Mechanism of interaction of the double-stranded RNA (dsRNA) binding domain of protein kinase R with short dsRNA sequences.

The dsRNA-activated protein kinase (PKR) plays a major role in the cellular response to viral infection. PKR contains an N-terminal dsRNA binding domain (dsRBD) and a C-terminal kinase domain. The dsRBD consists of two tandem copies of a conserved double-stranded RNA binding motif, dsRBM1 and dsRBM2. dsRNA binding is believed to activate PKR by inducing dimerization and subsequent autophosphorylation reactions. We have characterized the function of the dsRBD by assessing the binding of dsRBM1 and dsRBD to a series of dsRNA sequences ranging from 15 to 45 bp. For dsRBM1, the binding stoichiometries agree with an overlapping ligand binding model where the motif binds to multiple faces of the dsRNA duplex and overlaps along the helical axis. Similar behavior is observed for a dsRBD containing both dsRBM1 and dsRBM2 for sequences up to 30 bp; however, the binding affinity is enhanced 30-fold. Longer dsRNA sequences exhibit lower-than-expected stoichiometries, indicating a change in binding mode. NMR spectroscopy was used to define the regions of the dsRBD that interact with dsRNA. dsRNA binding induces exchange broadening of cross-peaks in 1H-15N HSQC spectra. For a 20 bp dsRNA, the resonances most affected map to the known dsRNA binding regions of dsRBM1 as well as the N-terminus of dsRBM2. For a longer 40 bp sequence, additional regions of dsRBM2 exhibit enhanced broadening. These data support a model in which dsRBM1 plays the dominant role in binding short dsRNA sequences and dsRBM2 makes additional interactions with the longer sequences capable of activating PKR.

The conformation of the cytoplasmic helix 8 of the CB1 cannabinoid receptor using NMR and circular dichroism.

The cytoplasmic helix domain (fourth cytoplasmic loop, helix 8) of numerous GPCRs such as rhodopsin and the beta-adrenergic receptor exhibits unique structural and functional characteristics. Computational models also predict the existence of such a structural motif within the CB1 cannabinoid receptor, another member of the G-protein coupled receptor superfamily. To gain insights into the conformational properties of this GPCR component, a peptide corresponding to helix 8 of the CB1 receptor with a small contiguous segment from transmembrane helix 7 (TM7) was chemically synthesized and its secondary structure determined by circular dichroism (CD) and solution NMR spectroscopy. Our studies in DPC and SDS micelles revealed significant alpha-helical structure while in an aqueous medium, the peptide exhibited a random coil configuration. The relative orientation of helix 8 within the CB1 receptor was obtained from intermolecular 31P-1H and 1H-1H NOE measurements. Our results suggest that in the presence of an amphipathic membrane environment, helix 8 assumes an alpha helical structure with an orientation parallel to the phospholipid membrane surface and perpendicular to TM7. In this model, positively charged side chains interact with the lipid headgroups while the other polar side chains face the aqueous region. The above observations may be relevant to the activation/deactivation of the CB1 receptor.

Structural studies of metarhodopsin II, the activated form of the G-protein coupled receptor, rhodopsin.

The structural changes that accompany activation of a G-protein coupled receptor (GPCR) are not well understood. To better understand the activation of rhodopsin, the GPCR responsible for visual transduction, we report studies on the three-dimensional structure for the activated state of this receptor, metarhodopsin II. Differences between the three-dimensional structure of ground state rhodopsin and metarhodopsin II, particularly in the cytoplasmic face of the receptor, suggest how the receptor is activated to couple with transducin. In particular, activation opens a groove on the surface of the receptor that could bind the N-terminal helix of the G protein, transducin alpha.