Domains involved in the in vivo function and oligomerization of apical growth determinant DivIVA in Streptomyces coelicolor

The coiled-coil protein DivIVA is a determinant of apical growth and hyphal branching in Streptomyces coelicolor . We have investigated the properties of this protein and the involvement of different domains in its essential function and subcellular targeting. In S. coelicolor cell extracts, DivIVA was present as large oligomeric complexes that were not strongly membrane associated. The purified protein could self-assemble into extensive protein filaments in vitro . Two large and conspicuous segments in the amino acid sequence of streptomycete DivIVAs not present in other homologs, an internal PQG-rich segment and a carboxy-terminal extension, are shown to be dispensable for the essential function in S. coelicolor . Instead, the highly conserved amino-terminal of 22 amino acids was required and affected establishment of new DivIVA foci and hyphal branches, and an essential coiled-coil domain affected oligomerization of the protein.     

A conserved charged single α-helix with a putative steric role in paraspeckle formation

Paraspeckles are subnuclear particles involved in the regulation of mRNA expression. They are formed by the association of DBHS family proteins and the NEAT1 long noncoding RNA. Here, we show that a recently identified structural motif, the charged single α-helix, is largely conserved in the DBHS family. Based on the available structural data and a previously suggested multimerization scheme of DBHS proteins, we built a structural model of a (PSPC1/NONO)_n multimer that might have relevance in paraspeckle formation. Our model contains an extended coiled-coil region that is followed by and partially overlaps with the predicted charged single α-helix. We suggest that the charged single α-helix can act as an elastic ruler governing the exact positioning of the dimeric core structures relative to each other during paraspeckle assembly along the NEAT1 noncoding RNA.     

Engineering of deltoid and reduced deltoid: Two chimeric proteins containing the oligomerization site of the hepatitis delta antigen

Summary Hepatitis delta antigen (HDAg) must form oligomers to be biologically active. Quandrin (HDAg-(12–60)-Tyr) is a 50-residue protein segment from the oligomerization domain of HDAg. The crystal structure of quadrin shows an octamer consisting of four identical copies of a dimer containing an antiparallel α-helical coiled coil. Each end of the dimer contains an oligomerization site that interacts isologously with the oligomerization site of another dimer to form a right-angled corner. The resulting quadrin octamer is a 400-residue square protein surrounding a large aqueous hole. We have designed, chemically synthesized, and characterized deltoid and reduced deltoid, two 51-residue chimeric proteins that structurally and functionally mimic one of the two oligomerization sites of the quadrin dimer. Dimerization of deltoid or reduced deltoid should emulate the dimerization of two quadrin dimers to form one right-angled corner of the square. Deltoid and reduced deltoid were designed by molecular modeling, mechanics, and dynamics and synthesized by the solid-phase method. The amino acid sequence of deltoid (GREDILEQWVSCRKKL+PKAPPEE+LRKLKKKCKKLEEDNPWLGNIKGIIGKY) is a chimera of three protein segments: HDAg-(12–28),Thermus thermophilus serine tRNA synthase-(59–65), and HDAg-(34–60)-Tyr. Cysteine (C) was introduced at two positions to explore the effects of the presence (deltoid) or absence (reduced deltoid) of an interhelical disulfide bond. Circular dichroic spectropolarimetry revealed that both synthetic proteins from an α-helical structure that is stable over a wide range of pH and KCl concentrations. Size-exclusion chromatography indicated that deltoid and reduced deltoid each form a dimer. Interconversion of these monomers and dimers should be useful model systems for studying the structural features of the right-angled corners of the quandrin octamer that contribute to HDAg oligomerization. If, like quadrin, deltoid or reduced deltoid interferes with HDAg oligomerization, it might serve as a lead compound for the design of potent HDV inhibitors.