Recognition of viral RNA stem–loops by the tandem double-stranded RNA binding domains of PKR

Abstract

In humans, the double-stranded RNA (dsRNA)-activated protein kinase (PKR) is expressed in late stages of the innate immune response to viral infection by the interferon pathway. PKR consists of tandem dsRNA binding motifs (dsRBMs) connected via a flexible linker to a Ser/Thr kinase domain. Upon interaction with viral dsRNA, PKR is converted into a catalytically active enzyme capable of phosphorylating a number of target proteins that often results in host cell translational repression. A number of high-resolution structural studies involving individual dsRBMs from proteins other than PKR have highlighted the key features required for interaction with perfectly duplexed RNA substrates. However, viral dsRNA molecules are highly structured and often contain deviations from perfect A-form RNA helices. By use of small-angle X-ray scattering (SAXS), we present solution conformations of the tandem dsRBMs of PKR in complex with two imperfectly base-paired viral dsRNA stemloops; HIV-1 TAR and adenovirus VA I -AS. Both individual components and complexes were purified by size exclusion chromatography and characterized by dynamic light scattering at multiple concentrations to ensure monodispersity. SAXS ab initio solution conformations of the individual components and RNA-protein complexes were determined and highlight the potential of PKR to interact with both stem and loop regions of the RNA. Excellent agreement between experimental and model-based hydrodynamic parameter determination heightens our confidence in the obtained models. Taken together, these data support and provide a framework for the existing biochemical data regarding the tolerance of imperfectly base-paired viral dsRNA by PKR.

Dynamic light scattering: a practical guide and applications in biomedical sciences

“…Similarly, the adenovirus VA I RNA lacking the terminal stem-loop RNA (VA I ΔTS, 32.3 kDa) was prepared by in-vitro transcription method followed by purification using SEC (Dzananovic et al 2014), whereas human protein kinase R (PKR) lacking the Cterminal kinase domain (PKR 1-169 18.8 kDa) was expressed into BL21(DE3) cells, purified by means of affinity and SEC followed by DLS data collection in 50 mM Tris (pH 7.50), 100 mM NaCl, and 5 mM 2-mercaptoethanol (Dzananovic et al 2013). DLS data for SEC purified VAIΔTS-PKR 1-169 complex was collected in 50 mM Tris (pH 7.50), 100 mM NaCl buffer (Dzananovic et al 2014) the literature.…”

The β-Lactamase Gene Regulator AmpR Is a Tetramer That Recognizes and Binds the d-Ala-d-Ala Motif of Its Repressor UDP-N-acetylmuramic Acid (MurNAc)-pentapeptide

“…The partial specific volume of 0.738 ml/g for AmpR was calculated using SEDNTERP from its amino acid sequence, whereas that for DNA was considered to be 0.57 ml/g (55). By using Equation 1 from Dzananovic et al (56) and considering the molecular mass of tetramer for protein (130.8 kDa) and dimer of DNA (25.7 kDa), the partial specific volume of 0.710 ml/g was obtained.…”

Characterization of the termini of the West Nile virus genome and their interactions with the small isoform of the 2′ 5′-oligoadenylate synthetase family

“…SAXS data for proteins (wild type and mutant versions of OAS1), RNAs including WNV 5'--TR, 3'--TR and 3'--SL RNA were collected using an in--house Rigaku instrument as described previously [272] . This in--house Rigaku S--MAX3000 is mg/mL) and 3'--TR RNA (1.5, 2.0, 2.5 and 3.0 mg/mL).…”

“…Twelve models using DAMMIF and ten models using BUNCH were generated which were then rotated, aligned and averaged using DAMAVER [377]. The program HYDROPRO [378] was employed to calculate solution properties such as hydrodynamic radius, radius of gyration and maximal particle dimension for each model calculated using SAXS data following a similar approach as outlined previously [379]. The input parameters included the density (1.0038 g/mL) and viscosity (0.01026 Poise) for buffer (50mM Tris, 100mM NaCl) as well as partial specific volume of OAS1 (0.7424 mL/g), obtained from the program SEDNTERP [380].…”

“…With the exception to the 5'--TR (twelve models) and the 5′/3′--TR complex (fifteen models), twenty models were generated for all other samples (RNA and OAS1+RNA complex), which were then rotated, aligned and averaged using DAMAVER [377]. The program HYDROPRO [378] was then employed to calculate solution hydrodynamic properties averaged--filtered model following a similar approach as outlined previously [272]. The input parameters included the density and viscosity of buffer (1.0038 g/mL and 0.01026 Poise pH 7.0 for buffer containing 50mM Tris, 100mM NaCl; 1.002 g/mL and 0.01023 Poise for buffer containing 50mM Tris, 40mM NaCl, 1mM EDTA, pH 7.5) as well as partial specific volume of OAS1 (0.7424 mL/g) [211], 3′--SL (0.5698 mL/g), 3′--TR RNA (0.567 mL/g), 5′--TR RNA (0.569 mL/g), OAS1+3′--TR RNA complex (0.660 mL/g) and OAS1+5′--TR RNA complex (0.649 mL/g) obtained from the program SEDNTERP [380].…”