Structure of mouse muskelin discoidin domain and biochemical characterization of its self-association

Muskelin is an intracellular kelch-repeat protein comprised of discoidin, LisH, CTLH and kelch-repeat domains. It is involved in cell adhesion and the regulation of cytoskeleton dynamics as well as being a component of a putative E3 ligase complex. Here, the first crystal structure of mouse muskelin discoidin domain (MK-DD) is reported at 1.55 Å resolution, which reveals a distorted eight-stranded β-barrel with two short α-helices at one end of the barrel. Interestingly, the N- and C-termini are not linked by the disulfide bonds found in other eukaryotic discoidin structures. A highly conserved MIND motif appears to be the determinant for MK-DD specific interaction together with the spike loops. Analysis of interdomain interaction shows that MK-DD binds the kelch-repeat domain directly and that this interaction depends on the presence of the LisH domain.

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Characterization of a Viral Phosphoprotein Binding Site on the Surface of the Respiratory Syncytial Nucleoprotein

Journal of Virology ◽

10.1128/jvi.00058-12 ◽

2012 ◽

Vol 86 (16) ◽

pp. 8375-8387 ◽

Cited By ~ 28

Author(s):

Marie Galloux ◽

Bogdan Tarus ◽

Ilfad Blazevic ◽

Jenna Fix ◽

Stéphane Duquerroy ◽

...

Keyword(s):

Biochemical Characterization ◽

Specific Interaction ◽

Viral Rna ◽

Site Directed Mutagenesis ◽

Luciferase Reporter ◽

Potential Candidate ◽

Luciferase Reporter Gene ◽

Interaction Domain ◽

Rna Complex

The human respiratory syncytial virus (HRSV) genome is composed of a negative-sense single-stranded RNA that is tightly associated with the nucleoprotein (N). This ribonucleoprotein (RNP) complex is the template for replication and transcription by the viral RNA-dependent RNA polymerase. RNP recognition by the viral polymerase involves a specific interaction between the C-terminal domain of the phosphoprotein (P) (PCTD) and N. However, the P binding region on N remains to be identified. In this study, glutathioneS-transferase (GST) pulldown assays were used to identify the N-terminal core domain of HRSV N (NNTD) as a P binding domain. A biochemical characterization of the PCTDand molecular modeling of the NNTDallowed us to define four potential candidate pockets on N (pocket I [PI] to PIV) as hydrophobic sites surrounded by positively charged regions, which could constitute sites complementary to the PCTDinteraction domain. The role of selected amino acids in the recognition of the N-RNA complex by P was first screened for by site-directed mutagenesis using a polymerase activity assay, based on an HRSV minigenome containing a luciferase reporter gene. When changed to Ala, most of the residues of PI were found to be critical for viral RNA synthesis, with the R132A mutant having the strongest effect. These mutations also reduced or abolishedin vitroandin vivoP-N interactions, as determined by GST pulldown and immunoprecipitation experiments. The pocket formed by these residues is critical for P binding to the N-RNA complex, is specific for pneumovirus N proteins, and is clearly distinct from the P binding sites identified so far for other nonsegmented negative-strand viruses.

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