand-induced conformational change in transferrins.

Proteins of the transferrin family, which contains serum transferrin and lactoferrin, control iron levels in higher animals through their very tight (Kapp approximately 10(20)) but reversible binding of iron. These bilobate molecules have two binding sites, one per lobe, each housing one Fe3+ and the synergistic CO3(2-) ion. Crystallographic studies of human lactoferrin and rabbit serum transferrin in their iron-bound forms have characterized their binding sites and protein structure. Physical studies show that a substantial conformational change accompanies iron binding and release. We have addressed this phenomenon through crystal structure analysis of human apolactoferrin at 2.8 A resolution. In this structure the N-lobe binding cleft is wide open, following a domain rotation of 53 degrees, mediated by the pivoting of two helices and flexing of two interdomain polypeptide strands. Remarkably, the C-lobe cleft is closed, but unliganded. These observations have implications for transferrin function and for binding proteins in general.

Structure of human lactoferrin: crystallographic structure analysis and refinement at 2.8 A resolution.

The structure of human lactoferrin has been refined crystallographically at 2.8 A (1 A = 0.1 nm) resolution using restrained least squares methods. The starting model was derived from a 3.2 A map phased by multiple isomorphous replacement with solvent flattening. Rebuilding during refinement made extensive use of these experimental phases, in combination with phases calculated from the partial model. The present model, which includes 681 of the 691 amino acid residues, two Fe3+, and two CO3(2-), gives an R factor of 0.206 for 17,266 observed reflections between 10 and 2.8 A resolution, with a root-mean-square deviation from standard bond lengths of 0.03 A. As a result of the refinement, two single-residue insertions and one 13-residue deletion have been made in the amino acid sequence, and details of the secondary structure and tertiary interactions have been clarified. The two lobes of the molecule, representing the N-terminal and C-terminal halves, have very similar folding, with a root-mean-square deviation, after superposition, of 1.32 A for 285 out of 330 C alpha atoms; the only major differences being in surface loops. Each lobe is subdivided into two dissimilar alpha/beta domains, one based on a six-stranded mixed beta-sheet, the other on a five-stranded mixed beta-sheet, with the iron site in the interdomain cleft. The two iron sites appear identical at the present resolution. Each iron atom is coordinated to four protein ligands, 2 Tyr, 1 Asp, 1 His, and the specific Co3(2-), which appears to bind to iron in a bidentate mode. The anion occupies a pocket between the iron and two positively charged groups on the protein, an arginine side-chain and the N terminus of helix 5, and may serve to neutralize this positive charge prior to iron binding. A large internal cavity, beyond the Arg side-chain, may account for the binding of larger anions as substitutes for CO3(2-). Residues on the other side of the iron site, near the interdomain crossover strands could provide secondary anion binding sites, and may explain the greater acid-stability of iron binding by lactoferrin, compared with serum transferrin. Interdomain and interlobe interactions, the roles of charged side-chains, heavy-atom binding sites, and the construction of the metal site in relation to the binding of different metals are also discussed.

Preliminary crystallographic studies on human apo-lactoferrin in its native and deglycosylated forms.

Human apo-lactoferrin in both native and deglycosylated forms has been purified, and crystals obtained by dialysis against low ionic strength buffer solutions. The crystals of native apo-lactoferrin are orthorhombic, space group P2(1)2(1)2(1) with cell dimensions a = 222.0 A, b = 115.6 A, c = 77.8 A and have two protein molecules per asymmetric unit. Two crystal forms of deglycosylated apo-lactoferrin have been obtained. One is orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 152.1 A, b = 94.6 A, c = 55.8 A. The second is tetragonal, space group I4, with cell dimensions a = b = 189.4 A, c = 55.1 A. Both of the latter have only one molecule per asymmetric unit, and are suitable for high-resolution X-ray structure analysis.