How a single residue in individual β-thymosin/WH2 domains controls their functions in actin assembly.

β-Thymosin (βT) 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 Thymosin-β4, or enhance motility by directing polarized filament assembly like Ciboulot βT. We combined mutational, functional or structural analysis by X-ray crystallography, SAXS (small angle X-ray scattering) and NMR on Thymosin-β4, Ciboulot, TetraThymosinβ and the long WH2 domain of WASP-interacting protein.

Control of actin assembly by the WH2 domains and their multifunctional tandem repeats in Spire and Cordon-Bleu.

The WASP-homology 2 (WH2) domain is a 5-kDa actin-binding protein module that attracts increasing interest by its multifunctional regulation of actin dynamics in motile and morphogenetic processes. Identified by a short consensus sequence LKKT/V originally found in the actin-sequestering ß-thymosin peptides, the ßT/WH2 domains are inserted in a large number of proteins, in particular, the WASP proteins involved in cell protrusions. WH2 are found in tandem repeats in proteins involved in early development and axis-patterning processes, like Spire and Cordon-Bleu.

Cordon-Bleu uses WH2 domains as multifunctional dynamizers of actin filament assembly.

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.

Specificity shifts in the rRNA and tRNA nucleotide targets of archaeal and bacterial m5U methyltransferases.

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.

Multifunctionality of the beta-thymosin/WH2 module: G-actin sequestration, actin filament growth, nucleation, and severing.

The beta-thymosin/WH2 actin-binding module shows an amazing adaptation to multifunctionality. The beta-thymosins are genuine G-actin sequesterers of moderate affinity for G-actin, allowing an efficient regulation of the G-actin/F-actin ratio in cells by amplifying changes in the critical concentration for filament assembly. In contrast, the first beta-thymosin domain of the protein Ciboulot makes with G-actin a complex that supports filament growth, such as profilin-actin.

Recognition elements in rRNA for the tylosin resistance methyltransferase RlmA(II).

The methyltransferase RlmA(II) (formerly TlrB) is found in many Gram-positive bacteria, and methylates the N-1 position of nucleotide G748 within the loop of hairpin 35 in 23S rRNA. Methylation of the rRNA by RlmA(II) confers resistance to tylosin and other mycinosylated 16-membered ring macrolide antibiotics. We have previously solved the solution structure of hairpin 35 in the conformation that is recognized by the RlmA(II) methyltransferase from Streptococcus pneumoniae.

Cysteine of sequence motif VI is essential for nucleophilic catalysis by yeast tRNA m5C methyltransferase.

Sequence comparison of several RNA m(5)C methyltransferases identifies two conserved cysteine residues that belong to signature motifs IV and VI of RNA and DNA methyltransferases. While the cysteine of motif IV is used as the nucleophilic catalyst by DNA m(5)C methyltransferases, this role is fulfilled by the cysteine of motif VI in Escherichia coli 16S rRNA m(5)C967 methyltransferase, but whether this conclusion applies to other RNA m(5)C methyltransferases remains to be verified. Yeast tRNA m(5)C methyltransferase Trm4p is a multisite-specific S-adenosyl-L-methionine-dependent enzyme that catalyzes the methylation of cytosine at C5 in several positions of tRNA.