Distance mapping of protein‐binding sites using spin‐labeled oligosaccharide ligands

Abstract

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...

Unidirectional Binding of Clostridial Collagenase to Triple Helical Substrates

“…2). The PROXYL group being paramagnetic enhances the relaxation rate of the NMR resonances of the protein under study via electron-nuclei dipolar coupling (10). An arginine residue was placed in the C-terminal triplet to increase solubility while stabilizing the triple helix to a similar extent as a hydroxyproline residue (25).…”

“…We have shown that CBD can be functional to anchor growth factors in local tissue. A 1 H- 15 N HSQC NMR titration study with three different tropocollagen analogues ((POG) 10 ) 3 , ((GPOG) 7 PRG) 3 , and (GPRG(POG) 7 C-carbamidomethyl) 3 , mapped a saddle-like binding cleft on CBD. NMR titrations with three nitroxide spin-labeled analogues of collagenous peptide, (PROXYL-G(POG) 7 PRG) 3 , (PROXYL-G(POG) 7 ) 3 , and (GPRG(POG) 7 C-PROXYL) 3 (where PROXYL represents 2,2,5,5-tetramethyl-L-pyrrolidinyloxy), unambiguously demonstrated unidirectional binding of CBD to the tropocollagen analogues.…”

“…To identify the binding direction, three different NMR titrations were performed with spinlabeled analogues of tropocollagen, where a nitroxide spin label 2,2,5,5-tetramethyl-L-pyrrolidinyloxy (PROXYL) was attached to either the N or C terminus of the collagenous peptide. The nitroxide moiety with an unpaired electron can cause enhancement in paramagnetic relaxation of NMR resonances of CBD via electron-nuclear dipolar coupling, thereby resulting in extreme line broadening of those resonances (10,11). Furthermore, small angle x-ray scattering (SAXS) was utilized to obtain the three-dimensional structure of the CBD-collagenous peptide complex.…”

“…Peptides-(POG) 10 was purchased from Peptide Institute, Inc. (Osaka, Japan). Other peptides were constructed manually based on the standard Fmoc (N-(9-fluorenyl)methoxycarbonyl)-based strategy on Rink-amide resins (Novabiochem, Darmstadt, Germany).…”

Practical aspects of 1H transverse paramagnetic relaxation enhancement measurements on macromolecules

“…PRE analysis for such systems is simple, since there are no pseudo-contact shifts, and Curie-spin relaxation that could potentially exhibit significant cross-correlation with other relaxation mechanisms (2,3), is negligible. Using this type of PRE data, macromolecular structures have been characterized for soluble proteins (4-10), protein-protein complexes (11)(12)(13)(14), protein-oligosaccharide complexes (15,16), protein-nucleic acid complexes (17)(18)(19)(20)(21), and membrane proteins (22,23). The PRE can also provide information relating to large-scale dynamics that accompany changes of paramagnetic center -1 H distances, for example in non-specific protein-DNA interactions (24,25) and interdomain motions (26).…”