X-ray structure of the curare alkaloid (+)-tubocurarine dibromide.

X-ray structure determination of the compound (C37H42N2O6)2+ .2Br-.4CH3OH, confirms that (+)-tubocurarine is a monoquaternary salt and has established that the molecule adopts different conformations in crystals of the dibromide and dichloride salts. The crystal structure is stabilised by a number of hydrogen bonds involving the two free hydroxyl groups and the tertiary nitrogen of the tubocurarine molecule, the bromide ions and the solvent molecules. The absolute configuration of the molecule, determined by X-ray anomalous scattering, confirms the configuration assigned earlier by chemical studies.

Conformational analysis of pentapeptide sequences matching a proposed recognition motif for lysosomal degradation.

A selective pathway for the degradation of specific long-lived cytosolic proteins is activated in response to starvation in vivo or to serum withdrawal from cultured cells. It involves recognition of a targeting motif by a member of the hsp70 family. A 5-residue targeting motif has been proposed on the basis of sequence comparisons. We investigate whether there is any structural basis for this motif being the true recognition signal. We examine the conformations of four motif peptides in proteins that are either known to be serum regulated or are from related vertebrate species, and two equivalent peptides in bacterial proteins that closely resemble other regulated proteins. Our studies show that all the motif sequences are located near the ends of surface helices with one or more of the residues buried in the structure, yet it is known that members of the hsp70 family tend to interact with extended peptide chains. Furthermore, recognition by these proteins generally requires a specific ordering of key residues, yet the motif implies a largely order-independent sequence characterized by residue type only. We conclude that the proposed motif is unlikely to be the true targeting signal for lysosomal degradation unless additional factors apply.

Evidence for the glycosylation of porcine serum transferrin at a single site located within the C-terminal lobe.

In this study we report the number and location of the glycans on PST. Urea PAGE and SDS-PAGE have been used to follow the enzymatic removal of sialic acids and of glycans from PST and the masses of native and deglycosylated PST have been determined by electrospray mass spectrometry. The results are consistent with the presence of a single biantennary glycan chain. As amino acid sequence analysis demonstrated the absence of a glycosylated asparagine at position 25, the glycosylation site is restricted to Asp-497.

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.

Identification of potential ferric binding residues in the iron-binding protein of pathogenic Neisseria meningitidis through structure-based multiple sequence alignments.

The ferric iron-binding proteins of pathogenic Neisseria display structural and metal-binding properties characteristic of the transferrin family. In the absence of structural data for the ferric iron-binding proteins, spacial folding templates have been derived for the meningococcal protein from complete and partial structure-based multiple sequence alignments with structurally related proteins. The templates have been used to identify a number of potential iron-binding residues. These include four residues that are identical with the iron coordinating ligands of transferrin, but only two reside within equivalent structural elements.

Identification of potential ferric binding residues in the iron-binding protein of pathogenic Neisseria meningitidis through structure-based multiple sequence alignments.

The ferric iron-binding proteins of pathogenic Neisseria display structural and metal-binding properties characteristic of the transferrin family. In the absence of structural data for the ferric iron-binding proteins, spacial folding templates have been derived for the meningococcal protein from complete and partial structure-based multiple sequence alignments with structurally related proteins. The templates have been used to identify a number of potential iron-binding residues. These include four residues that are identical with the iron coordinating ligands of transferrin, but only two reside within equivalent structural elements.

Characterisation of the meningococcal transferrin binding protein complex by photon correlation spectroscopy.

Photon correlation spectroscopy demonstrated for the first time that co-purified meningococcal TbpA+B form a complex in solution. This structure bound hTf and the resultant species underwent partial dissociation after exposure to additional hTf or following prolonged incubation. Purified TbpA and TbpB had similar apparent sizes but showed distinctive size profiles suggesting that TbpA forms a largely homogeneous population while TbpB may produce more variable particle sizes under these conditions.

Transferrin-binding protein B isolated from Neisseria meningitidis discriminates between apo and diferric human transferrin.

Neisseria meningitidis utilization of human serum transferrin (hTF)-bound iron is an important pathogenicity determinant. The efficiency of this system would clearly be increased through preferential binding of diferric hTF over the iron-free form. To characterize this process, functionally active meningococcal transferrin-binding protein A (TbpA) and TbpB have been purified from N. meningitidis using a novel purification procedure. The association of isolated Tbps and Tbps in the presence of hTF was investigated by gel filtration. Co-purified TbpA+B formed a complex of molecular mass 300 kDa which bound 1-2 molecules of hTF. Purified TbpA formed a complex of 200 kDa, indicating association as a dimer, whereas TbpB aggregated to form multimers of variable sizes. On recombining TbpA and TbpB, a stable complex of equivalent size to co-purified TbpA+B was formed. This complex may be composed of a single TbpA dimer and 1 molecule of TbpB. The technique of surface plasmon resonance (SPR) was used to demonstrate clearly that TbpB of either high (85 kDa) or low (68 kDa) molecular-mass preferentially bound diferric hTF in comparison with iron-free hTF. This selectivity was not observed with TbpA, but was found at low levels with co-purified TbpA+B. Individual TbpA and TbpB, recombined in a 1:1 molecular ratio, showed iron-mediated discriminatory binding at an intermediate level. SPR was also used to show that TbpA and TbpB bound to distinct regions of hTF, and that prior saturation with TbpB reduced subsequent TbpA binding. The results demonstrated that hTF bound more TbpA than TbpB, with an approximate ratio of 2:1. We have demonstrated that in vitro, TbpA+B exists as a receptor complex composed of a TbpA dimer and one molecule of TbpB, and that TbpB selectively binds diferric hTF. We propose that, in vivo, TbpA and TbpB also exist as a receptor complex, with TbpB selectively binding diferric hTF, bringing it close to TbpA, the transmembrane component, where the ferric iron can be transported to the periplasm.

Evidence for the bilobal nature of diferric rabbit plasma transferrin.

Plasma transferrin is involved in iron transport within the circulatory system of vertebrates, and provides an iron source for haemoglobin synthesis and other metabolic requirements. However, despite extensive studies by spectroscopic, biochemical and physiological techniques, the nature of iron binding and the mechanisms of uptake and release of iron are not fully understood. Plasma transferrins are monomeric glycoproteins with a molecular weight of approximately 80,000 (ref. 2); they have two similar and very strong binding sites for Fe(III), together with two associated anion binding sites. Fragmentation studies on various transferrins have shown that the polypeptide chain is composed of two domains formed from the N-terminal and C-terminal halves of the polypeptide chain. Each domain contains one metal binding site. The marked sequence similarities which exist between the two halves may reflect a doubling of an ancestral structural gene during the phylogenetic development of the protein. Preliminary crystallographic investigations of diferric rabbit plasma transferrin have been reported from this laboratory. We now report initial studies of the X-ray structure determination of dife-ric rabbit plasma transferrin which have led to a 6-A resolution electron density map.

Molecular structure of serum transferrin at 3.3-A resolution.

Serum transferrin is a metal-binding glycoprotein, molecular weight ca. 80,000, whose primary function is the transport of iron in the plasma of vertebrates. The X-ray crystallographic structure of diferric rabbit serum transferrin has been determined to a resolution of 3.3 A. The molecule has a beta alpha structure of similar topology to human lactoferrin and is composed of two homologous lobes that each bind a single ferric ion. Each lobe is further divided into two dissimilar domains, and the iron-binding site is located within the interdomain cleft. The iron is bound by two tyrosines, a histidine, and an aspartic acid residue. The location of the 19 disulfide bridges is described, and their possible structural roles are discussed in relation to the transferrin family of proteins. Mapping of the intron/exon splice junctions onto the molecule provides some topological evidence in support of the putative secondary role for transferrin in stimulating cell proliferation.

Purified meningococcal transferrin-binding protein B interacts with a secondary, strain-specific, binding site in the N-terminal lobe of human transferrin.

Neisseria meningitidis, grown in iron-limited conditions, produces two transferrin-binding proteins (TbpA and TbpB) that independently and specifically bind human serum transferrin (hTF) but not bovine serum transferrin (bTF). We have used surface plasmon resonance to characterize the interaction between individual TbpA and TbpB and a series of full-length human-bovine chimaeric transferrins (hbTFs) under conditions of variable saturation with iron. A comparative analysis of hTF and hbTF chimaera-binding data confirmed that the major features involved in Tbp binding are located in the C-terminal lobe of hTF and that isolated TbpA can recognize distinct sites present in, or conformationally influenced by, residues 598-679. Binding by TbpB was maintained at a significant but decreased level after replacement of the entire hTF C-terminal lobe by the equivalent bovine sequence. The extent of this binding difference was dependent on the meningococcal strain and on the presence of hTF residues 255-350. This indicated that TbpB from strain SD has a secondary, strain-specific, binding site located within this region, whereas TbpB from strain B16B6 does not share this recognition site. Binding of TbpA was influenced primarily by sequence substitutions in the hTF C-terminal lobe, and co-purified TbpA and TbpB (TbpA+B) was functionally distinct from either of its components. The limited divergence between hTF and bTF has been related to observed differences in binding by Tbps and has been used to delineate those regions of hTF that are important for such interactions.