The attachment of paramagnetic lanthanide ions to proteins can lead to a range of useful effects that are observable by nuclear magnetic resonance (NMR) spectroscopy, including pseudocontact shifts (PCSs) and residual dipolar coupling (RDC). The analysis of these parameters, obtained from measurement in simple and sensitive NMR experiments, provides valuable restraints spanning large distances that facilitate the refinement of the 3D structure of macromolecules including protein-protein and protein-drug complexes . Herein we report several new synthetic lanthanide binding tags (LBTs) which can be attached to solvent-exposed thiols on proteins to facilitate paramagnetic NMR spectroscopy in structural biology.
The first LBT, Cys-NTA, generates useful PCSs when attached to the a-helix in human Ubiquitin bearing an aspartic acid at the i + 4 position . Attachment of two copies of Cys-NTA in an i and i + 4 arrangement results in a highly rigid complex as evidenced by substantially larger PCSs and RDCs. The second LBT, Cys-IDA, generates very large PCSs and RDCs when attached to the same a-helix. However, this strategy yields a highly rigid complex yet exhibits sufficiently fast lanthanide exchange to give cross-peaks in heteronuclear longitudinal (15Nzz) exchange spectra recorded with a mixture of paramagnetic and diamagnetic lanthanide ions . Analysis of exchange spectra enables the unambiguous and facile assignment of paramagnetic NMR spectra without recourse to an initial protein model or multiple lanthanides. Furthermore, unprecedentedly large PCS for amides close to the metal centre that are usually broadened beyond detection in regular 15N HSQC spectra are readily observed via the heteronuclear exchange experiment. Lastly we have developed complementary, enantiomeric pairs of high affinity cyclen based tags for wide range application to biomolecules of unknown structure . The 8 or 9 coordinated lanthanide metal is fully encapsulated by virtue of chiral arms branching out from a dota macrocyclic ring. The utility of these tags is illustrated for the antimicrobial 20kDa target, HPPK, in complex with an inhibitor and the cofactor AMPCPP to show the conformation of a critical active site loop during the binding of the inhibitor that could not be achieved by other NMR methods.