How reliable are pseudocontact shifts induced in proteins and ligands by mobile paramagnetic metal tags? A modelling study

Abstract

The anisotropic component of the magnetic susceptibility tensor (Δχ tensor) associated with various paramagnetic metal ions can induce pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs) in proteins, yielding valuable restraints in structural studies. In particular, PCSs have successfully been used to study ligands that bind to proteins tagged with a paramagnetic metal ion, which is of great interest in fragment-based drug design. To create easy-to-interpret PCSs, the metal ion must be attached to the protein in a rigid manner. Most of the existing methods for site-specific attachment of a metal tag, however, result in tethers with residual flexibility. Here we present model calculations to quantify the extent, to which mobility of the metal-binding tag can compromise the quality of the Δχ tensor that can be determined from the PCSs observed in the protein. Assuming that the protein can be approximated by a sphere and the tag is attached by a single tether, the results show that a single effective ∆χ tensor can describe the PCSs and RDCs of the protein spins very well even in the presence of substantial tag mobility, implying that PCSs of ligands in binding pockets of the protein can be predicted with similar accuracy. In contrast, the quality of the PCS prediction for nuclear spins positioned above the surface of the protein is significantly poorer, with implications for studies of protein-protein complexes. The simulations probed the sensitivity of the effective Δχ tensor to different parameters, including length of the tether between protein and metal ion, protein size, type and amplitude of tag motion, tensor orientation relative to the protein and direction of tag motion. Tether length and amplitude of motion were identified as two key parameters. It is shown that the amplitude of tag motions cannot be quantified by simple comparisons of the effective Δχ tensor with the alignment tensor determined from RDCs.

Model-free extraction of spin label position distributions from pseudocontact shift data

“…14 Even DOTA-M8, 15 which uses a sterically overcrowded – and therefore rigid – metal cage, 16 still has a flexible linker. The conformational mobility of lanthanide tags is visible in the distance distributions measured by double electron resonance, 17 and in molecular dynamics simulations.…”

Sequence-specific assignment of methyl groups from the neuronal SNARE complex using lanthanide-induced pseudocontact shifts

“…However, the perturbations caused by the mutations used in our study did not really cause difficulties, as they were very limited, and in fact helped to confirm these assignments. A more important problem associated with the choice of tag location is the potential for tag mobility, which can lead to complete absence of PCSs (because they are averaged to zero) or averaging of the Δχ tensor that can complicate the interpretation of the measured PCSs (Shishmarev and Otting 2013). In our previous study, the Dy 3+ -loaded C2 tag led to the observation of PCSs that could be fit to a unique Δχ tensor when placed at four out of ten sites in the SNARE complex (Brewer et al 2015).…”

How to tackle protein structural data from solution and solid state: An integrated approach

“…from Dṽ axðdomain 2Þ =Dv axðdomain 1Þ . This is not valid in principle for PCSs, which cannot be represented by any mean tensor in the case of motion, unless all nuclei have a fixed distance from the metal ion during the motion [121], which is never the case in practice.…”

“…For this reason, in principle they cannot be used in the structure refinement of the protein domain if the latter is mobile with respect to the position of the metal ion, unless the geometry of the system suggests that the variability in the metal position is small compared to the metal-nuclear distances, and some inaccuracy in the results can be tolerated [121].…”