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This study investigated the antimicrobial activities of peptides derived from the N-terminal region of human lactoferrin, and examined the contributions of individual residues to the activity of the most potent peptide. Two regions of antimicrobial activity were identified, the first corresponding to a weakly active peptide, HLP-9, comprising residues 1-9, and a second corresponding to a more potent peptide, HLP-10, comprising residues 18-26 and containing the hexapeptide motif, FQWQRN. Inhibitory studies on peptides from the first region confirm the importance of tryptophan residues in enhancing and broadening peptide activity. Inhibitory studies with glycine-substituted homologues of the more potent peptide showed that F21/G and R25/G substitutions resulted in a major reduction or complete loss of activity, while increased peptide cationicity or flexibility had little effect. Our findings demonstrate that F21 and R25 are critical determinants of potency for HLP-10, and that the second aromatic residue may act synergistically with W23 in developing and enhancing the activity of this cationic peptide.

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.

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.

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.

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.

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.

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.