Cryo-EM and X-ray crystallographic studies of the citrate transporter INDY from D.melanogaster and the β-carbonic anhydrase CafA and CafB from A.fumigatus

Abstract

Part I: The single particle cryo-EM study of the citrate transporter INDY from D.melanogaster The citrate is an essential molecule in various metabolic signaling, such as energy generation, body fat accumulation, bone and teeth formation, a precursor of the neurotransmitter, and etc. The citrate is transported by SLC13A5 into the cell. The INDY (I’m Not Dead Yet) from Drosophila melanogaster is the fly homology SLC13A5. The dmINDY is highly expressed in the cell plasma membrane of the midgut, fat body, and oenocytes (fly liver) in the fly. Unlike other SLC13 transporters, dmINDY transports the TCA cycle intermediate across cell membranes independently of the cations. When the indy gene was mutated in flies, the life span of adult flies was extended through a mechanism similar to calorie restriction without reducing food intake. Also, It is affected to reduce reactive oxygen species production and increase mitochondrial biogenesis. Here, I present the cryo-EM structures of asymmetric conformation dmINDY without citrate in peptidisc at 3.18 Å resolution and inward-facing conformation dmINDY with citrate in nanodisc at 4.01 Å resolution. Comparison of two structures reveals that the dmINDY is a homodimer and the scaffold domain is important for dimeric structure and can help to keep the structural rigidity of the substrate-binding site, which consists of four loops, from various aromatic side chains interaction. The citrate is firmly fixed by essential hydrogen bonds and ionic interactions on the dmINDY in nanodisc. F119 may play a key role as a gatekeeper to prevent the substrate binding on the other side where the substrate binding site is open. Taken together, my results suggest that the dmINDY is transported substrate across the cell membrane through a rigid body elevator-type mechanism.

Part II: X-ray crystallographic study of the major β-carbonic anhydrase CafA and CafB from the A.fumigatus In fungi the β-class of carbonic anhydrases (β-CAs) are zinc metalloenzymes that are essential for growth, survival, differentiation, and virulence. Aspergillus fumigatus is the most important pathogen responsible for invasive aspergillosis and possesses two major β-CAs, CafA and CafB. Both enzymes exhibit apparent in vitro CO2 hydration activity. Despite the overall similarities in structures, there are notable differences in the catalytic active sites. Here, I report a crystallographic analysis of CafA and CafB revealing the mechanism of enzyme catalysis and establish the relationship of this enzyme to other β-CAs. The high-resolution crystal structure of CafA revealed which the catalytic zinc ion is tetrahedrally coordinated by three conserved residues (C119, H175, C178) and an acetate anion presumably acquired from the crystallization solution, indicating a freely accessible ″open″ conformation. Furthermore, the structure of CafA revealed complex with the potent inhibitor acetazolamide. CafB, when exposed to acidic pH and/or an oxidative environment, has a novel type of active site in which a disulfide bond is formed between two zinc-ligating cysteines (C57 and C116), expelling the zinc ion and stabilizing the inactive form of the enzyme. Based on the structural data, I generated an oxidation-resistant mutant (Y159A) of CafB. The crystal structure of the mutant under reducing conditions retains catalytic zinc at the expected position, tetrahedrally coordinated by three residues (C57, H113, and C116) and an aspartic acid (D59), and replacing the zinc-bound water molecule in the closed-form. Furthermore, the active site of CafB crystals grown under zinc-limiting conditions has a novel conformation in which the solvent-exposed catalytic cysteine (C116) is flipped out of the metal coordination sphere, facilitating the release of the zinc ion. Taken together, our results suggest that A. fumigatus use sophisticated activity-inhibiting strategies to enhance its survival during infection and inhibitor data could be exploited to develop new antifungal agents for the treatment of invasive aspergillosis.