Characterization of structural and functional domains of the anillin-related protein Mid1p that contribute to cytokinesis in fission yeast

Fission yeast cells depend on the anillin-related protein Mid1p for reliable cytokinesis. Insolubility limits the purification of full-length Mid1p for biophysical analysis, and lack of knowledge about the structural domains of Mid1p limits functional analysis. We addressed these limitations by identifying in a bacterial expression screen of random Mid1p fragments five soluble segments that can be purified and one insoluble segment. Using complementation experiments in Δmid1 cells, we tested the biological functions of these six putative domains that account for full-length Mid1p. The N-terminal domain (residues 1–149) is essential for correct positioning and orientation of septa. The third domain (residues 309–452) allows the construct composed of the first three domains (residues 1-452) to form hydrodynamically well-behaved octamers. Constructs consisting of residues 1–452 or 1–578 carry out most functions of full-length Mid1p, including concentration at the equatorial cortex in nodes that accumulate myosin-II and other contractile ring proteins during mitosis. However, cells depending on these constructs without the insoluble domain (residues 579–797) form equatorially located rings slowly from strands rather than by direct condensation of nodes. We conclude that residues 1–578 assemble node components myosin-II, Rng2p, and Cdc15p, and the insoluble domain facilitates the normal, efficient condensation of nodes into rings.     

A novel dimer-tetramer transition captured by the crystal structure of the HIV-1 Nef

HIV-1 Nef modulates disease progression through interactions with over 30 host proteins. Individual chains fold into membrane-interacting N-terminal and C-terminal core (Nef_core) domains respectively. Nef exists as small oligomers near membranes and associates into higher oligomers such as tetramers or hexadecamers in the cytoplasm. Earlier structures of the Nef_core in apo and complexed forms with the Fyn-kinase SH3 domain revealed dimeric association details and the role of the conserved PXXP recognition motif (residues 72–78) of Nef in SH3-domain interactions. The crystal structure of the tetrameric Nef reported here corresponds to the elusive cytoplasmic stage. Comparative analyses show that subunits of Nef_core dimers (open conformation) swing out with a relative displacement of ∼22 Å and rotation of ∼174° to form the ‘closed’ tetrameric structure. The changes to the association are around Asp125, a conserved residue important for viral replication and the important XR motif (residues 107–108). The tetramer associates through C4 symmetry instead of the 222 symmetry expected when two dimers associate together. This novel dimer-tetramer transition agrees with earlier solution studies including small angle X-ray scattering, analytical ultracentrifugation, dynamic laser light scattering and our glutaraldehyde cross-linking experiments. Comparisons with the Nef_core—Fyn-SH3 domain complexes reveal that the PXXP motif that interacts with the SH3-domain in the dimeric form is sterically occluded in the tetramer. However the 151–180 loop that is distal to the PXXP motif and contains several protein interaction motifs remains accessible. The results suggest how changes to the oligomeric state of Nef can help it distinguish between protein partners.