SUMMARY Superfamily ATPases in Type IV pili (T4P), Type 2 secretion (T2S), and archaella (formerly archaeal flagella) employ similar sequences for distinct biological processes. Here we structurally and functionally characterize prototypical superfamily ATPase FlaI from Sulfolobus acidocaldarius showing FlaI activities in archaeal swimming organelle assembly and movement. FlaI solution X-ray scattering and crystal structures with and without nucleotide reveal a hexameric crown assembly with key cross-subunit interactions: rigid building blocks form between N-terminal domains (points) and neighboring subunit C-terminal domains (crown ring). Upon nucleotide binding, these six cross-subunit blocks move with respect to each other distinctly from secretion and pilus ATPases. Crown interactions and conformations regulate assembly, motility and force direction by a basic-clamp switching mechanism driving conformational changes between stable, backbone-interconnected moving blocks. Collective structural and mutational results identify in vivo functional components for assembly and motility, phosphate triggered rearrangements by ATP-hydrolysis, and molecular predictors for distinct ATPase superfamily functions.
SummaryThe motor of the membrane‐anchored archaeal motility structure, the archaellum, contains FlaX, FlaI and FlaH. FlaX forms a 30 nm ring structure that acts as a scaffold protein and was shown to interact with the bifunctional ATPase FlaI and FlaH. However, the structure and function of FlaH has been enigmatic. Here we present structural and functional analyses of isolated FlaH and archaellum motor subcomplexes. The FlaH crystal structure reveals a RecA/Rad51 family fold with an ATP bound on a conserved and exposed surface, which presumably forms an oligomerization interface. FlaH does not hydrolyze ATP in vitro, but ATP binding to FlaH is essential for its interaction with FlaI and for archaellum assembly. FlaH interacts with the C‐terminus of FlaX, which was earlier shown to be essential for FlaX ring formation and to mediate interaction with FlaI. Electron microscopy reveals that FlaH assembles as a second ring inside the FlaX ring in vitro. Collectively these data reveal central structural mechanisms for FlaH interactions in mediating archaellar assembly: FlaH binding within the FlaX ring and nucleotide‐regulated FlaH binding to FlaI form the archaellar basal body core.
The Bunyavirales order contains several emerging viruses with high epidemic potential, including Severe fever with thrombocytopenia syndrome virus (SFTSV). The lack of medical countermeasures, such as vaccines and antivirals, is a limiting factor for the containment of any virus outbreak. To develop such antivirals a profound understanding of the viral replication process is essential. The L protein of bunyaviruses is a multi-functional and multi-domain protein performing both virus transcription and genome replication and, therefore, is an ideal drug target. We established expression and purification procedures for the full-length L protein of SFTSV. By combining single-particle electron cryo-microscopy and X-ray crystallography, we obtained 3D models covering ∼70% of the SFTSV L protein in the apo-conformation including the polymerase core region, the endonuclease and the cap-binding domain. We compared this first L structure of the Phenuiviridae family to the structures of La Crosse peribunyavirus L protein and influenza orthomyxovirus polymerase. Together with a comprehensive biochemical characterization of the distinct functions of SFTSV L protein, this work provides a solid framework for future structural and functional studies of L protein–RNA interactions and the development of antiviral strategies against this group of emerging human pathogens.
Rift Valley fever virus (RVFV) belongs to the family of Phenuiviridae within the order of Bunyavirales . The virus may cause fatal disease both in livestock and humans, and therefore, is of great economical and public health relevance. In analogy to the influenza virus polymerase complex, the bunyavirus L protein is assumed to bind to and cleave off cap structures of cellular mRNAs to prime viral transcription. However, even though the presence of an endonuclease in the N-terminal domain of the L protein has been demonstrated for several bunyaviruses, there is no evidence for a cap-binding site within the L protein. We solved the structure of a C-terminal 117 amino acid-long domain of the RVFV L protein by X-ray crystallography. The overall fold of the domain shows high similarity to influenza virus PB2 cap-binding domain and the putative non-functional cap-binding domain of reptarenaviruses. Upon co-crystallization with m 7 GTP, we detected the cap-analogue bound between two aromatic side chains as it has been described for other cap-binding proteins. We observed weak but specific interaction with m 7 GTP rather than GTP in vitro using isothermal titration calorimetry. The importance of m 7 GTP-binding residues for viral transcription was validated using a RVFV minigenome system. In summary, we provide structural and functional evidence for a cap-binding site located within the L protein of a virus from the Bunyavirales order.
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