Engineering Encodable Lanthanide-Binding Tags into Loop Regions of Proteins

Lanthanide-binding tags (LBTs) are valuable tools for investigation of protein structure, function, and dynamics by NMR spectroscopy, X-ray crystallography, and luminescence studies. We have inserted LBTs into three different loop positions (denoted L, R, and S) of the model protein interleukin-1 beta (IL1 beta) and varied the length of the spacer between the LBT and the protein (denoted 1-3). Luminescence studies demonstrate that all nine constructs bind Tb3+ tightly in the low nanomolar range. No significant change in the fusion protein occurs from insertion of the LBT, as shown by two X-ray crystallographic structures of the IL1 beta-S1 and IL1 beta-L3 constructs and for the remaining constructs by comparing the H-1-N-15 heteronuclear single-quantum coherence NMR spectra with that of the wild-type IL1 beta. Additionally, binding of LBT-loop IL1 beta proteins to their native binding partner in vitro remains unaltered. X-ray crystallographic phasing was successful using only the signal from the bound lanthanide. Large residual dipolar couplings (RDCs) could be determined by NMR spectroscopy for all LBT-loop constructs and revealed that the LBT-2 series were rigidly incorporated into the interleukin-1 beta structure. The paramagnetic NMR spectra of loop-LBT mutant IL1 beta-R2 were assigned and the Delta chi tensor components were calculated on the basis of RDCs and pseudocontact shifts. A structural model of the IL1 beta-R2 construct was calculated using the paramagnetic restraints. The current data provide support that encodable LBTs serve as versatile biophysical tags when inserted into loop regions of proteins of known structure or predicted via homology modeling.