Cordon-Bleu is, like Spire, a member of the growing family of WH2 repeat proteins, which emerge as versatile regulators of actin dynamics. They are expressed in morphogenetic and patterning processes and nucleate actin assembly in vitro. Here, we show that Cordon-Bleu works as a dynamizer of actin assembly by combining many properties of profilin with weak filament nucleating and powerful filament severing activities and sequestration of ADP-actin, which altogether generate oscillatory polymerization kinetics. A short lysine-rich sequence, N-terminally adjacent to the three WH2 domains, is required for nucleation and severing. In this context, nucleation requires only one WH2 domain, but filament severing requires two adjacent WH2 domains. A model integrating the multiple activities of Cordon-Bleu and quantitatively fitting the multiphasic polymerization curves is derived. Hence, with similar structural organization of WH2 repeats, Cordon-Bleu and Spire display different functions by selecting different sets of the multifunctional properties of WH2 domains.

b-Thymosin (bT) and WH2 domains are widespread, intrinsically disordered actin-binding peptides that display significant sequence variability and different regulations of actin self-assembly in motile and morphogenetic processes. Here, we reveal the structural mechanisms by which, in their 1:1 stoichiometric complexes with actin, they either inhibit assembly by sequestering actin monomers like Thymosinb4, or enhance motility by directing polarized filament assembly like Ciboulot bT. We combined mutational, functional or structural analysis by X-ray crystallography, SAXS (small angle X-ray scattering) and NMR on Thymosin-b4, Ciboulot, TetraThymosinb and the long WH2 domain of WASP-interacting protein. The latter sequesters G-actin with the same molecular mechanisms as Thymosin-b4. Functionally different bT/WH2 domains differ by distinct dynamics of their C-terminal half interactions with G-actin pointed face. These C-terminal interaction dynamics are controlled by the strength of electrostatic interactions with G-actin. At physiological ionic strength, a single salt bridge with actin located next to their central LKKT/V motif induces G-actin sequestration in both isolated long bT and WH2 domains. The results open perspectives for elucidating the functions of bT/WH2 domains in other modular proteins.

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Methyltransferase enzymes that use S-adenosylmethionine as a cofactor to catalyze 5-methyl uridine (m 5 U) formation in tRNAs and rRNAs are widespread in Bacteria and Eukaryota, but are restricted to the Thermococcales and Nanoarchaeota groups amongst the Archaea. The RNA m 5 U methyltransferases appear to have arisen in Bacteria and were then dispersed by horizontal transfer of an rlmD-type gene to the Archaea and Eukaryota. The bacterium Escherichia coli has three gene paralogs and these encode the methyltransferases TrmA that targets m 5 U54 in tRNAs, RlmC (formerly RumB) that modifies m 5 U747 in 23S rRNA, and RlmD (formerly RumA) the archetypical enzyme that is specific for m 5 U1939 in 23S rRNA. The thermococcale archaeon Pyrococcus abyssi possesses two m 5 U methyltransferase paralogs, PAB0719 and PAB0760, with sequences most closely related to the bacterial RlmD. Surprisingly, however, neither of the two P. abyssi enzymes displays RlmD-like activity in vitro. PAB0719 acts in a TrmA-like manner to catalyze m 5 U54 methylation in P. abyssi tRNAs, and here we show that PAB0760 possesses RlmC-like activity and specifically methylates the nucleotide equivalent to U747 in P. abyssi 23S rRNA. The findings indicate that PAB0719 and PAB0760 originated as RlmD-type m 5 U methyltransferases and underwent changes in target specificity after their acquisition by a Thermococcales ancestor from a bacterial source.