Crystal Structure of the Hypoxia-inducible Form of 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3)

Abstract

The hypoxia-inducible form of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) plays a crucial role in the progression of cancerous cells by enabling their glycolytic pathways even under severe hypoxic conditions. To understand its structural architecture and to provide a molecular scaffold for the design of new cancer therapeutics, the crystal structure of the human form was determined. The structure at 2.1 Å resolution shows that the overall folding and functional dimerization are very similar to those of the liver (PFKFB1) and testis (PFKFB4) forms, as expected from sequence homology. However, in this structure, the N-terminal regulatory domain is revealed for the first time among the PFKFB isoforms. With a ␤-hairpin structure, the N terminus interacts with the 2-Pase domain to secure binding of fructose-6-phosphate to the active pocket, slowing down the release of fructose-6-phosphate from the phosphoenzyme intermediate product complex. The C-terminal regulatory domain is mostly disordered, leaving the active pocket of the fructose-2,6-bisphosphatase domain wide open. The active pocket of the 6-phosphofructo-2-kinase domain has a more rigid conformation, allowing independent bindings of substrates, fructose-6-phosphate and ATP, with higher affinities than other isoforms. Intriguingly, the structure shows an EDTA molecule bound to the fructose-6-phosphate site of the 6-phosphofructo-2-kinase active pocket despite its unfavorable liganding concentration, suggesting a high affinity. EDTA is not removable from the site with fructose-6-P alone but is with both ATP and fructose-6-P or with fructose-2,6-bisphosphate. This finding suggests that a molecule in which EDTA is covalently linked to ADP is a good starting molecule for the development of new cancer-therapeutic molecules.

Nuclear Targeting of 6-Phosphofructo-2-kinase (PFKFB3) Increases Proliferation via Cyclin-dependent Kinases

“…In the recently solved crystal structure of PFKFB3, the carboxyl terminus appears flexible and disordered (28,42), suggesting a discrete function. We have found that the dominantly expressed splice variant of PFKFB3 localizes to the nucleus and that the carboxyl terminus is required for this localization.…”

PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3)

“…Residues 4-15 of the kinase domain form a β-hairpin structure and the rest is used as an arm connecting the hairpin to the bisphosphatase domain. Additionally, the contacting area of the bisphosphatase domain is functionally very sensitive due to the residues critical for binding of both product and substrate are located very close (Kim et al, 2006). The low bisphosphatase activity of PFKFB3, which is lower than that of other isoforms by an order of magnitude, is due to the presence of a serine at residue 302 instead of an arginine as conserved in the other isoforms.…”

“…The conformations of the substrate loops in the kinase domain are different from those of other isoforms (Hasemann et al, 1996;Lee et al, 2003), giving a structural explanation for the higher kinase activity. Moreover, the N-terminus binds to the bisphosphatase domain to produce a conformational change in the active pocket to enhance inhibitory binding of product (Kim et al, 2006). Residues 4-15 of the kinase domain form a β-hairpin structure and the rest is used as an arm connecting the hairpin to the bisphosphatase domain.…”

“…The low bisphosphatase activity of PFKFB3, which is lower than that of other isoforms by an order of magnitude, is due to the presence of a serine at residue 302 instead of an arginine as conserved in the other isoforms. This residue is said to interact with the 2-phosphate and further stabilizes the transitions state (Cavalier et al, 2012;Kim et al, 2006).…”

“…This isoform, which has a native activity ratio of roughly 700-fold kinase-to-phosphatase activity, dramatically increases upon phosphorylation of Ser461 by protein kinase A (PKA), AMP-dependent protein kinase (AMPK) or other kinases. The low bisphosphatase activity of PFKFB3, which is lower than that of other isoforms by an order of magnitude, is solely due to the presence of a serine at residue 302 instead of an arginine as conserved in the other isoforms (Cavalier et al, 2012;Kim et al, 2006). PFKFB3 gene was cloned from a fetal brain library (Manzano et al, 1998;Ventura et al, 1995), human placenta (Sakai et al, 1996) and breast cancer cells (Hamilton et al, 1997).…”