The structure of IP-10 was solved by NMR spectroscopy and represents the first structure from the class of agonists toward the receptor CXCR3. CXCR3 binding chemokines are unique in their ability to ...bind receptors from both the CC and CXC classes of chemokine receptors. An unusual structural feature of IP-10 was identified that may provide the basis for the ability of IP-10 to bind both CXCR3 and CCR3. The surface of IP-10 that interacts with the N-terminus of CXCR3 was defined by monitoring changes in the NMR spectrum of IP-10 upon addition of a CXCR3 N-terminal peptide. These studies indicated that the interaction involves a hydrophobic cleft, formed by the N-loop and 40s-loop region of IP-10, similar to the interaction surface observed for other chemokines such as IL-8. An additional region of interaction was observed that consists of a hydrophobic cleft formed by the N-terminus of IP-10 and 30s-loop of IP-10.
The malarial surface antigen apical membrane antigen (AMA1), from
Plasmodium falciparum, is a leading candidate for inclusion in a vaccine against malaria. AMA1 is synthesised by mature blood-stages ...of the parasite and is located initially in the apical organelles of the merozoite. Prior to merozoite invasion of host erythrocytes, it is processed into a 66
kDa type 1 integral membrane protein on the merozoite surface. The pattern of disulphide bonds in AMA1 has been the basis for separation of the ectodomain into three domains, with three, two and three disulphide bonds, respectively. We have determined the solution structure of a 16
kDa construct corresponding to the putative second domain of AMA1. While circular dichroism and hydrodynamic data were consistent with a folded structure for domain II, its NMR spectra were characterised by broad lines and significant peak overlap, more typical of a molten globule. Consistent with this, domain II bound the fluorescent dye 8-anilino-1-naphthalene sulphonate (ANS). We have nonetheless determined a structure, which defines the secondary structure elements and global fold. The two disulphide bonds link the N and C-terminal regions of the molecule, which come together to form a four-stranded β-sheet linked to a short helix. A long loop linking the N and C-terminal regions contains four other α-helices, the locations of which are not fixed relative to the β-sheet core, even though they are well-defined locally. Very recently this region of domain II has been shown to contain the epitope recognised by the invasion-inhibitory antibody 4G2, even though it does not contain any of the polymorphisms that are regarded as having arisen in response to the pressure of immune recognition.
SmIIIA is a new μ-conotoxin isolated recently from Conus stercusmuscarum. Although it shares several biochemical characteristics with other μ-conotoxins (the arrangement of cysteine residues and a ...conserved arginine believed to interact with residues near the channel pore), it has several distinctive features, including the absence of hydroxyproline, and is the first specific antagonist of tetrodotoxin-resistant voltage-gated sodium channels to be characterized. It therefore represents a potentially useful tool to investigate the functional roles of these channels. We have determined the three-dimensional structure of SmIIIA in aqueous solution. Consistent with the absence of hydroxyprolines, SmIIIA adopts a single conformation with all peptide bonds in the trans configuration. The spatial orientations of several conserved Arg and Lys side chains, including Arg14 (using a consensus numbering system), which plays a key role in sodium channel binding, are similar to those in other μ-conotoxins but the N-terminal regions differ, reflecting the trans conformation for the peptide bond preceding residue 8 in SmIIIA, as opposed to the cis conformation in μ-conotoxins GIIIA and GIIIB. Comparison of the surfaces of SmIIIA with other μ-conotoxins suggests that the affinity of SmIIIA for TTX-resistant channels is influenced by the Trp15 side chain, which is unique to SmIIIA. Arg17, which replaces Lys in the other μ-conotoxins, may also be important. Consistent with these inferences from the structure, assays of two chimeras of SmIIIA and PIIIA in which their N- and C-terminal halves were recombined, indicated that residues in the C-terminal half of SmIIIA confer affinity for tetrodotoxin-resistant sodium channels in the cell bodies of frog sympathetic neurons. SmIIIA and the chimera possessing the C-terminal half of SmIIIA also inhibit tetrodotoxin-resistant sodium channels in the postganglionic axons of sympathetic neurons, as indicated by their inhibition of C-neuron compound action potentials that persist in the presence of tetrodotoxin.
Insulin-like growth factor-binding protein-2 (IGFBP-2) is the largest member of a family of six proteins (IGFBP-1 to 6) that bind insulin-like growth factors I and II (IGF-I/II) with high affinity. ...In addition to regulating IGF actions, IGFBPs have IGF-independent functions. The C-terminal domains of IGFBPs contribute to high-affinity IGF binding, and confer binding specificity and have overlapping but variable interactions with many other molecules. Using nuclear magnetic resonance (NMR) spectroscopy, we have determined the solution structure of the C-terminal domain of IGFBP-2 (C-BP-2) and analysed its backbone dynamics based on
15N relaxation parameters. C-BP-2 has a thyroglobulin type 1 fold consisting of an α-helix, a three-stranded anti-parallel β-sheet and three flexible loops. Compared to C-BP-6 and C-BP-1, structural differences that may affect IGF binding and underlie other functional differences were found. C-BP-2 has a longer disordered loop I, and an extended C-terminal tail, which is unstructured and very mobile. The length of the helix is identical with that of C-BP-6 but shorter than that of C-BP-1. Reduced spectral density mapping analysis showed that C-BP-2 possesses significant rapid motion in the loops and termini, and may undergo slower conformational or chemical exchange in the structured core and loop II. An RGD motif is located in a solvent-exposed turn. A pH-dependent heparin-binding site on C-BP-2 has been identified. Protonation of two histidine residues, His271 and His228, seems to be important for this binding, which occurs at slightly acidic pH (6.0) and is more significant at pH 5.5, but is largely suppressed at pH 7.4. Possible preferential binding of IGFBP-2 and its C- domain fragments to glycosaminoglycans in the acidic extracellular matrix (ECM) of tumours may be related to their roles in cancer.
The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical ...properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional
H,
N and
H,
C NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-
N, 20%-
C-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics.
IGFs are important mediators of growth. IGF binding proteins (IGFBPs) 1-6 regulate IGF actions and have IGF-independent actions. The C-terminal domains of IGFBPs contribute to high-affinity IGF ...binding and modulation of IGF actions and confer some IGF-independent properties, but understanding how they achieve this has been constrained by the lack of a three-dimensional structure. We therefore determined the solution structure of the C-domain of IGFBP-6 using nuclear magnetic resonance (NMR). The domain consists of a thyroglobulin type 1 fold comprising an alpha-helix followed by a loop, a three-stranded antiparallel beta-sheet incorporating a second loop, and finally a disulfide-bonded flexible third loop. The IGF-II binding site on the C-domain was identified by examining NMR spectral changes upon complex formation. It consists of a largely hydrophobic surface patch involving the alpha-helix, the first beta-strand, and the first and second loops. The site was confirmed by mutagenesis of several residues, which resulted in decreased IGF binding affinity. The IGF-II binding site lies adjacent to surfaces likely to be involved in glycosaminoglycan binding of IGFBPs, which might explain their decreased IGF affinity when bound to glycosaminoglycans, and nuclear localization. Our structure provides a framework for understanding the roles of IGFBP C-domains in modulating IGF actions and conferring IGF-independent actions, as well as ultimately for the development of therapeutic IGF inhibitors for diseases including cancer.
Insulin-like growth factors (IGFs) are important mediators of growth and IGF-binding proteins (IGFBPs) 1–6 regulate IGF actions. As IGFBP C-terminal domains contribute to high-affinity IGF binding, ...we have defined the binding site for the C-domain of IGFBP-6 on IGF-II using NMR. This site lies adjacent to and between the binding sites for the IGFBP N-domain and IGF-I receptor (IGFIR), which have previously been found on opposite sides of the IGF molecule. The C-domain is therefore likely to interfere with IGF binding to the IGFIR, providing a structural basis for the potent inhibitory effects of intact IGFBPs on IGF actions.
Apical membrane antigen 1 (AMA1) of the human malaria parasite Plasmodium falciparum is synthesized by schizont stage parasites and has been implicated in merozoite invasion of host erythrocytes. ...Phage-display techniques have recently been used to identify two 15-residue peptides, F1 and F2, which bind specifically to P. falciparum AMA1 and inhibit parasite invasion of erythrocytes Li, F., et al. (2002) J. Biol. Chem. 277, 50303−50310. We have synthesized F1, F2, and three peptides with high levels of sequence identity, determined their relative binding affinities for P. falciparum AMA1 with a competition ELISA, and investigated their solution structures by NMR spectroscopy. The strongest binding peptide, F1, contains a β-turn that includes residues identified via an alanine scan as being critical for binding to AMA1 and inhibition of merozoite invasion of erythrocytes. The three F1 analogues include a 10-residue analogue of F1 truncated at the C-terminus (tF1), a partially scrambled 15-mer (sF1), and a disulfide-constrained 14-mer (F1tbp) which is related to F1 but has a sequence identical to that of a disulfide-constrained loop in the first epidermal growth factor module of the latent transforming growth factor-β binding protein. tF1 and F1tbp bound competitively with F1 to AMA1, and all three contain a type I β-turn encompassing key residues involved in F1 binding. In contrast, sF1 lacked this structural motif, and did not compete for binding to AMA1 with F1; rather, sF1 contained a type III β-turn involving a different part of the sequence. Although F2 was able to bind to AMA1, it was unstructured in solution, consistent with its weak invasion inhibitory effects. Thus, the secondary structure elements observed for these peptides in solution correlate well with their potency in binding to AMA1 and inhibiting merozoite invasion. The structures provide a valuable starting point for the development of peptidomimetics as antimalarial antagonists directed at AMA1.
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