In vitro biochemical and thermodynamic characterization of nucleocapsid protein of SARS

Luo, Hai‐Bin; Ye, Fei; Sun, Tao; Yue, Liduo; Peng, Sean; Chen, Jing; Li, Guowei; Du, Yi; Xie, Youhua; Yang, Yuxiang; Shen, Jianhua; Wang, Yuan; Shen, Xu; Jiang, Hualiang

doi:10.1016/j.bpc.2004.06.008

Cited by 58 publications

(72 citation statements)

References 35 publications

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“…As shown in Figure 1B, the R-galactosidase activity of cells with pGADT7-N and pGBKT7-N is over 2.5 milliunits, which follows the fact that the SARS-CoV N protein could selfassociate as indicated by the published results (16)(17)(18), further confirming that our yeast two-hybrid system is reliable enough. As also indicated in Figure 1B, the ∆N N / ∆N N interaction could not be detected and ∆N C /∆N C showed a stronger interaction than even N/N.…”

Section: Yeast Two-hybrid Technology-based Analysis Of the Interactiosupporting

confidence: 88%

“…To compare the multimerization capability of N11 with that of the full-length SARS-CoV N, we applied these two proteins to a gel-filtration column using the same molar concentration. On the basis of the standard proteins as markers ( Figure 5A), the full-length SARS-CoV N protein was found to exist mostly as dimers ( Figure 5B, ---), according to the recently reported results (16,17). However, N11 typically forms multimers as shown in Figure 5B (s).…”

Section: C-terminal 140 Amino Acids (Amino Acids 283-422 N11) Of Sarmentioning

confidence: 79%

“…The SASR-CoV N protein was recently reported to self-associate in forming homotypic dimers or multimers by different independent methods (13,(16)(17)(18). It is believed that self-association is also the fundamental function for the SARS-CoV N protein in coating the viral genomic RNA.…”

Section: Disscussionmentioning

confidence: 98%

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SR-Rich Motif Plays a Pivotal Role in Recombinant SARS Coronavirus Nucleocapsid Protein Multimerization

Luo

Chen

et al. 2005

Biochemistry

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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.

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Section: Yeast Two-hybrid Technology-based Analysis Of the Interactiosupporting

confidence: 88%

Section: C-terminal 140 Amino Acids (Amino Acids 283-422 N11) Of Sarmentioning

confidence: 79%

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SR-Rich Motif Plays a Pivotal Role in Recombinant SARS Coronavirus Nucleocapsid Protein Multimerization

Luo

Chen

et al. 2005

Biochemistry

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“…The C-terminal structural domain coincides with the dimerization domain identified in previous studies, and our biochemical studies showed that it exists as a dimer in solution. Denaturation studies have shown that dissociation of the SARS-CoV N protein is coupled with loss of structure, implying a structure-dependent mechanism for self-association [9]. Understanding this mechanism would not only provide insights into the viral assembly process, but also identify additional targets for drugs to combat SARS through disruption of N protein self-association.…”

Section: Introductionmentioning

confidence: 99%

The dimer interface of the SARS coronavirus nucleocapsid protein adapts a porcine respiratory and reproductive syndrome virus‐like structure

Chang

Sue

et al. 2005

FEBS Letters

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We have employed NMR to investigate the structure of SARS coronavirus nucleocapsid protein dimer. We found that the secondary structure of the dimerization domain consists of five a helices and a b-hairpin. The dimer interface consists of a continuous four-stranded b-sheet superposed by two long a helices, reminiscent of that found in the nucleocapsid protein of porcine respiratory and reproductive syndrome virus. Extensive hydrogen bond formation between the two hairpins and hydrophobic interactions between the b-sheet and the a helices render the interface highly stable. Sequence alignment suggests that other coronavirus may share the same structural topology.

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“…In addition, the plasmids for glutathione S-transferase (GST) fusions PGST-ESAT-6 was described previously (Lwabuchi et al, 2004). The recombinant ADAM9 protein of M. tb was cloned, expressed and purified according to the published procedure reported by Luo et al (2004) for GST pull-down assay. For surface plasmon resonance binding assay, ADAM9 was subcloned into pENTR/D-TOPO (Invitrogen) according to the manufacturer's protocol.…”

Section: Plasmids and Construction Of Recombinant Vectorsmentioning

confidence: 99%

Interaction of Mycobacterium tuberculosis ESAT-6 protein with ADAM9 protein

Liang¹,

Chen²

2011

Afr. J. Microbiol. Res.

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The ESAT-6 protein of Mycobacterium tuberculosis (M. tb) is an important structural and functional protein, which has been known to be involved in the virulence, pathogenesis as well as proliferation of the pathogen; however, how ESAT-6 protein interact with host protein is still unclear. In order to study the function of the M. tuberculosis protein ESAT-6 in the infection process, we searched for host proteins that interact with this secreted mycobacterial protein. Using a yeast two-hybrid system we identified the ADAM9 (a disintegrin and metalloprotease) protein as a candidate to interact with ESAT-6. This interaction was further confirmed by protein overlay and surface plasmon resonance binding assay using recombinant ESAT-6 and ADAM9, and by GST pull-down analysis of the mycobacterial expressed ESAT-6 and ADAM9. The interaction domains were localized by yeast two-hybrid studies using truncated derivatives of ESAT-6 protein. The C-terminus of ESAT-6 binds to the ADAM9, Thus, the host protein ADAM9 represents a possible cellular receptor for the mycobacterial protein ESAT-6. This is the first report demonstrating the interaction of ADAM9 with a structural protein of M. tb.

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In vitro biochemical and thermodynamic characterization of nucleocapsid protein of SARS

Cited by 58 publications

References 35 publications

SR-Rich Motif Plays a Pivotal Role in Recombinant SARS Coronavirus Nucleocapsid Protein Multimerization

SR-Rich Motif Plays a Pivotal Role in Recombinant SARS Coronavirus Nucleocapsid Protein Multimerization

The dimer interface of the SARS coronavirus nucleocapsid protein adapts a porcine respiratory and reproductive syndrome virus‐like structure

Interaction of Mycobacterium tuberculosis ESAT-6 protein with ADAM9 protein

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