Archaeal GPN-loop GTPases involve a lock-switch-rock mechanism for GTP hydrolysis.

IMPORTANCE: GPN-loop GTPases have been found to be crucial for eukaryotic RNA polymerase II assembly and nuclear trafficking. Despite their ubiquitous occurrence in eukaryotes and archaea, the mechanism by which these GTPases mediate their function is unknown. Our study on an archaeal representative from Sulfolobus acidocaldarius showed that these dimeric GTPases undergo large-scale conformational changes upon GTP hydrolysis, which can be summarized as a lock-switch-rock mechanism. The observed requirement of SaGPN for motility appears to be due to its large footprint on the archaeal proteome.

The archaeal triphosphate tunnel metalloenzyme SaTTM defines structural determinants for the diverse activities in the CYTH protein family.

CYTH proteins make up a large superfamily that is conserved in all three domains of life. These enzymes have a triphosphate tunnel metalloenzyme (TTM) fold, which typically results in phosphatase functions, e.g., RNA triphosphatase, inorganic polyphosphatase, or thiamine triphosphatase. Some CYTH orthologs cyclize nucleotide triphosphates to 3',5'-cyclic nucleotides. So far, archaeal CYTH proteins have been annotated as adenylyl cyclases, although experimental evidence to support these annotations is lacking. To address this gap, we characterized a CYTH ortholog, SaTTM, from the crenarchaeote Sulfolobus acidocaldarius. Our in silico studies derived ten major subclasses within the CYTH family implying a close relationship between these archaeal CYTH enzymes and class IV adenylyl cyclases. However, initial biochemical characterization reveals inability of SaTTM to produce any cyclic nucleotides. Instead, our structural and functional analyses show a classical TTM behavior, i.e., triphosphatase activity, where pyrophosphate causes product inhibition. The Ca2+-inhibited Michaelis complex indicates a two-metal-ion reaction mechanism analogous to other TTMs. Cocrystal structures of SaTTM further reveal conformational dynamics in SaTTM that suggest feedback inhibition in TTMs due to tunnel closure in the product state. These structural insights combined with further sequence similarity network-based in silico analyses provide a firm molecular basis for distinguishing CYTH orthologs with phosphatase activities from class IV adenylyl cyclases.

Crystal structure of an Lrs14-like archaeal biofilm regulator from Sulfolobus acidocaldarius.

The small winged helix-turn-helix (wHTH) proteins of the Lrs14 family are major transcriptional regulators and act as archaeal biofilm regulators (AbfRs) in the crenarchaeote Sulfolobus acidocaldarius. Here, the first crystal structure of an AbfR ortholog, AbfR2, the deletion of which is known to impair biofilm formation, is presented. Like most other wHTH orthologs, AbfR2 is dimeric in solution as well as in its 2.45 Šresolution crystal structure. Given the presence of three independent AbfR2 dimers in the asymmetric unit, the crystal structure shows a considerable degree of conformational variation within the dimer, the antiparallel orientations of which are stabilized by coiled-coil interaction between H4 helices. Conserved anchor interactions between helices H0 and H4 of AbfR2 further contribute to dimer stabilization. The combined structural and bioinformatic analysis reveals cluster-specific structural differences between different members of the Lrs14 protein family.

Structural and mechanistic divergence of the small (p)ppGpp synthetases RelP and RelQ.

The nutritional alarmones ppGpp and pppGpp (collectively: (p)ppGpp) are nucleotide-based second messengers enabling bacteria to respond to environmental and stress conditions. Several bacterial species contain two highly homologous (p)ppGpp synthetases named RelP (SAS2, YwaC) and RelQ (SAS1, YjbM). It is established that RelQ forms homotetramers that are subject to positive allosteric regulation by pppGpp, but structural and mechanistic insights into RelP lack behind. Here we present a structural and mechanistic characterization of RelP. In stark contrast to RelQ, RelP is not allosterically regulated by pppGpp and displays a different enzyme kinetic behavior. This discrepancy is evoked by different conformational properties of the guanosine-substrate binding site (G-Loop) of both proteins. Our study shows how minor structural divergences between close homologues result in new functional features during the course of molecular evolution.