Cryo-EM structures of peripherin-2 and ROM1 suggest multiple roles in photoreceptor membrane morphogenesis.

Mammalian peripherin-2 (PRPH2) and rod outer segment membrane protein 1 (ROM1) are retina-specific tetraspanins that partake in the constant renewal of stacked membrane discs of photoreceptor cells that enable vision. Here, we present single-particle cryo-electron microscopy structures of solubilized PRPH2-ROM1 heterodimers and higher-order oligomers. High-risk PRPH2 and ROM1 mutations causing blindness map to the protein-dimer interface. Cysteine bridges connect dimers forming positive-curved oligomers, whereas negative-curved oligomers were observed occasionally. Hexamers and octamers exhibit a secondary micelle that envelopes four carboxyl-terminal helices, supporting a potential role in membrane remodeling. Together, the data indicate multiple structures for PRPH2-ROM1 in creating and maintaining compartmentalization of photoreceptor cells.

NMR secondary structure and interactions of recombinant human MOZART1 protein, a component of the gamma-tubulin complex.

Mitotic-spindle organizing protein associated with a ring of γ-tubulin 1 (MOZART1) is an 8.5 kDa protein linked to regulation of γ-tubulin ring complexes (γTuRCs), which are involved in nucleation of microtubules. Despite its small size, MOZART1 represents a challenging target for detailed characterization in vitro. We described herein a protocol for efficient production of recombinant human MOZART1 in Escherichia coli and assessed the properties of the purified protein using a combination of size exclusion chromatography coupled with multiangle light scattering (SEC-MALS), dynamic light scattering (DLS), and nuclear magnetic resonance (NMR) experiments. MOZART1 forms heterogeneous oligomers in solution. We identified optimal detergent and buffer conditions for recording well resolved NMR experiments allowing nearly full protein assignment and identification of three distinct alpha-helical structured regions. Finally, using NMR, we showed that MOZART1 interacts with the N-terminus (residues 1-250) of GCP3 (γ-tubulin complex protein 3). Our data illustrate the capacity of MOZART1 to form oligomers, promoting multiple contacts with a subset of protein partners in the context of microtubule nucleation.