Enzyme structure: Crystallography of ubiquitous protein

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  • Published: Jan 15, 2015
  • Author: David Bradley
  • Channels: X-ray Spectrometry
thumbnail image: Enzyme structure: Crystallography of ubiquitous protein


The crystal structure of transhydrogenase (TH) shows two copies of the molecule, each of which contains three domains: domain I, domain II and domain III. Black spheres in domain III represent NADP(H). Structural asymmetry is observed in domain III: one is facing up (green) to catalyze the production of NADPH; the other is facing down (magenta) towards the transmembrane domain II to facilitate the transit of a proton. Labels “in” and “out” respectively denote mitochondrial matrix or the space outside the inner mitochondrial membrane. c/o Scripps

US scientists have used X-ray crystallography to focus on an almost ubiquitous metabolic enzyme, nicotinamide nucleotide transhydrogenase, found in most life forms but with medically relevant links to diseases as disparate as diabetes and cancer.

Josephine Leung, Mutsuo Yamaguchi, Charles Stout, Arne Moeller and Jeffrey Speir of The Scripps Research Institute, La Jolla, California, Lici Schurig-Briccio and Robert Gennis of the University of Illinois, Urbana, have improved our understanding of the complex molecular mechanisms that underpin the behaviour of nicotinamide nucleotide transhydrogenase, which is considered an ancient evolutionary enzyme involved in the maintenance of healthy cells.

In humans and other "higher" organisms, TH enzymes work within mitochondria. TH enzymes, which are embedded in the inner membrane of the mitochondrion allow a single-lane flow of protons back through the membrane within the matrix as electrons are shed through metabolism. This process is linked to NADPH (nicotinamide adenine dinucleotide phosphate) production, a crucial component in mopping up reactive oxygen radicals.

Mitochondrial membrane

"Despite its importance, TH has been one of the least-studied of mitochondrial enzymes," explains Stout. "Our new study helps clear up some mysteries - suggesting how the enzyme structure might harness protons and indicating that its two sides are able to alternate functions, always staying in balance."

In earlier work, Stout's team and others obtained structures of the portion of TH that protrudes from the mitochondrial membrane but they had not pinned down a precise mechanism for the enzyme's mode of action, partly because of the well-known difficulty in carrying out crystallography on membrane proteins. "Key details we've been lacking include the structure of TH's transmembrane portion, and the way in which the parts assemble into the whole enzyme," Leung explains.

Role reversal

Leung and colleagues used new technology developed by Vadim Cherezov now at the University of Southern California to crystallise TH for the first time allowing them to obtain a crystal structure at a resolution of 2.8 angstroms for the partial enzyme. A lower resolution was achievable for their crystal structure of the whole TH enzyme, 6.9 angstroms. Electron microscopy data also confirmed that TH exists as a "dimer" in its natural state. The team also showed that lying above TH's transmembrane structure, just inside the mitochondrial matrix, is the so-called domain III structure the binding site for the NADPH precursor molecule, NADP+. Working out how these two units can exist side by side without interfering with each other in the dimer has now been laid bare by the team's study. "Our most striking finding was that the two domain III structures are not symmetric - one of them faces up while the other faces down," Leung says. One site appears to catalyse NADPH production, the other may facilitate transport of a proton. As a proton passes through the two structures seem to flip, switching roles.

Related Links

Science, 2015, 347, 178-181: "Division of labor in transhydrogenase by alternating proton translocation and hydride transfer"

Article by David Bradley

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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