Restructuring of the dinucleotide-binding fold in an NADP(H) sensor protein

Abstract

Rossmann fold ͉ signal transduction N AD and its phosphorylated form, NADP, is a universal cofactor in a large number of redox reactions carried out by a variety of enzymes, especially dehydrogenases. It is widely recognized that NAD(P) is an essential molecule in energy metabolism in all organisms. More recently, mounting evidence suggests that, in addition to its central role in energy metabolism, NAD(P) is also involved in signaling pathways that regulate fundamental processes of cellular functions, including gene transcription and apoptosis (1,2). This realization generated a link between the energy metabolism and the regulated networks of biological processes.One of the NAD signaling mechanisms is through gene silencer proteins known as sirtuins, such as Sir2 in yeast, a NAD-dependent histone deacetylase. Increased activities of Sir2 induced by an increase in NAD production extended lifespan (3). It was shown that a product of NAD-dependent deacetylation by Sir2, nicotinamide, strongly inhibits the activity of Sir2 (4, 5). Similar mechanisms are present in other eukaryotes, including humans (6). NAD is also directly related to transcription regulation. PolyADP-ribose polymerases (PARPs) can modify the acceptor proteins by synthesizing a polyADP-ribose molecule using NAD ϩ as the substrate. The transcription factors that can be modified by PARPs include p53, YY1, NF-B, and TATA-binding protein (1). In other cases, transcription regulation is not carried out by any enzymatic reaction. For instance, the C-terminal-binding protein CtBP is a corepressor that has an increased affinity to its partners, such as adenovirus E1A or cellular repressor ZEB, when NADH binds to CtBP (7). Crystal structures showed that NADH binding to CtBP induced a conformational switch that stabilizes the dimerization of CtBP, which in turn promotes its binding to the repressors (8). In the case of the negative transcriptional regulator NmrA, NAD ϩ binding to NmrA controls the rate of nuclear entry of the GATA transcription-activating protein AreA (9, 10). NmrA has a similar structure to short-chain dehydrogenase/reductase (SDR) family proteins but no enzyme activities, because of the lack of conserved active-site residues (9). NAD(P) exerts its functions by association with proteins. The protein fold that binds NAD(P) was discovered by Rossmann when the crystal structure of lactate dehydrogenase was determined (11). The Rossmann fold is the most common fold, based on its predicted occurrence from the genes known today (12). This motif consists of six ␤-strands connected by ␣-helices with the NAD(P) molecule bound at the top [supporting information (SI) Fig. 5]. The Rossmann fold has always been present as a rigid-body domain in all other known crystal structures until the structure of HSCARG was determined. We found that the common ␣E that connects ␤5 to ␤6 in the Rossmann fold is deformed as an extended loop when NADP is not bound with HSCARG. This allows the formation of an asymmetric dimer between a subunit with NADP bound and an emp...

HSCARG inhibits activation of NF-κB by interacting with IκB kinase-β

“…The human protein HSCARG (NmrA-like family domaincontaining protein 1 or NMRAL1) has been identified as a novel NADPH sensor Zheng et al, 2007). Our previous studies have shown that HSCARG restructures and redistributes in response to changes of intracellular NADPH/NADP + levels Zheng et al, 2007) and found that HSCARG is involved in the regulation of nitric oxide (NO) production through repression of the activity of argininosuccinate synthetase (AS), a rate-limiting enzyme in NO synthesis .…”

“…Our previous studies have shown that HSCARG restructures and redistributes in response to changes of intracellular NADPH/NADP + levels Zheng et al, 2007) and found that HSCARG is involved in the regulation of nitric oxide (NO) production through repression of the activity of argininosuccinate synthetase (AS), a rate-limiting enzyme in NO synthesis . The interaction between HSCARG and AS is enhanced when the intracellular NADPH/NADP + levels decreased and leads to a stronger inhibition by HSCARG on AS and NO production.…”

The transcription repressor NmrA is subject to proteolysis by three Aspergillus nidulans proteases

“…27 In this respect we note that the Human protein HSCARG, which belongs to the NmrA-like structural family, binds NADPH with a 360-fold lower K D than that for NADP þ . 15 Given these observations, a redox sensing role for NmrA is less likely.…”

“…13 HSCARG is essential for epithelial cell viability, binds NADPH much more strongly than NADP þ and may link changes in the NADP þ : NADPH ratio to modulation of the production of the important cell signaling molecule nitric oxide. [14][15][16] Microcalorimetry experiments showed that NmrA binds NAD þ and NADP þ with similar affinity but has a greatly reduced affinity for NADH and NADPH. The structure of NmrA in a complex with NADP þ has shown how repositioning a His 37 sidechain allows the different conformations of NAD þ and NADP þ to be accommodated.…”

Novel resistance mechanism of barley chlorina f104 antenna mutant against photoinhibition: possible role of new identified chloroplastic cpNrp protein

“…It also shows close homology with UDP-galactose 4-epimerase. It is known that NmrA protein binds oxidized NAD which has probably certain regulating function (Zheng et al 2007). Its dehydrogenase and reductase activity probably does not work in this case ?…”