Search

Coronavirus nucleocapsid (N) protein envelops the genomic RNA to form long helical nucleocapsid during virion assembly. Since N protein oligomerization is usually a crucial step in this process, characterization of such an oligomerization will help in the understanding of the possible mechanisms for nucleocapsid formation. The N protein of severe acute respiratory syndrome coronavirus (SARS-CoV) was recently discovered to self-associate by its carboxyl terminus. In this study, to further address the detailed understanding of the association feature of this C-terminus, its oligomerization was systematically investigated by size exclusion chromatography and chemical cross-linking assays. Our results clearly indicated that the C-terminal domain of SARS-CoV N protein could form not only dimers but also trimers, tetramers, and hexamers. Further analyses against six deletion mutants showed that residues 343-402 were necessary and sufficient for this C-terminus oligomerization. Although this segment contains many charged residues, differences in ionic strength have no effects on its oligomerization, indicating the absence of electrostatic force in SARS-CoV N protein C-terminus self-association. Gel shift assay results revealed that the SARS-CoV N protein C-terminus is also able to associate with nucleic acids and residues 363-382 are the responsible interaction partner, demonstrating that this fragment might involve genomic RNA binding sites. The fact that nucleic acid binding could promote the SARS-CoV N protein C-terminus to form high-order oligomers implies that the oligomeric SARS-CoV N protein probably combines with the viral genomic RNA in triggering long nucleocapsid formation.

Abeta peptides cleaved from the amyloid precursor protein are the main components of senile plaques in Alzheimer's disease. Abeta peptides adopt a conformation mixture of random coil, beta-sheet, and alpha-helix in solution, which makes it difficult to design inhibitors based on the 3D structures of Abeta peptides. By targeting the C-terminal beta-sheet region of an Abeta intermediate structure extracted from molecular dynamics simulations of Abeta conformational transition, a new inhibitor that abolishes Abeta fibrillation was discovered using virtual screening in conjunction with thioflavin T fluorescence assay and atomic force microscopy determination. Circular dichroism spectroscopy demonstrated that the binding of the inhibitor increased the beta-sheet content of Abeta peptides either by stabilizing the C-terminal beta-sheet conformation or by inducing the intermolecular beta-sheet formation. It was proposed that the inhibitor prevented fibrillation by blocking interstrand hydrogen bond formation of the pleated beta-sheet structure commonly found in amyloid fibrils. The study not only provided a strategy for inhibitor design based on the flexible structures of amyloid peptides but also revealed some clues to understanding the molecular events involved in Abeta aggregation.

Spike glycoprotein of SARS coronavirus (S protein) plays a pivotal role in SARS coronavirus (SARS_CoV) infection. The immunological fragment of the S protein (Ala251-His641, SARS_S1b) is believed to be essential for SARS_CoV entering the host cell through S protein-ACE-2 interaction. We have quantitatively characterized the thermally induced and GuHCl-induced unfolding features of SARS_S1b using circular dichroism (CD), tryptophan fluorescence, and stopped-flow spectral techniques. For the thermally induced unfolding at pH 7.4, the apparent activation energy (E(app)) and transition midpoint temperature (Tm) were determined to be 16.3 +/- 0.2 kcal/mol and 52.5 +/- 0.4 degrees C, respectively. The CD spectra are not dependent on temperature, suggesting that the secondary structure of SARS_S1b has a relatively high thermal stability. GuHCl strongly affected SARS_S1b structure. Both the CD and fluorescent spectra resulted in consistent values of the transition middle concentration of the denaturant (Cm, ranging from 2.30 to 2.45 M) and the standard free energy change (deltaG(o), ranging from 2.1 to 2.5 kcal/mol) for the SARS_S1b unfolding reaction. Moreover, the kinetic features of the chemical unfolding and refolding of SARS_S1b were also characterized using a stopped-flow CD spectral technique. The obvious unfolding reaction rates and relaxation times were determined at various GuHCl concentrations, and the Cm value was obtained, which is very close to the data that resulted from CD and fluorescent spectral determinations. Secondary and three-dimensional structural predictions by homology modeling indicated that SARS_S1b folded as a globular-like structure by beta-sheets and loops; two of the four tryptophans are located on the protein surface, which is in agreement with the tryptophan fluorescence result. The three-dimensional model was also used to explain the recently published experimental results of S1-ACE-2 binding and immunizations.

The nucleocapsid (N) protein of SARS coronavirus (SARS-CoV) is reported to function in encapsidating the viral genomic RNA into helical nucleocapsid, and its self-association is believed to be vital in coating the viral genomic RNA. Characterization of SARS-CoV N multimerization may thereby help us better understand the coronavirus assembly. In the current work, using the yeast two-hybrid technique, an unexpected interaction between residues 1-210 and 211-290 (central region) of the SARS-CoV N protein was detected, and SPR results further revealed that the SR-rich motif (amino acids 183-197) of SARS-CoV N protein is responsible for such an interaction. Chemical cross-linking and gel-filtration analyses indicated that the residues 283-422 of the SARS-CoV N protein have multimeric ability, although the full-length N protein is prone to exist predominantly as dimers. In addition, the multimeric ability of the C-terminal domain of SARS-CoV N protein could be weakened by the SR-rich motif interaction with the central region (amino acids 211-290). All of these data suggested that the SR-rich motif of the SARS-CoV N protein might play an import role in the transformation of the SARS-CoV N protein between the dimer and multimer during its binding to its central region for self-association or dissociation. This current paper will hopefully provide some new ideas in studying SARS-CoV N multimerization.