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. 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.

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

ß-Thymosin (ßT) 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-ß4, or enhance motility by directing polarized filament assembly like Ciboulot ßT. We combined mutational, functional or structural analysis by X-ray crystallography, SAXS (small angle X-ray scattering) and NMR on Thymosin-ß4, Ciboulot, TetraThymosinß and the long WH2 domain of WASP-interacting protein. The latter sequesters G-actin with the same molecular mechanisms as Thymosin-ß4. Functionally different ßT/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 ßT and WH2 domains. The results open perspectives for elucidating the functions of ßT/WH2 domains in other modular proteins.

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. 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.

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. It was shown that while essential recognition elements are located in hairpin 35, the interactions between RlmA(II) and hairpin 35 are insufficient on their own to support the methylation reaction. Here we use biochemical techniques in conjunction with heteronuclear/homonuclear nuclear magnetic resonance spectroscopy to define the RNA structures that are required for efficient methylation by RlmA(II). Progressive truncation of the rRNA substrate indicated that multiple contacts occur between RlmA(II) and nucleotides in stem-loops 33, 34 and 35. RlmA(II) appears to recognize its rRNA target through specific surface shape complementarity at the junction formed by these three helices. This means of recognition is highly similar to that of the orthologous Gram-negative methyltransferase, RlmA(I) (formerly RrmA), which also interacts with hairpin 35, but methylates at the adjacent nucleotide G745.

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. These intrinsically disordered proteins regulate actin assembly in an adaptive and versatile fashion by a fine control of local interaction dynamics within the WH2-actin complex. Versatility is amplified by the protein environment in which the WH2 domain is inserted and by synergy with other adjacent actin-binding modules. Multifunctional activities emerge in WH2 repeats, including filament nucleation, dramatic severing, and barbed end capping or tracking. WH2 domains thus are instrumental in designing customized actin regulators.

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. We illustrate how the use of engineered chimeric proteins, actin-binding and polymerization assays, crystallographic, NMR, and SAXS structural approaches complement each other to decipher the molecular basis for the functional versatility of these intrinsically disordered domains when they form various 1:1 complexes with G-actin. Multifunctionality is expanded in tandem repeats of WH2 domains present in WASP family proteins and proteins involved in axis patterning like Cordon-Bleu and Spire. The tandem repeats generate new functions such as filament nucleation and severing, as well as barbed end binding, which add up to the G-actin sequestering activity. Novel regulation pathways in actin assembly emerge from these additional activities.

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. Here, we confirm that Cys310 of motif VI in Trm4p is essential for nucleophilic catalysis, presumably by forming a covalent link with carbon 6 of cytosine. Indeed, the enzyme is able to form a stable covalent adduct with the 5-fluorocytosine-containing RNA substrate analog, whereas the C310A mutant protein is inactive and unable to form the covalent complex.