Research Article: T-to-R switch of muscle fructose-1,6-bisphosphatase involves fundamental changes of secondary and quaternary structure

Date Published: April 01, 2016

Publisher: International Union of Crystallography

Author(s): Jakub Barciszewski, Janusz Wisniewski, Robert Kolodziejczyk, Mariusz Jaskolski, Dariusz Rakus, Andrzej Dzugaj.


When crystallized in the absence of the allosteric inhibitor AMP, human muscle fructose-1,6-bisphosphatase has a totally unexpected quaternary structure of its active R form, with the two dimers of the homotetrameric molecule in a perpendicular orientation, in stark contrast to the coplanar arrangement of the closely related liver isozyme. The T-to-R switch of the muscle enzyme also involves a highly unusual α→β refolding of the N-terminus.

Partial Text

Fructose-1,6-bisphosphatase (FBPase; EC, which catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate, is a key enzyme of gluconeogenesis and glyconeogenesis (Tejwani, 1983 ▸) and, more generally, of the control of energy metabolism and glucose homeostasis. Vertebrate genomes contain two distinct genes, FBP1 and FBP2, coding for two FBPase isozymes. Liver FBPase, the protein product of the FBP1 gene, is expressed mainly in gluconeogenic organs, where it functions as a regulator of glucose synthesis from noncarbohydrates (Al-Robaiy & Eschrich, 1999 ▸). The activity of liver FBPase is regulated by fructose 2,6-bisphosphate, a compound whose concentration is under hormonal control (Bartrons et al., 1983 ▸; Pilkis et al., 1995 ▸) and which inhibits the liver enzyme synergistically with AMP (Van Schaftingen & Hers, 1981 ▸; Benkovic & deMaine, 1982 ▸; Liu & Fromm, 1988 ▸). Unlike liver FBPase, the muscle isoform, encoded by FBP2, is expressed in all vertebrate cells, both in glyconeogenic cells (e.g. muscle fibres) as well as in cells such as neurons which are not supposed to synthesize glycogen from carbohydrate precursors (Löffler et al., 2001 ▸). Muscle FBPase is also present in cells that predominantly express the liver isozyme, e.g. in the liver itself (Al-Robaiy & Eschrich, 1999 ▸). Recent studies have demonstrated that the physiological role of muscle FBPase goes beyond its enzymatic function, as this isozyme was localized inside cell nuclei (Gizak et al., 2009 ▸) and was shown to interact with mitochondria (Gizak et al., 2012 ▸), where it is involved in regulation of the cell cycle (Gizak et al., 2006 ▸; Mamczur et al., 2012 ▸) and apoptosis (Gizak et al., 2012 ▸; Pirog et al., 2014 ▸), respectively. This moonlighting function of muscle FBPase does not rely on the catalytic activity of the enzyme but on its ability to interact with various nuclear and mitochondrial proteins, such as helicases, Myb-binding protein 1A, ribonucleoproteins (Mamczur et al., 2012 ▸), ATP synthase sub­units, VDACs (voltage-dependent anion channels) and Slc25a5 (solute carrier family 25; adenine nucleotide translocator) (Gizak et al., 2012 ▸).

The most important finding of this study is the observation that muscle FBPase adopts a unique, cross-like quaternary arrangement of its subunits in the active R state, in which the upper dimer is rotated by nearly −90° with respect to the lower dimer. This unexpected quaternary conformation has also been confirmed by SAXS experiments conducted under more physiological conditions (using 50 mM HEPES buffer and in the absence of salt), which demonstrated that in the absence of AMP the enzyme exists in the cruciform arrangement of the subunits, whereas the addition of AMP induced the formation of a T-like state (Szpotkowski, personal communication). Thus, whereas the quaternary structure of the inactive T form of muscle FBPase closely resembles the corresponding conformation of the liver isozyme, the cruciform R state of muscle FBPase is dramatically different from the planar arrangement observed for the active liver homotetramer.

In the present paper, we have demonstrated that the R state of mammalian muscle FBPase is significantly different from the corresponding form of the liver isozyme and that this novel quaternary structure may explain the complex role of muscle FBPase in cell physiology. The presented structural description of the new protein surfaces exposed in the R state may serve as a starting point for the identification of the cellular binding partners of muscle FBPase and for the design of small-molecule competitors for the interactions.