Convenient method for resolving degeneracies due to symmetry of the magnetic susceptibility tensor and its application to pseudo contact shift-based protein-protein complex structure determination.

Yoshihiro Kobashigawa,Tomohide Saio,Masahiro Ushio,Kenji Ogura,Masashi Yokochi,Mitsuhiro Sekiguchi,Fuyuhiko Inagaki

doi:10.1007/s10858-012-9623-8

Yoshihiro Kobashigawa, Tomohide Saio + Show 5 more

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https://doi.org/10.1007/s10858-012-9623-8

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Abstract

Pseudo contact shifts (PCSs) induced by paramagnetic lanthanide ions fixed in a protein frame provide long-range distance and angular information, and are valuable for the structure determination of protein–protein and protein–ligand complexes. We have been developing a lanthanide-binding peptide tag (hereafter LBT) anchored at two points via a peptide bond and a disulfide bond to the target proteins. However, the magnetic susceptibility tensor displays symmetry, which can cause multiple degenerated solutions in a structure calculation based solely on PCSs. Here we show a convenient method for resolving this degeneracy by changing the spacer length between the LBT and target protein. We applied this approach to PCS-based rigid body docking between the FKBP12-rapamycin complex and the mTOR FRB domain, and demonstrated that degeneracy could be resolved using the PCS restraints obtained from two-point anchored LBT with two different spacer lengths. The present strategy will markedly increase the usefulness of two-point anchored LBT for protein complex structure determination.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-012-9623-8) contains supplementary material, which is available to authorized users.

Highlights

Long-range distance and angular information is useful for the structural analysis of large proteins, multidomain proteins and protein complexes (Gaponenko et al 2002, 2004; Battiste and Wagner 2000; Vlasie et al 2007; Tang and Clore 2006; Rumpel et al 2007; Bertini et al 2009)
We prepared constructs containing one- (H-), two- (H-M), three- (H-MG), four- (H-M-S-G) and five-residue (H-M-G-S-G) linkers, named L1, L2, L3, L4- and L5-FKBP12, respectively (Fig. 1a). These constructs were first screened for their suitability for NMR experiments, based on melting temperature (Tm) measured using differential scanning fluorometry (DSF; Niesen et al 2007) in the presence of Lu3?, since we assumed that Tm was sensitive to the structural distortion and/or hindrance
The residues indicating spectral shifts on the attachment of the two-point anchored LBT were located very close to the anchoring points in the case of L3- to L5FKBP12-rapamycin, while a large shift was observed for G62 on the a-helix region close to the N-terminal anchoring point in the case of L1- and L2FKBP12-rapamycin

Summary

Introduction

Long-range distance and angular information is useful for the structural analysis of large proteins, multidomain proteins and protein complexes (Gaponenko et al 2002, 2004; Battiste and Wagner 2000; Vlasie et al 2007; Tang and Clore 2006; Rumpel et al 2007; Bertini et al 2009). Paramagnetic lanthanide ions are useful probes for solution structure determination by NMR and have been applied successfully to metalloproteins (Bertini et al 2001, 2004, 2007; Barbieri et al 2002; Pintacuda et al 2006, 2007; Allegrozzi et al 2000). Ions can be replaced by lanthanide ions as they share similar chemical properties These approaches are limited, in principle, to metalloproteins

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