Structural basis for ligand binding modes of CTP synthase

Cytidine triphosphate synthase (CTPS), which comprises an ammonia ligase domain and a glutamine amidotransferase domain, catalyzes the final step of de novo CTP biosynthesis. The activity of CTPS is regulated by the binding of four nucleotides and glutamine. While glutamine serves as an ammonia donor for the ATP-dependent conversion of UTP to CTP, the fourth nucleotide GTP acts as an allosteric activator. Models have been proposed to explain the mechanisms of action at the active site of the ammonia ligase domain and the conformational changes derived by GTP binding. However, actual GTP/ATP/UTP binding modes and relevant conformational changes have not been revealed fully. Here, we report the discovery of binding modes of four nucleotides and a glutamine analog 6-diazo-5-oxo-L-norleucine in Drosophila CTPS by cryo–electron microscopy with near-atomic resolution. Interactions between GTP and surrounding residues indicate that GTP acts to coordinate reactions at both domains by directly blocking ammonia leakage and stabilizing the ammonia tunnel. Additionally, we observe the ATP-dependent UTP phosphorylation intermediate and determine interacting residues at the ammonia ligase. A noncanonical CTP binding at the ATP binding site suggests another layer of feedback inhibition. Our findings not only delineate the structure of CTPS in the presence of all substrates but also complete our understanding of the underlying mechanisms of the allosteric regulation and CTP synthesis.

Role of allosteric switches and adaptor domains in long-distance cross-talk and transient tunnel formation

Transient tunnels that assemble and disassemble to facilitate passage of unstable intermediates in enzymes containing multiple reaction centers are controlled by allosteric cues. Using the 140-kDa purine biosynthetic enzyme PurL as a model system and a combination of biochemical and x-ray crystallographic studies, we show that long-distance communication between ~25-Å distal active sites is initiated by an allosteric switch, residing in a conserved catalytic loop, adjacent to the synthetase active site. Further, combinatory experiments seeded from molecular dynamics simulations help to delineate transient states that bring out the central role of nonfunctional adaptor domains. We show that carefully orchestrated conformational changes, facilitated by interplay of dynamic interactions at the allosteric switch and adaptor-domain interface, control reactivity and concomitant formation of the ammonia tunnel. This study asserts that substrate channeling is modulated by allosteric hotspots that alter protein energy landscape, thereby allowing the protein to adopt transient conformations paramount to function.

Different ways to transport ammonia in human and Mycobacterium tuberculosis NAD+ synthetases

NAD+ synthetase is an essential enzyme of de novo and recycling pathways of NAD+ biosynthesis in Mycobacterium tuberculosis but not in humans. This bifunctional enzyme couples the NAD+ synthetase and glutaminase activities through an ammonia tunnel but free ammonia is also a substrate. Here we show that the Homo sapiens NAD+ synthetase (hsNadE) lacks substrate specificity for glutamine over ammonia and displays a modest activation of the glutaminase domain compared to tbNadE. We report the crystal structures of hsNadE and NAD+ synthetase from M. tuberculosis (tbNadE) with synthetase intermediate analogues. Based on the observed exclusive arrangements of the domains and of the intra- or inter-subunit tunnels we propose a model for the inter-domain communication mechanism for the regulation of glutamine-dependent activity and NH3 transport. The structural and mechanistic comparison herein reported between hsNadE and tbNadE provides also a starting point for future efforts in the development of anti-TB drugs.

Regulation of the intersubunit ammonia tunnel in Mycobacterium tuberculosis glutamine-dependent NAD+ synthetase

Glutamine-dependent NAD+ synthetase is an essential enzyme and a validated drug target in Mycobacterium tuberculosis (mtuNadE). It catalyses the ATP-dependent formation of NAD+ from NaAD+ (nicotinic acid–adenine dinucleotide) at the synthetase active site and glutamine hydrolysis at the glutaminase active site. An ammonia tunnel 40 Å (1 Å=0.1 nm) long allows transfer of ammonia from one active site to the other. The enzyme displays stringent kinetic synergism; however, its regulatory mechanism is unclear. In the present paper, we report the structures of the inactive glutaminase C176A variant in an apo form and in three synthetase–ligand complexes with substrates (NaAD+/ATP), substrate analogue {NaAD+/AMP-CPP (adenosine 5′-[α,β-methylene]triphosphate)} and intermediate analogues (NaAD+/AMP/PPi), as well as the structure of wild-type mtuNadE in a product complex (NAD+/AMP/PPi/glutamate). This series of structures provides snapshots of the ammonia tunnel during the catalytic cycle supported also by kinetics and mutagenesis studies. Three major constriction sites are observed in the tunnel: (i) at the entrance near the glutaminase active site; (ii) in the middle of the tunnel; and (iii) at the end near the synthetase active site. Variation in the number and radius of the tunnel constrictions is apparent in the crystal structures and is related to ligand binding at the synthetase domain. These results provide new insight into the regulation of ammonia transport in the intermolecular tunnel of mtuNadE.