Chemical denaturant sensitivity of the dimeric main protease from severe acute respiratory syndrome (SARS) coronavirus to guanidinium chloride was examined in terms of fluorescence spectroscopy, circular dichroism, analytical ultracentrifuge, and enzyme activity change. The dimeric enzyme dissociated at guanidinium chloride concentration of <0.4 M, at which the enzymatic activity loss showed close correlation with the subunit dissociation. Further increase in guanidinium chloride induced a reversible biphasic unfolding of the enzyme. The unfolding of the C-terminal domain-truncated enzyme, on the other hand, followed a monophasic unfolding curve. Different mutants of the full-length protease (W31 and W207/W218), with tryptophanyl residue(s) mutated to phenylalanine at the C-terminal or N-terminal domain, respectively, were constructed. Unfolding curves of these mutants were monophasic but corresponded to the first and second phases of the protease, respectively. The unfolding intermediate of the protease thus represented a folded C-terminal domain but an unfolded N-terminal domain, which is enzymatically inactive due to loss of regulatory properties. The various enzyme forms were characterized in terms of hydrophobicity and size-and-shape distributions. We provide direct evidence for the functional role of C-terminal domain in stabilization of the catalytic N-terminal domain of SARS coronavirus main protease.

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The main protease of the coronavirus causing severe acute respiratory syndrome performs proteolytic processing of the viral polyproteins. The active form of the enzyme is a homodimer with each subunit consisting of three structural domains. Domains I and II, hosting the complete catalytic machinery, constitute the N-terminal chymotrypsin-like folding scaffold and connect to the extra C-terminal domain III by a long loop. Previously, the domain III-truncated enzyme was demonstrated to fold independently into an intact chymotrypsin-like fold, but it showed no enzyme activity. To further delineate the structure-function relationships of the domain III and the long loop, we generated some truncated and mutated M(pro) forms bearing various combinations of the loop with other structural parts of the enzyme. Their conformational and association properties were investigated in detail. Far-ultraviolet circular dichroism (CD) measurements revealed that these fragments could fold independently. The secondary, tertiary and quaternary structures of these mixtures were monitored by CD, fluorescence spectroscopy and analytical ultracentrifugation. However, no enzyme activity was observed for any mutant or mixtures. These observations indicate that the covalent linkage between the chymotrypsin like and the extra domain is essential for enzymatic activity of the main coronavirus protease and for the integrity of its quaternary structure.

J. Neurochem. (2010) 112, 1562–1573. Abstract Calcium/calmodulin‐dependent serine kinase (CASK), a causative gene in X‐linked mental retardation, acts as a multi‐domain scaffold protein and interacts with more than 20 cellular proteins in different subcellular regions of neurons. It is of interest, therefore, to explore whether post‐translational modification regulates CASK’s protein–protein interactions. Here, we provide evidence that CASK is phosphorylated by protein kinase A (PKA), identifying residue S562 in the PSD‐95‐Dlg‐ZO‐1 domain and residue T724 in the guanylate kinase domain as PKA sites by an in vitro PKA kinase reaction and site‐directed mutagenesis. Although the role of S562 phosphorylation is not clear, T724 phosphorylation up‐regulates the interaction between CASK and T‐box transcription factor T‐brain‐1 (Tbr‐1). NMDAR2b, a downstream target of the CASK‐Tbr‐1 complex, was then used to explore the significance of CASK phosphorylation by PKA. In cultured cortical neurons, the PKA pathway stimulates both the protein expression and the promoter activity of NMDAR2b. Deletion of the Tbr‐1‐binding sites greatly reduces the 3′‐5′‐cyclic AMP responsiveness of the NMDAR2b promoter, and the CASK T724A mutation does not promote the 3′‐5′‐cyclic AMP responsiveness of NMDAR2b. In conclusion, our data provide evidence that PKA phosphorylates CASK, regulates the nuclear function of CASK, and consequently modulates NMDAR2b expression.

a b s t r a c tThe dipeptidyl peptidase (DPP) family members, including DPP-IV, DPP8, DPP9 and others, cleave the peptide bond after the penultimate proline residue and are drug target rich. The dimerization of DPP-IV is required for its activity. A propeller loop located at the dimer interface is highly conserved within the family. Here we carried out site-directed mutagenesis on the loop of DPPIV and identified several residues important for dimer formation and enzymatic activity. Interestingly, the corresponding residues on DPP9 have a different impact whereby the mutations decrease activity without changing dimerization. Thus the propeller loop seems to play a varying role in different DPPs. Structured summary of protein interactions:DPP-IV and DPP-IV physically interact by comigration in gel electrophoresis (View interaction: 1, 2, 3, 4) DPP9 and DPP9 bind by circular dichroism (View interaction) DPP-IV and DPP-IV bind by circular dichroism (View interaction: 1, 2, 3, 4, 5) DPP-IV and DPP-IV bind by cosedimentation in solution (View interaction: 1, 2, 3, 4, 5) ADA binds to DPP-IV by surface plasmon resonance (View interaction: 1, 2, 3, 4, 5, 6) DPP9 and DPP9 bind by cosedimentation in solution (View interaction)