Conformational control of small GTPases by AMPylation.

Posttranslational modifications (PTMs) are important physiological means to regulate the activities and structures of central regulatory proteins in health and disease. Small GTPases have been recognized as important molecules that are targeted by PTMs during infections of mammalian cells by bacterial pathogens. The enzymes DrrA/SidM and AnkX from Legionella pneumophila AMPylate and phosphocholinate Rab1b during infection, respectively. Cdc42 is AMPylated by IbpA from Histophilus somni at tyrosine 32 or by VopS from Vibrio parahaemolyticus at threonine 35. These modifications take place in the important regulatory switch I or switch II regions of the GTPases. Since Rab1b and Cdc42 are central regulators of intracellular vesicular trafficking and of the actin cytoskeleton, their modifications by bacterial pathogens have a profound impact on the course of infection. Here, we addressed the biochemical and structural consequences of GTPase AMPylation and phosphocholination. By combining biochemical experiments and NMR analysis, we demonstrate that AMPylation can overrule the activity state of Rab1b that is commonly dictated by binding to guanosine diphosphate or guanosine triphosphate. Thus, PTMs may exert conformational control over small GTPases and may add another previously unrecognized layer of activity control to this important regulatory protein family.

Conformational dynamics and alignment properties of loop lanthanide-binding-tags (LBTs) studied in interleukin-1ß.

Encodable lanthanide binding tags (LBTs) have become an attractive tool in modern structural biology as they can be expressed as fusion proteins of targets of choice. Previously, we have demonstrated the feasibility of inserting encodable LBTs into loop positions of interleukin-1ß (Barthelmes et al. in J Am Chem Soc 133:808-819, 2011). Here, we investigate the differences in fast dynamics of selected loop-LBT interleukin-1ß constructs by measuring 15N nuclear spin relaxation experiments. We show that the loop-LBT does not significantly alter the dynamic motions of the host protein in the sub-τc-timescale and that the loop-LBT adopts a rigid conformation with significantly reduced dynamics compared to the terminally attached encodable LBT leading to increased paramagnetic alignment strength. We further analyze residual dipolar couplings (RDCs) obtained by loop-LBTs and additional liquid crystalline media to assess the applicability of the loop-LBT approach for RDC-based methods to determine structure and dynamics of proteins, including supra-τc dynamics. Using orthogonalized linear combinations (OLCs) of RDCs and Saupe matrices, we show that the combined use of encodable LBTs and external alignment media yields up to five linear independent alignments.

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

We recently engineered encodable lanthanide binding tags (LBTs) into proteins and demonstrated their applicability in Nuclear Magnetic Resonance (NMR) spectroscopy, X-ray crystallography and luminescence studies. Here, we engineered two-loop-LBTs into the model protein interleukin-1ß (IL1ß) and measured (1)H, (15)N-pseudocontact shifts (PCSs) by NMR spectroscopy. We determined the Δχ-tensors associated with each Tm(3+)-loaded loop-LBT and show that the experimental PCSs yield structural information at the interface between the two metal ion centers at atomic resolution. Such information is very valuable for the determination of the sites of interfaces in protein-protein-complexes. Combining the experimental PCSs of the two-loop-LBT construct IL1ß-S2R2 and the respective single-loop-LBT constructs IL1ß-S2, IL1ß-R2 we additionally determined the distance between the metal ion centers. Further, we explore the use of two-loop LBTs loaded with Gd(3+) as a novel tool for distance determination by Electron Paramagnetic Resonance spectroscopy and show the NMR-derived distances to be remarkably consistent with distances derived from Pulsed Electron-Electron Dipolar Resonance.

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-1ß (IL1ß) 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 IL1ß-S1 and IL1ß-L3 constructs and for the remaining constructs by comparing the 1H−15N heteronuclear single-quantum coherence NMR spectra with that of the wild-type IL1ß. Additionally, binding of LBT-loop IL1ß 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-1ß structure. The paramagnetic NMR spectra of loop-LBT mutant IL1ß-R2 were assigned and the Δχ tensor components were calculated on the basis of RDCs and pseudocontact shifts. A structural model of the IL1ß-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.