An NMR strategy for fragment-based ligand screening utilizing a paramagnetic lanthanide probe

Abstract

A nuclear magnetic resonance-based ligand screening strategy utilizing a paramagnetic lanthanide probe is presented. By fixing a paramagnetic lanthanide ion to a target protein, a pseudo-contact shift (PCS) and a paramagnetic relaxation enhancement (PRE) can be observed for both the target protein and its bound ligand. Based on PRE and PCS information, the bound ligand is then screened from the compound library and the structure of the ligand–protein complex is determined. PRE is an isotropic paramagnetic effect observed within 30 Å from the lanthanide ion, and is utilized for the ligand screening in the present study. PCS is an anisotropic paramagnetic effect providing long-range (~40 Å) distance and angular information on the observed nuclei relative to the paramagnetic lanthanide ion, and utilized for the structure determination of the ligand–protein complex. Since a two-point anchored lanthanide-binding peptide tag is utilized for fixing the lanthanide ion to the target protein, this screening method can be generally applied to non-metal-binding proteins. The usefulness of this strategy was demonstrated in the case of the growth factor receptor-bound protein 2 (Grb2) Src homology 2 (SH2) domain and its low- and high-affinity ligands.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-011-9566-5) contains supplementary material, which is available to authorized users.

Information content of long-range NMR data for the characterization of conformational heterogeneity

“…However, the presence of an anisotropic magnetic susceptibility also gives rise to pseudocontact shifts (PCS) (Kurland and McGarvey, 1970), which are reporters on the positions of the nuclei in the principal axis frame of the magnetic susceptibility tensor centered on the paramagnetic site, and therefore contain information about the structure/shape of a molecule. The use of paramagnetism-induced restraints (Gochin and Roder, 1995a; Gochin and Roder, 1995b; Banci et al, 1996; Banci et al, 1998; Bertini et al, 2001a; Gaponenko et al, 2004; Bertini et al, 2005; Diaz-Moreno et al, 2005; Jensen et al, 2006; Bertini et al, 2008; Schmitz et al, 2012; Yagi et al, 2013b) is becoming increasingly popular because of the introduction of lanthanide binding tags (Barthelmes et al, 2011; Wöhnert et al, 2003; Rodriguez-Castañeda et al, 2006; Su et al, 2006; John and Otting, 2007; Pintacuda et al, 2007; Zhuang et al, 2008; Su et al, 2008b; Su et al, 2008a; Keizers et al, 2008; Häussinger et al, 2009; Su and Otting, 2010; Hass et al, 2010; Man et al, 2010; Das Gupta et al, 2011; Saio et al, 2011; Swarbrick et al, 2011b; Swarbrick et al, 2011a; Bertini et al, 2012a; Liu et al, 2012; Kobashigawa et al, 2012; Cerofolini et al, 2013; Yagi et al, 2013a; Gempf et al, 2013; Loh et al, 2013), that extend the range of applications from paramagnetic metalloproteins (Banci et al, 1996; Banci et al, 1997) (or proteins in which the naturally occurring metal can be replaced by a paramagnetic one (Allegrozzi et al, 2000; Bertini et al, 2001a; Bertini et al, 2001c; Bertini et al, 2001b; Bertini et al, 2003; Bertini et al, 2004b; Balayssac et al, 2008; Bertini et al, 2010a; Luchinat et al, 2012b)) to, in principle, any protein.…”

FANTEN: a new web-based interface for the analysis of magnetic anisotropy-induced NMR data

“…The advantages offered by paramagnetic restraints are more and more exploited thanks to the development of paramagnetic tags that can be attached to diamagnetic proteins, so that PCSs and RDCs can be easily measured also for molecules without a natural metal binding site (Wöhnert et al 2003;Rodriguez-Castañeda et al 2006;Su et al 2006;Su et al 2008a;Keizers et al 2008;Su et al 2008b;Zhuang et al 2008;Häussinger et al 2009;Su and Otting 2010;Hass et al 2010;Man et al 2010;Das Gupta et al 2011;Liu et al 2012;Cerofolini et al 2013;Yagi et al 2013a;Gempf et al 2013;Kobashigawa et al 2012;Saio et al 2011;Watanabe et al 2010;Loh et al 2013;Swarbrick et al 2011aSwarbrick et al , 2011b.…”

Identification of the Glycosaminoglycan Binding Site of Interleukin-10 by NMR Spectroscopy

“…PCS assignments were made following the displacement of the individual HSQC cross-peaks along diagonal lines in the spectrum (Fig. 6a) (Table 2) of each lanthanide ion were determined using the Numbat program as described under "Experimental Procedures" and were found to be comparable with those values reported in the literature (23,64). The effect of residual chemical shift anisotropy, which leads to additional shifts between the diamagnetic and the paramagnetic sample, was also considered and corrected in Numbat.…”

“…Second, the lack of NOEs as structural constraints for the IL-10⅐GAG system motivated the application of paramagnetic NMR experiments because these take advantage of a strong electron/nucleus dipolar interaction that can be quantified over long distances (up to 30 -40 Å). Recently, pseudocontact shifts (PCSs) were successfully applied to the structural investigation of proteins interacting with small ligands (23,24), including carbohydrates (25,26). PCSs, which are caused by paramagnetic metal ions with an anisotropic magnetic susceptibility (e.g.…”