Abstract
Phosphorylation-dependent interactions play crucial regulatory roles in all domains of life. Forkhead-associated (FHA) and von Willebrand type A (vWA) domains are involved in several phosphorylation-dependent processes of multiprotein complex assemblies. Although well-studied in eukaryotes and bacteria, the structural and functional contexts of these domains are not yet understood in Archaea. Here, we report the structural base for such an interacting pair of FHA and vWA domain-containing proteins, ArnA and ArnB, in the thermoacidophilic archaeon Sulfolobus acidocaldarius, where they act synergistically and negatively modulate motility. The structure of the FHA domain of ArnA at 1.75 Å resolution revealed that it belongs to the subclass of FHA domains, which recognizes double-pSer/pThr motifs. We also solved the 1.5 Å resolution crystal structure of the ArnB paralog vWA2, disclosing a complex topology comprising the vWA domain, a β-sandwich fold, and a C-terminal helix bundle. We further show that ArnA binds to the C terminus of ArnB, which harbors all the phosphorylation sites identified to date and is important for the function of ArnB in archaellum regulation. We also observed that expression levels of the archaellum components in response to changes in nutrient conditions are independent of changes in ArnA and ArnB levels and that a strong interaction between ArnA and ArnB observed during growth on rich medium sequentially diminishes after nutrient limitation. In summary, our findings unravel the structural features in ArnA and ArnB important for their interaction and functional archaellum expression and reveal how nutrient conditions affect this interaction.
Highlights
Phosphorylation-dependent interactions play crucial regulatory roles in all domains of life
Levels of ArnA and ArnB do not change during starvation In S. acidocaldarius, expression of the archaellum is induced upon nutrient limitation [23]
To test whether repression of expression levels of the components of the archaellum occurs via changes in the levels of arnA and arnB gene products, the protein levels of ArnA, ArnB, and FlaB were determined during exponential growth in rich conditions and under nutrient depletion (Fig. 1)
Summary
Phosphorylation-dependent interactions play crucial regulatory roles in all domains of life. The overall structure of the FHA domain is similar in both eukaryotes and bacteria: FHA domains comprise a fold consisting of two -sheets that are connected via loops of variable length to form a twisted -sandwich These loops are the main interaction sites with other proteins because they harbor the important pThr-binding motifs [8]. We show that the C-terminal four helix bundle of ArnB is the main site of phosphorylation of ArnB, and deletion of this domain results in a strong reduction of the interaction with ArnA and reduced motility To our knowledge, this is the first example of interaction between a FHA domain and a vWA domain– containing protein that is phosphorylation-dependent and affects motility
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