Quadruple space-group ambiguity owing to rotational and translational noncrystallographic symmetry in human liver fructose-1,6-bisphosphatase

Abstract

Fructose-1,6-bisphosphatase (FBPase) is a key regulator of gluconeogenesis and a potential drug target for type 2 diabetes. FBPase is a homotetramer of 222 symmetry with a major and a minor dimer interface. The dimers connected via the minor interface can rotate with respect to each other, leading to the inactive T-state and active R-state conformations of FBPase. Here, the first crystal structure of human liver FBPase in the R-state conformation is presented, determined at a resolution of 2.2 Å in a tetragonal setting that exhibits an unusual arrangement of noncrystallographic symmetry (NCS) elements. SelfPatterson function analysis and various intensity statistics revealed the presence of pseudo-translation and the absence of twinning. The space group is P4 1 2 1 2, but structure determination was also possible in space groups P4 3 2 1 2, P4 1 22 and P4 3 22. All solutions have the same arrangement of three C 2 -symmetric dimers spaced by 1/3 along an NCS axis parallel to the c axis located at (1/4, 1/4, z), which is therefore invisible in a self-rotation function analysis. The solutions in the four space groups are related to one another and emulate a body-centred lattice. If all NCS elements were crystallographic, the space group would be I4 1 22 with a c axis three times shorter and a single FBPase subunit in the asymmetric unit. I4 1 22 is a minimal, non-isomorphic supergroup of the four primitive tetragonal space groups, explaining the space-group ambiguity for this crystal.

Triple-Stage Mass Spectrometry Unravels the Heterogeneity of an Endogenous Protein Complex

“…The profile of the deconvoluted spectra fitted the observed peaks very closely, as shown in Figure S3. The analysis indicated that a fraction of the complex is associated with magnesium ions, in accordance with the tendency of this complex to bind three divalent metal ions per subunit. , The stoichiometry of the FBP1 complex remained stable despite the exposure of yeast cells to heat shock or to the glucose-containing medium, suggesting that stoichiometry is not used to regulate the complex. However, a distinct pattern of modifications could be identified under each growth condition.…”

“…S3. The analysis indicated that a fraction of the complex is associated with magnesium ions, in accordance with the tendency of this complex to bind three divalent metal ions per subunit30–31. The stoichiometry of the FBP1 complex remained stable despite the exposure of yeast cells to heat shock or to the glucose-containing medium, suggesting that stoichiometry is not used to regulate the complex.…”

FBP1 is a nonenzymatic safety valve that curtails AKT activation to prevent insulin hyperresponsiveness

“…The catalytically inactive FBP1 E98A variant binds ALDOB and PP2A-C and inhibits AKT activation as effectively as the native protein, but human FBP1 missense mutants that are not severely destabilized, L329P, R158W, G164S and N213K, are devoid of AKT inhibitory activity. One of the residues affected by these mutations, L329, is not a part of the catalytic pocket (Huang et al, 2020; Ruf et al, 2016) and is associated with only a modest decrease in FBPase activity, underscoring the disconnect between loss of GNG and insulin hyperresponsiveness. Mass spectrometry, IP, gel filtration and PLA demonstrated that FBP1 is the lynchpin responsible for formation of the FBP1:AKT:PP2A-C:ALDOB regulatory complex that is destabilized by insulin to facilitate AKT activation.…”

Characterizing pathological imperfections in macromolecular crystals: lattice disorders and modulations

“…Fructose-1,6-bisphosphatase (FBPase) with bound adenosine monophosphate (AMP) in the T (inactive) state resulted in a crystal with four possible space groups P4 1 22, P4 3 22, P4 1 2 1 2 and P4 3 2 1 2 [89]. For this crystal, tNCS related elements resulted in a unit cell that was 3x longer in the c dimension than what may have been initially estimated.…”