Structural Dynamics of the C-terminal X Domain of Nipah and Hendra Viruses Controls the Attachment to the C-terminal Tail of the Nucleocapsid Protein.

To understand the dynamic interactions between the phosphoprotein (P) and the nucleoprotein (N) within the transcription/replication complex of the Paramyxoviridae and to decipher their roles in regulating viral multiplication, we characterized the structural properties of the C-terminal X domain (PXD) of Nipah (NiV) and Hendra virus (HeV) P protein. In crystals, isolated NiV PXD adopted a two-helix dimeric conformation, which was incompetent for binding its partners, but in complex with the C-terminal intrinsically disordered tail of the N protein (NTAIL), it folded into a canonical 3H bundle conformation. In solution, SEC-MALLS, SAXS and NMR spectroscopy experiments indicated that both NiV and HeV PXD were larger in size than expected for compact proteins of the same molecular mass and were in conformational exchange between a compact three-helix (3H) bundle and partially unfolded conformations, where helix α3 is detached from the other two. Some measurements also provided strong evidence for dimerization of NiV PXD in solution but not for HeV PXD. Ensemble modeling of experimental SAXS data and statistical-dynamical modeling reconciled all these data, yielding a model where NiV and HeV PXD exchanged between different conformations, and where NiV but not HeV PXD formed dimers. Finally, recombinant NiV comprising a chimeric P carrying HeV PXD was rescued and compared with parental NiV. Experiments carried out in cellula demonstrated that the replacement of PXD did not significantly affect the replication dynamics while caused a slight virus attenuation, suggesting a possible role of the dimerization of NiV PXD in viral replication.

Structure and Functions of Microtubule Associated Proteins Tau and MAP2c: Similarities and Differences.

The stability and dynamics of cytoskeleton in brain nerve cells are regulated by microtubule associated proteins (MAPs), tau and MAP2. Both proteins are intrinsically disordered and involved in multiple molecular interactions important for normal physiology and pathology of chronic neurodegenerative diseases. Nuclear magnetic resonance and cryo-electron microscopy recently revealed propensities of MAPs to form transient local structures and long-range contacts in the free state, and conformations adopted in complexes with microtubules and filamentous actin, as well as in pathological aggregates. In this paper, we compare the longest, 441-residue brain isoform of tau (tau40), and a 467-residue isoform of MAP2, known as MAP2c. For both molecules, we present transient structural motifs revealed by conformational analysis of experimental data obtained for free soluble forms of the proteins. We show that many of the short sequence motifs that exhibit transient structural features are linked to functional properties, manifested by specific interactions. The transient structural motifs can be therefore classified as molecular recognition elements of tau40 and MAP2c. Their interactions are further regulated by post-translational modifications, in particular phosphorylation. The structure-function analysis also explains differences between biological activities of tau40 and MAP2c.

Distinct ubiquitin binding modes exhibited by SH3 domains: molecular determinants and functional implications.

SH3 domains constitute a new type of ubiquitin-binding domains. We previously showed that the third SH3 domain (SH3-C) of CD2AP binds ubiquitin in an alternative orientation. We have determined the structure of the complex between first CD2AP SH3 domain and ubiquitin and performed a structural and mutational analysis to decipher the determinants of the SH3-C binding mode to ubiquitin. We found that the Phe-to-Tyr mutation in CD2AP and in the homologous CIN85 SH3-C domain does not abrogate ubiquitin binding, in contrast to previous hypothesis and our findings for the first two CD2AP SH3 domains. The similar alternative binding mode of the SH3-C domains of these related adaptor proteins is characterised by a higher affinity to C-terminal extended ubiquitin molecules. We conclude that CD2AP/CIN85 SH3-C domain interaction with ubiquitin constitutes a new ubiquitin-binding mode involved in a different cellular function and thus changes the previously established mechanism of EGF-dependent CD2AP/CIN85 mono-ubiquitination.