No abstract

ClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies of the E. coli enzyme that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel, as observed for other AAA+ proteases and protein-remodeling machines. Strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been inferred from these structural features for other AAA+ unfoldases, but biochemical and single-molecule biophysical studies indicate that ClpXP operates by a probabilistic mechanism in which five to eight residues are translocated for each ATP hydrolyzed. We propose structure-based models that could account for the functional results.

Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV), a positive-strand RNA virus that belongs to the Arteriviridae family of Nidovirales, has been identified as the causative agent of PRRS. Nsp1␣ is the amino (N)-terminal protein in a polyprotein encoded by the PRRSV genome and is reported to be crucial for subgenomic mRNA synthesis, presumably by serving as a transcription factor. Before functioning in transcription, nsp1␣ proteolytically releases itself from nsp1␤. However, the structural basis for the selfreleasing and biological functions of nsp1␣ remains elusive. Here we report the crystal structure of nsp1␣ of PRRSV (strain XH-GD) in its naturally self-processed form. Nsp1␣ contains a ZF domain (which may be required for its biological function), a papain-like cysteine protease (PCP) domain with a zinc ion unexpectedly bound at the active site (which is essential for proteolytic self-release of nsp1␣), and a carboxyl-terminal extension (which occupies the substrate binding site of the PCP domain). Furthermore, we determined the exact location of the nsp1␣ self-processing site at Cys-Ala-Met1802Ala-Asp-Val by use of crystallographic data and N-terminal amino acid sequencing. The crystal structure also suggested an in cis self-processing mechanism for nsp1␣. Furthermore, nsp1␣ appears to have a dimeric architecture both in solution and as a crystal, with a hydrophilic groove on the molecular surface that may be related to nsp1␣'s biological function. Compared with existing structure and function data, our results suggest that PRRSV nsp1␣ functions differently from other reported viral leader proteases, such as that of foot-and-mouth disease.