Date Published: November 01, 2016
Publisher: International Union of Crystallography
Author(s): Armin Ruf, Tim Tetaz, Brigitte Schott, Catherine Joseph, Markus G. Rudolph.
The crystal structure of the liver isoform of human fructose-1,6-bisphosphatase in the active R-state conformation was determined by molecular replacement using data from a crystal with noncrystallographic rotational symmetry and pseudo-translation. Owing to an almost perfect placement of noncrystallographic symmetry elements, quadruple space-group ambiguity within the same Laue symmetry arises, including two enantiogenic pairs. The origins of space-group ambiguity, the assignment of the correct space group, refinement and model properties are discussed.
Glucose is the main energy source for the brain. In mammals, blood glucose homeostasis is maintained mainly by the balance of catabolic glycolysis on the one hand and (with respect to glucose) anabolic glycogenolysis and gluconeogenesis on the other. Increased glucose production is the predominant cause of high blood glucose levels in type 2 diabetes, which can lead to kidney, neurological and cardiovascular damage. In humans, high glucose levels arise from excessive gluconeogenesis in the liver rather than from glycogenolysis of hepatic glycogen stores. Fructose 1,6-bisphosphatase (FBPase) is a major control point in gluconeogenesis, catalyzing the hydrolysis of fructose 1,6-bisphosphate (F-1,6-P2) to fructose 6-phosphate (F6P) and inorganic phosphate (Fig. 1 ▸a). This step in gluconeogenesis is synergistically down-regulated by fructose 2,6-bisphosphate (F-2,6-P2) and AMP, which bind to the active site and an allosteric site of FBPase, respectively. While the cellular level of AMP seems to be constant (Xue et al., 1994 ▸), the concentration of F-2,6-P2 is controlled by the glucagon-sensitive enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. A small change in the F-2,6-P2 concentration thus has a large effect on AMP-mediated FBPase inhibition. During times of glucose demand, F-2,6-P2 levels are reduced, leading to increased activity of FBPase. An aberrant up-regulation of gluconeogenesis, especially when coupled with decreased uptake and metabolism of glucose from the blood into cells, may lead to type 2 diabetes (Visinoni et al., 2012 ▸). Inhibition of the liver isoform of FBPase (hlFBPase; the other isoform being the muscle isoform) is therefore an attractive avenue for disease treatment.
While a model for the T state of hlFBPase is sufficient for structure-based drug-design purposes, the conformational picture of human liver FBPase has hitherto been incomplete. The structure of hlFBPase in the R state fills this gap. It shows that the R state of hlFBPase is similar to the R states of the rabbit, porcine and human muscle FBPases and that the human liver isoform engages in conformational changes similar in magnitude to those of porcine FBPase. The hlFBPase structure exhibits a number of interesting properties, including a metal ion in a comparatively rare trigonal bipyramidal coordination bound via a water molecule to a sulfate ion that mimics the leaving inorganic phosphate after hydrolysis of the substrate. From a crystallographic point of view, the hlFBPase structure is interesting for its peculiar arrangement of NCS elements, which emulate I-centred symmetry while the true symmetry is primitive with a c axis that is three times longer. A search in the PDB for structures with more than two molecules per asymmetric unit in the four space groups P4122, P4322, P41212 and P43212 that exhibit pseudo-translation returned 86 instances with a vector >20% of the Patterson origin peak. Of these, 33 emulate I-centring with a peak at (1/2, 1/2, w), but only three structures, PDB entries 4gqc, 2g6z and 3gfb, have w at rational fractions of the c axis (w = 1/2, w = 1/6 and w = 1/4, respectively). In none of these cases is the rNCS axis at a position to emulate crystallographic symmetry, so the case of hlFBPase seems unique at present. However, given the high prevalence of pseudo-symmetry in macromolecular crystal structures of ∼8% (Zwart, Grosse-Kunstleve et al., 2008 ▸), similar cases are to be expected.