Site-specific modification and RNA crosslinking of the RNA-binding domain of PKR

Abstract

RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates and inhibits the function of the translation initiation factor eIF-2. PKR is activated in vitro by binding RNA molecules with extensive duplex structure. To further define the nature of the RNA regulation of PKR, we have prepared and characterized site-specifically modified proteins consisting of the PKR 20 kDa RNA-binding domain (RBD). Here we show that the two cysteines found naturally in this domain can be altered by site-directed mutagenesis without loss of RNA binding affinity or the RNA-regulated kinase activity. Introduction of cysteine residues at other sites in the PKR RBD allows for site-specific modification with thiol-selective reagents. PKR RBD mutants reacted selectively with a maleimide to introduce a photoactivatable cross-linking aryl azide at three different positions in the protein. RNA crosslinking efficiency was found to be dependent on the amino acid modified, suggesting differences in access to the RNA from these positions in the protein. One of the amino acid modifications that led to crosslinking of the RNA is located at a residue known to be an autophosphorylation site, suggesting that autophosphorylation at this site could influence the RNA binding properties of PKR. The PKR RBD conjugates described here and other similar reagents prepared via these methods are applicable to future studies of PKR-RNA complexes using techniques such as photocrosslinking, fluorescence resonance energy transfer and affinity cleaving.

Figure 1

Domain structure of PKR. The dsRBMs and the kinase subdomains of PKR are shown as shaded and black boxes, respectively. Amino acid numbering is given above the diagram.

Figure 2

In vitro activation of PKR (C121V, C135V). (A) Shown is a storage phosphor autoradiogram of a 10% SDS–polyacrylamide gel used to resolve PKR (C121V, C135V) autophosphorylated in response to various added concentrations of poly(I)·poly(C). Lanes 1–6, 0.012, 0.12, 1.2, 12, 120 and 0 µg/ml, respectively. (B) Plot of relative extents of PKR (C121V, C135V) autophosphorylation in the presence of different poly(I)·poly(C) concentrations.

Figure 3

Native gel mobility shifts for the wild-type PKR RBD and PKR RBD (E29C) binding to a 92 nt RNA ligand. (A) Proposed secondary structure of the PKR RNA ligand (8). (B) (Top) Storage phosphor autoradiogram of a representative gel used to separate wild-type PKR RBD-bound from free RNA. Lanes 1–17, 0, 0.01, 0.03, 0.05, 0.075, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0 and 10.0 µM wild-type PKR RBD added, respectively. (Bottom) Representative plot of fraction RNA bound by wild-type PKR RBD as a function of protein concentration. The data were fitted to the equation: fraction bound = Θ {[RBD]/([RBD] + K_d)} using the least squares method of KaleidaGraph. (C) (Top) Storage phosphor autoradiogram of a representative gel used to separate PKR RBD (E29C)-bound from free RNA. Lanes 1–17, 0, 0.01, 0.03, 0.05, 0.075, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0 and 10.0 µM PKR RBD (E29C) added, respectively. (Bottom) Representative plot of fraction RNA bound by RBD (E29C) as a function of protein concentration.

Figure 4

Structure of the PKR RBD (amino acids 1–179) showing the three amino acids selected for mutagenesis along with the structure of the photocrosslinking reagent TFPAM-3 used to modify the different cysteine residues. Using Chem3D (CambridgeSoft), the distance between the reactive maleimide carbon and the putative nitrene was estimated to be ~11 Å. The protein structure shown was generated using Insight II (Biosym) running on a Silicon Graphics O₂ workstation and coordinates kindly provided by Qin and co-workers (Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH) (16).

Figure 5

Electrospray mass spectra of the PKR RBD with a cysteine residue introduced at amino acid position 13. PKR RBD was manually infused on a Micromass Quattro II triple quadrupole mass spectrometer and molecular masses were calculated using MassLynx software. (A) Unmodified PKR RBD (E13C). The minor peak in the spectrum shown occurs at a mass of 21 031. (B) TFPAM-3-modified PKR RBD (E13C).

Figure 6

Protein–RNA binding and crosslinking. (A) Native mobility shift experiments of the wild-type PKR RBD and TFPAM-3-modified PKR RBD binding to a 92 nt RNA ligand. Lanes 1–10, 0, 0.005, 0.1, 0.8, 1.0, 2.0, 4.0, 6.0, 12.0 and 20.0 µM wild type or TFPAM-3-modified PKR RBD added, respectively. (B) Denaturing gel experiments resolving the PKR RBD/RNA ligand crosslinked complexes. Lanes 1–8, 0, 0.005, 0.1, 1.0, 2.0, 4.0, 6.0 and 12.0 µM TFPAM-3-modified PKR RBD added, respectively.

Figure 7

Crosslinking of the PKR RBD to an RNA ligand. See Materials and Methods for photocrosslinking procedures. (A) Characterization of the crosslinked species. Lane 1, no protein added and no irradiation; lane 2, UV irradiation with no protein added; lane 3, 6 µM PKR RBD (E29C) with no UV irradiation; lane 4, PKR RBD (E29C) with 18 min UV irradiation; lane 5, TFPAM-3-modified PKR RBD (E29C) with no UV irradiation; lane 6, TFPAM-3-modified PKR RBD (E29C) with 18 min UV irradiation; lane 7, TFPAM-3-modified PKR RBD (E29C) with 18 min UV irradiation followed by proteinase K treatment. (B) Crosslinking as a function of the amino acid modified. All samples were UV irradiated for 18 min. Lane 1, no protein added; lane 2, PKR RBD (E13C); lane 3, TFPAM-modified PKR RBD (E13C); lane 4, PKR RBD (E29C); lane 5, TFPAM-modified PKR RBD (E29C); lane 6, PKR RBD (S33C); lane 7, TFPAM-modified PKR RBD (S33C).

Figure 8

Structure of dsRBM I of the PKR RBD displaying the putative RNA-binding residues (N15, T16, P36, H37, R39, R58, S59, K60, K61 and K64) (16,17). The amino acids selected for modification are also highlighted in color.