Selective isotope labeling for NMR structure determination of proteins in complex with unlabeled ligands

Konstantinos Tripsianes,Michael Sattler,Leonidas Emmanouilidis,Gerd Gemmecker,Ulrike Schütz

doi:10.1007/s10858-019-00241-9

Abstract

The physiological role of proteins is frequently linked to interactions with non-protein ligands or posttranslational modifications. Structural characterization of these complexes or modified proteins by NMR may be difficult as the ligands are usually not available in an isotope-labeled form and NMR spectra may suffer from signal overlap. Here, we present an optimized approach that uses specific NMR isotope-labeling schemes for overcoming both hurdles. This approach enabled the high-resolution structure determination of the farnesylated C-terminal domain of the peroxisomal protein PEX19. The approach combines specific 13C, 15N and 2H isotope labeling with tailored NMR experiments to (i) unambiguously identify the NMR frequencies and the stereochemistry of the unlabeled 15-carbon isoprenoid, (ii) resolve the NMR signals of protein methyl groups that contact the farnesyl moiety and (iii) enable the unambiguous assignment of a large number of protein-farnesyl NOEs. Protein deuteration was combined with selective isotope-labeling and protonation of amino acids and methyl groups to resolve ambiguities for key residues that contact the farnesyl group. Sidechain-labeling of leucines, isoleucines, methionines, and phenylalanines, reduced spectral overlap, facilitated assignments and yielded high quality NOE correlations to the unlabeled farnesyl. This approach was crucial to enable the first NMR structure of a farnesylated protein. The approach is readily applicable for NMR structural analysis of a wide range of protein–ligand complexes, where isotope-labeling of ligands is not well feasible.

Highlights

Detailed understanding of the mechanism for protein–ligand interactions or the effects of posttranslational modifications requires structural information about the ligand-bound protein of interest on a molecular level
The farnesyl moiety is a linear grouping of three isoprene units that is attached enzymatically to the cysteine of the C-terminal CaaX box of PEX19
We report an optimized protocol for NMR structure calculation of proteins, where ligands or covalently attached modifications cannot be readily obtained in isotope-labeled form

Summary

Introduction

Detailed understanding of the mechanism for protein–ligand interactions or the effects of posttranslational modifications requires structural information about the ligand-bound protein of interest on a molecular level. These complexes or modified proteins often possess intrinsic flexibility or low ligand affinity and are difficult to crystallize in the native conformation. Structural analysis by NMR on the other hand, can be problematic as isotope-labeling of the ligands may not be readily available, narrowing down the options to derive NOE-based distance restraints to 1H only homonuclear experiments. It is estimated that over 100 human proteins are potential substrates for farnesylation and geranylgeranylation (McTaggart 2006). The combination of the various labeling protocols with individually adapted NMR experiments enabled us to circumvent the restrictions for the unlabeled ligand, to obtain complete resonance assignments for both protein and the attached lipid and to determine the first NMR structure of a farnesylated protein (Emmanouilidis et al 2017) (Fig. 1)

Results

CH2 11 CH 13 CH3

Conclusion