Engineering Encodable Lanthanide-Binding Tags into Loop Regions of Proteins

Abstract

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-1β and varied the length of the spacer between the LBT and the protein (denoted 1-3). Luminescence studies demonstrate that all nine constructs bind Tb 3+ 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 IL1β-S1 and IL1β-L3 constructs and for the remaining constructs by comparing 1 H-15 N-HSQC NMR spectra with wild-type IL1β. Additionally, binding of LBT-loop IL1β 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-loopconstructs and revealed that the LBT-2 series were rigidly incorporated into the interleukin-1β structure. The paramagnetic NMR spectra of loop-LBT mutant IL1β-R2 were assigned and the Δχ tensor components were calculated based on RDCs and pseudocontact shifts (PCSs). A structural model of the IL1β-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 modelling.

Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy

“…or paramagnetic Tm 3? at a field strength of B 0 = 14.1 T. The 1 H, 15 N resonance assignments of diamagnetic IL1b-S2R2 were inferred from the assignments of the respective single-loop LBT constructs IL1b-R2 and IL1b-S2 (Barthelmes et al 2011 and employed a bootstrapping assignment procedure as described previously (Barthelmes et al 2011). Despite considerable line broadening resulting from the two paramagnetic metal ion centers, a total of 61 cross peaks in the 15 N-HSQC spectrum of IL1b-S2R2 loaded with Tm 3?…”

“…4) confirming the additivity of both Dv-tensors in the two-loop-LBT construct. To obtain the lanthanide positions and the Dv-tensors in IL1b-S2R2, we fitted the PCS against the previously refined IL1b wild-type structure (Barthelmes et al 2011) under the assumption that the Dvtensors of Tm 3? in the R2-loop and the S2-loop are additive using a Mathematica (Wolfram Research Inc., Champaign 2014) script developed in-house (see SI).…”

“…1a, b) that specifically binds trivalent lanthanide (Ln) ions with low nM afffinity and the fusion LBT-protein can be produced in any suitable expression platform. Herein, we extend our approach by inserting the LBT sequence into both the R2 and S2 loop regions of the model protein IL1b (Barthelmes et al 2011) forming the two-loop-LBT tagged protein termed IL1b-S2R2 (Fig. 1c) and show the applicability of the two-loop LBT approach for PELDOR measurements and paramagnetic NMR-spectroscopy.…”

“…The determination of the Dv-tensors and metal positions is described in the Supporting Information (SI). b Structure representation of the folded LBT complexing a lanthanide-ion (green sphere) (Barthelmes et al 2011). c Schematic sequence representation of the two-loop-LBT construct of IL1b with the LBT sequences highlighted in red.…”

“…Recently, a genetically encodable lanthanide-binding tag (LBT) was introduced, which was initially attached to the protein termini (Wöhnert et al 2003), then extended to a double LBT (Silvaggi et al 2007;Martin et al 2007) and then further rigidified by insertion into loop regions of interleukin-1-beta (IL1b) (Barthelmes et al 2011). We demonstrated the applicability of LBTs for obtaining structural restraints by paramagnetic NMR spectroscopy.…”