Galectin-3 is an adhesion/growth-regulatory protein with a modular design comprising an N-terminal tail (NT, residues 1-111) and the conserved carbohydrate recognition domain (CRD, residues 112-250). The chimera-type galectin interacts with both glycan and peptide motifs. Complete (13)C/(15)N-assignment of the human protein makes NMR-based analysis of its structure beyond the CRD possible. Using two synthetic NT polypeptides covering residues 1-50 and 51-107, evidence for transient secondary structure was found with helical conformation from residues 5 to 15 as well as proline-mediated, multi-turn structure from residues 18 to 32 and around PGAYP repeats. Intramolecular interactions occur between the CRD F-face (the 5-stranded β-sheet behind the canonical carbohydrate-binding 6-stranded β-sheet of the S-face) and NT in full-length galectin-3, with the sequence P(23)GAW(26)…P(37)GASYPGAY(45) defining the primary binding epitope within the NT. Work with designed peptides indicates that the PGAX motif is crucial for self-interactions between NT/CRD. Phosphorylation at position Ser6 (and Ser12) (a physiological modification) and the influence of ligand binding have minimal effect on this interaction. Finally, galectin-3 molecules can interact weakly with each other via the F-faces of their CRDs, an interaction that appears to be assisted by their NTs. Overall, our results add insight to defining binding sites on galectin-3 beyond the canonical contact area for β-galactosides.

The binding of a nitroxide spin-labeled analog of N-acetyllactosamine to galectin-3, a mammalian lectin of 26 kD size, is studied to map the binding sites of this small oligosaccharide on the protein surface. Perturbation of intensities of cross-peaks in the 15 N heteronuclear single quantum coherence (HSQC) spectrum of full-length galectin-3 owing to the bound spin label is used qualitatively to identify protein residues proximate to the binding site for N-acetyllactosamine. A protocol for converting intensity measurements to a more quantitative determination of distances between discrete protein amide protons and the bound spin label is then described. This protocol is discussed as part of a drug design strategy in which subsequent perturbation of chemical shifts of distance mapped amide cross-peaks can be used effectively to screen a library of compounds for other ligands that bind to the target protein at distances suitable for chemical linkage to the primary ligand. This approach is novel in that it bypasses the need for structure determination and resonance assignment of the target protein.Keywords: Nitroxide spin label; galectin-3; drug design; drug screening; distance mapping Knowing the geometric relationship of various small molecules that bind to protein surfaces can be an important point of reference in the design of effective inhibitors of protein function (Shuker et al. 1996;Hadjuk et al. 1997). Here, we illustrate a method for providing intermolecular distance information based on the use of spin-labeled analogs of known protein ligands to perturb cross-peaks in a 15 N-1 H heteronuclear single quantum coherence (HSQC) spectrum in a distance-dependent fashion. Labeling crosspeaks in terms of distance from a primary ligand binding site allows use of those cross-peaks in subsequent chemical shift perturbation screens for secondary ligands that bind at appropriate distances for chemical linkage to the primary ligand.There are precedents in the literature for using a spinlabeled ligand to gain information about protein-ligand interactions (Kosen 1989;Johnson et al. 1999). The most common application employs nitroxide spin labels incorporated in chemically stable molecules such as 2,2,6,6-tetramethyl-1-piperidine-1-oxyl (TEMPO). The route to distance information makes use of the paramagnetic relaxation of NMR resonances caused by these spin-labeled compounds. The relaxation rate enhancement depends on the distance of the nuclei from the unpaired electron, a property that can be used to map distances of nuclei from the spin label. The unpaired electron-nucleus interaction is fairly long-range, unlike the nuclear Overhauser effect (NOE), which is short range in nature (<5 Å). Hence, distances up to 20 Å can be mapped. This general procedure has been used recently to characterize the nature of the interaction between a cellulose-derived ligand and the cellulose binding domains of ␤-1,4 glucanase CenC from Cellulomonas fimi (Johnson et al. 1999). Also, there is some related work published recently t...

Kinetics of interchange between boric acid and tetrahydroxyborate has been studied by the band shape analysis of nB NMR signals in aqueous solution of pH 8-10 at 20-52 °C and 0.1-250 MPa. The system was analyzed as two-site exchange B(OH)3 -B(OH)4-The observed first-order rate constant toward the right (kx) is related to the second-order exchange rate constant (kex) by the equation kx = fccxCi[OH-] /(Zcb + [OH-]), where kh = [B(OH)3]-[OH"]/[B(OH)4-], and Cj is the total concentration of boron. Under the given conditions kex is (2-7) X 106 dm* 123 mol-1 s-1 and AH*, AS*, and AV* are 20.1 ± 1.0 kJ moH, -55.0 ± 3.1 J mol"1 K"1, and -9.9 ± 0.5 cm3 mol"1, respectively. The activation parameters reflect the transition state forming dimeric [( )3 (µ-) ( )3]-.

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The conformation of the carbohydrate recognition domain of Galectin-3, a lectin known to bind galactose containing oligosaccharides in mammalian systems, has been investigated in the absence of ligand and in the presence of N-acetylactosamine. A new methodology based on the measurement of residual dipolar couplings from NMR spectra has been used to characterize differences in protein structure along the backbone in the presence and absence of ligand, as well as the binding geometry of the ligand itself. The data on the ligand are consistent with the ligand binding geometry found in a crystal structure of the complexed state. However, a significant rearrangement of backbone loops near the binding site appears to occur in the absence of ligand. The implications for ligand specificity and protein functionality are discussed.

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Combining three units of one of H-(alanyl)n-beta-(HO)alanyl peptides (n = 1-3) with nitrilotriacetic acid affords tripodal peptide hydroxamate ligands (1L, 1D, 2LL, 2DL, and 3LLL, where each L or D denotes the L- or D-alanyl residue). These ligands form six-coordinate octahedral complexes (Fe-1L, Fe-1D, Fe-2LL, Fe-2DL, and Fe-3LLL) with iron(III) in aqueous near neutral pH solution, and the stability and the chirality of the complexes formed depend on the alanyl residues incorporated. Thus Fe-2LL is the most stable against attack of H+ and OH- ions and the least labile in the iron(III) removal by EDTA. The CD spectra show a predominance of the A configuration for Fe-1D, Fe-2LL, Fe-2DL, and Fe-3LLL, but the opposite delta configuration for Fe-1L. These ligands and their gallium(III) complexes are studied by 1H NMR spectroscopy in DMSO-d6 solution. CD and NMR spectral analysis, aided by molecular model examinations, indicates that critical factors in controlling the configuration and the stability of the complexes are (1) the hydroxamate-carrying alanyl residue, (2) the expanse of an interior space in the ligand, and (3) an interstrand amide NH hydrogen bond; the latter bonding is possible with ligands 2LL and 2DL. A microbial growth promotion activity test shows that ligands 1L, 2LL, and 3LLL all act as iron-transporting agents.