Insights into FlaI Functions in Archaeal Motor Assembly and Motility from Structures, Conformations, and Genetics

Abstract

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.

The Type IV Pilus Assembly ATPase PilB of Myxococcus xanthus Interacts with the Inner Membrane Platform Protein PilC and the Nucleotide-binding Protein PilM

“…Crystal structures of T2SS assembly ATPases (42)(43)(44)(45), T4P retraction ATPases (46,47), and the motor ATPase of the archaellum (the archaeal T4P-like motility system) (48) revealed that these proteins consist of a conserved C-terminal ATPase domain and a much more variable N-terminal domain. These crystal structures and other data also revealed that PilB-like ATPases function as hexamers and that conformational changes upon hydrolysis of ATP most likely drive the extrusion or retraction of the pilus (48,49). Several studies have suggested an interaction between PilB and PilC homologs.…”

Investigation of the malE Promoter and MalR, a Positive Regulator of the Maltose Regulon, for an Improved Expression System in Sulfolobus acidocaldarius

“…Heterologous expression of the T. tenax PP i -dependent PFK from the new vector (pSVA1450) resulted in a yield of recombinant protein significantly higher than that with pCmalLacS. Therefore, pSVA1450 is now routinely used for the overexpression of proteins in S. acidocaldarius and has been successfully used for the expression of different proteins (34)(35)(36)(37)(38).…”

The Salmonella Type III Secretion System Virulence Effector Forms a New Hexameric Chaperone Assembly for Export of Effector/Chaperone Complexes

“…Although one study has suggested that formation of the EscN ATPase hexameric ring may occur independently of ATP binding, as mutation of the ATP binding site does not affect oligomerization (5), separate studies on the homologous bacterial flagellar ATPase FliI and type IV pili ATPase FlaI show that formation of the hexamer occurs in an ATP-dependent manner (17,18). Based on homology to the ␥ subunit of F 1 -ATPase and flagellar central stalk FliJ, EscO is predicted to form an extended coil-coil that penetrates into the center of the ATPase hexameric ring and connects to the membrane or export gate (4).…”