A Lumenal Loop Associated with Catalytic Asymmetry in Plant Vacuolar H+-Translocating Pyrophosphatase

Membrane-integral inorganic pyrophosphatases (mPPases) couple pyrophosphate hydrolysis with H+ and Na+ pumping in plants and microbes. mPPases are homodimeric transporters with two catalytic sites facing the cytoplasm and demonstrating highly different substrate-binding affinities and activities. The structural aspects of the functional asymmetry are still poorly understood because the structure of the physiologically relevant dimer form with only one active site occupied by the substrate is unknown. We addressed this issue by molecular dynamics (MD) simulations of the H+-transporting mPPase of Vigna radiata, starting from its crystal structure containing a close substrate analog (imidodiphosphate, IDP) in both active sites. The MD simulations revealed pre-existing subunit asymmetry, which increased upon IDP binding to one subunit and persisted in the fully occupied dimer. The most significant asymmetrical change caused by IDP binding is a ‘rigid body’-like displacement of the lumenal loop connecting α-helices 2 and 3 in the partner subunit and opening its exit channel for water. This highly conserved 14–19-residue loop is found only in plant vacuolar mPPases and may have a regulatory function, such as pH sensing in the vacuole. Our data define the structural link between the loop and active sites and are consistent with the published structural and functional data.

Poly(A) binding KPAF4/5 complex stabilizes kinetoplast mRNAs in Trypanosoma brucei

In Trypanosoma brucei, mitochondrial pre-mRNAs undergo 3′-5′ exonucleolytic processing, 3′ adenylation and uridylation, 5′ pyrophosphate removal, and, often, U-insertion/deletion editing. The 3′ modifications are modulated by pentatricopeptide repeat (PPR) Kinetoplast Polyadenylation Factors (KPAFs). We have shown that KPAF3 binding to the 3′ region stabilizes properly trimmed transcripts and stimulates their A-tailing by KPAP1 poly(A) polymerase. Conversely, poly(A) binding KPAF4 shields the nascent A-tail from uridylation and decay thereby protecting pre-mRNA upon KPAF3 displacement by editing. While editing concludes in the 5′ region, KPAF1/2 dimer induces A/U-tailing to activate translation. Remarkably, 5′ end recognition and pyrophosphate hydrolysis by the PPsome complex also contribute to mRNA stabilization. Here, we demonstrate that KPAF4 functions as a heterodimer with KPAF5, a protein lacking discernable motifs. We show that KPAF5 stabilizes KPAF4 to enable poly(A) tail recognition, which likely leads to mRNA stabilization during the editing process and impedes spontaneous translational activation of partially-edited transcripts. Thus, KPAF4/5 represents a poly(A) binding element of the mitochondrial polyadenylation complex. We present evidence that RNA editing substrate binding complex bridges the 5′ end-bound PPsome and 3′ end-bound polyadenylation complexes. This interaction may enable mRNA circularization, an apparently critical element of mitochondrial mRNA stability and quality control.

ATP-based therapy prevents vascular calcification and extends longevity in a mouse model of Hutchinson–Gilford progeria syndrome

Pyrophosphate deficiency may explain the excessive vascular calcification found in children with Hutchinson–Gilford progeria syndrome (HGPS) and in a mouse model of this disease. The present study found that hydrolysis products of ATP resulted in a <9% yield of pyrophosphate in wild-type blood and aortas, showing that eNTPD activity (ATP → phosphate) was greater than eNPP activity (ATP → pyrophosphate). Moreover, pyrophosphate synthesis from ATP was reduced and pyrophosphate hydrolysis (via TNAP; pyrophosphate → phosphate) was increased in both aortas and blood obtained from mice with HGPS. The reduced production of pyrophosphate, together with the reduction in plasma ATP, resulted in marked reduction of plasma pyrophosphate. The combination of TNAP inhibitor levamisole and eNTPD inhibitor ARL67156 increased the synthesis and reduced the degradation of pyrophosphate in aortas and blood ex vivo, suggesting that these combined inhibitors could represent a therapeutic approach for this devastating progeroid syndrome. Treatment with ATP prevented vascular calcification in HGPS mice but did not extend longevity. By contrast, combined treatment with ATP, levamisole, and ARL67156 prevented vascular calcification and extended longevity by 12% in HGPS mice. These findings suggest a therapeutic approach for children with HGPS.

Nucleotide-mediated allosteric regulation of bifunctional Rel enzymes

Bifunctional Rel stringent factors, the most broadly distributed class of RSHs, are ribosome-associated enzymes that transfer a pyrophosphate group from ATP onto the 3′ of GTP or GDP to synthesize (p)ppGpp and also catalyse the 3′ pyrophosphate hydrolysis of the alarmone to degrade it. The precise regulation of these enzymes seems to be a complex allosteric mechanism, and despite decades of research, it is unclear how the two opposing activities of Rel are controlled at the molecular level. Here we show that a stretch/recoil guanosine-switch mechanism controls the catalytic cycle of T. thermophilus Rel (RelTf). The binding of GDP/ATP stretches apart the NTD catalytic domains of RelTf (RelTtNTD) activating the synthetase domain and allosterically blocking the hydrolase active site. Conversely, binding of ppGpp unlocks the hydrolase domain and triggers recoil of both NTDs, which partially buries the synthetase active site and precludes the binding of synthesis precursors. This allosteric mechanism acts as an activity switch preventing futile cycles of alarmone synthesis and degradation.

Role of the potassium/lysine cationic center in catalysis and functional asymmetry in membrane-bound pyrophosphatases

Membrane-bound pyrophosphatases (mPPases), which couple pyrophosphate hydrolysis to transmembrane transport of H+ and/or Na+ ions, are divided into K+,Na+-independent, Na+-regulated, and K+-dependent families. The first two families include H+-transporting mPPases (H+-PPases), whereas the last family comprises one Na+-transporting, two Na+- and H+-transporting subfamilies (Na+-PPases and Na+,H+-PPases, respectively), and three H+-transporting subfamilies. Earlier studies of the few available model mPPases suggested that K+ binds to a site located adjacent to the pyrophosphate-binding site, but is substituted by the ε-amino group of an evolutionarily acquired lysine residue in the K+-independent mPPases. Here, we performed a systematic analysis of the K+/Lys cationic center across all mPPase subfamilies. An Ala → Lys replacement in K+-dependent mPPases abolished the K+ dependence of hydrolysis and transport activities and decreased these activities close to the level (4–7%) observed for wild-type enzymes in the absence of monovalent cations. In contrast, a Lys → Ala replacement in K+,Na+-independent mPPases conferred partial K+ dependence on the enzyme by unmasking an otherwise conserved K+-binding site. Na+ could partially replace K+ as an activator of K+-dependent mPPases and the Lys → Ala variants of K+,Na+-independent mPPases. Finally, we found that all mPPases were inhibited by excess substrate, suggesting strong negative co-operativity of active site functioning in these homodimeric enzymes; moreover, the K+/Lys center was identified as part of the mechanism underlying this effect. These findings suggest that the mPPase homodimer possesses an asymmetry of active site performance that may be an ancient prototype of the rotational binding-change mechanism of F-type ATPases.