RESUMO
Transposon-encoded IscB family proteins are RNA-guided nucleases in the OMEGA (obligate mobile element-guided activity) system, and likely ancestors of the RNA-guided nuclease Cas9 in the type II CRISPR-Cas adaptive immune system. IscB associates with its cognate ωRNA to form a ribonucleoprotein complex that cleaves double-stranded DNA targets complementary to an ωRNA guide segment. Although IscB shares the RuvC and HNH endonuclease domains with Cas9, it is much smaller than Cas9, mainly due to the lack of the α-helical nucleic-acid recognition lobe. Here, we report the cryo-electron microscopy structure of an IscB protein from the human gut metagenome (OgeuIscB) in complex with its cognate ωRNA and a target DNA, at 2.6-Å resolution. This high-resolution structure reveals the detailed architecture of the IscB-ωRNA ribonucleoprotein complex, and shows how the small IscB protein assembles with the ωRNA and mediates RNA-guided DNA cleavage. The large ωRNA scaffold structurally and functionally compensates for the recognition lobe of Cas9, and participates in the recognition of the guide RNA-target DNA heteroduplex. These findings provide insights into the mechanism of the programmable DNA cleavage by the IscB-ωRNA complex and the evolution of the type II CRISPR-Cas9 effector complexes.
Assuntos
Sistemas CRISPR-Cas , RNA Guia de Cinetoplastídeos , Humanos , Microscopia Crioeletrônica , RNA Guia de Cinetoplastídeos/metabolismo , Endonucleases/metabolismo , RNA/metabolismo , DNA/metabolismo , Ribonucleoproteínas/metabolismoRESUMO
Linker of nucleoskeleton and cytoskeleton (LINC) complexes are molecular tethers that span the nuclear envelope (NE) and physically connect the nucleus to the cytoskeleton. They transmit mechanical force across the NE in processes such as nuclear anchorage, nuclear migration, and homologous chromosome pairing during meiosis. LINC complexes are composed of KASH proteins traversing the outer nuclear membrane, and SUN proteins crossing the inner nuclear membrane. Humans have several SUN- and KASH-containing proteins, yet what governs their proper engagement is poorly understood. To investigate this question, we solved high resolution crystal structures of human SUN2 in complex with the KASH-peptides of Nesprin3, Nesprin4, and KASH5. In comparison to the published structures of SUN2-KASH1/2 we observe alternative binding modes for these KASH peptides. While the core interactions between SUN and the C-terminal residues of the KASH peptide are similar in all five complexes, the extended KASH-peptide adopts at least two different conformations. The much-improved resolution allows for a more detailed analysis of other elements critical for KASH interaction, including the KASH-lid and the cation loop, and a possible self-locked state for unbound SUN. In summary, we observe distinct differences between the examined SUN-KASH complexes. These differences may have an important role in regulating the SUN-KASH network.
Assuntos
Proteínas de Ciclo Celular/ultraestrutura , Proteínas de Membrana/ultraestrutura , Proteínas dos Microfilamentos/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Nucléolo Celular/genética , Nucléolo Celular/ultraestrutura , Pareamento Cromossômico/genética , Cristalografia por Raios X , Citoesqueleto/genética , Citoesqueleto/ultraestrutura , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Meiose/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Membrana Nuclear/genética , Membrana Nuclear/ultraestrutura , Matriz Nuclear/genética , Matriz Nuclear/ultraestrutura , Peptídeos/química , Peptídeos/genética , Conformação ProteicaRESUMO
Molecular tethers span the nuclear envelope to mechanically connect the cytoskeleton and nucleoskeleton. These bridge-like tethers, termed linkers of nucleoskeleton and cytoskeleton (LINC) complexes, consist of SUN proteins at the inner nuclear membrane and KASH proteins at the outer nuclear membrane. LINC complexes are central to a variety of cell activities including nuclear positioning and mechanotransduction, and LINC-related abnormalities are associated with a spectrum of tissue-specific diseases, termed laminopathies or envelopathies. Protocols used to study the biochemical and structural characteristics of core elements of SUN-KASH complexes are described here to facilitate further studies in this new field of cell biology.