Large-scale conformational changes and redistribution of surface negative charge upon sugar binding dictate the fidelity of phosphorylation in Vibrio cholerae fructokinase

Abstract

Fructokinase (FRK) catalyzes the first step of fructose metabolism i.e., D-fructose to D-fructose-6-phosphate (F6P), however, the mechanistic insights of this reaction are elusive yet. Here we demonstrate that the putative Vibrio cholerae fructokinase (VcFRK) exhibit strong fructose-6-kinase activity allosterically modulated by K⁺/Cs⁺. We have determined the crystal structures of apo-VcFRK and its complex with fructose, fructose-ADP-Ca²⁺, fructose-ADP-Ca²⁺-BeF₃^-. Collectively, we propose the catalytic mechanism and allosteric activation of VcFRK in atomistic details explaining why K⁺/Cs⁺ are better activator than Na⁺. Structural results suggest that apo VcFRK allows entry of fructose in the active site, sequester it through several conserved H-bonds and attains a closed form through large scale conformational changes. A double mutant (H108C/T261C-VcFRK), that arrests the closed form but unable to reopen for F6P release, is catalytically impotent highlighting the essentiality of this conformational change. Negative charge accumulation around ATP upon fructose binding, is presumed to redirect the γ-phosphate towards fructose for efficient phosphotransfer. Reduced phosphotransfer rate of the mutants E205Q and E110Q supports this view. Atomic resolution structure of VcFRK-fructose-ADP-Ca²⁺-BeF₃^-, reported first time for any sugar kinase, suggests that BeF₃^- moiety alongwith R176, Ca²⁺ and 'anion hole' limit the conformational space for γ-phosphate favoring in-line phospho-transfer.

Figure 1

Structure of VcFRK and its dimerization. (a) Cartoon representation of VcFRK monomer, ‘Lid’ domains (yellow), important loops involved in phosphorylation, helices (cadbury) and central β-sheet (sand) are labeled. (b) Superposition of apo (cadbury shades) and fructose-ADP bound (green shades) dimers of VcFRK in cartoon representation. (c) Involvement of lid domains in dimerization of apo and (d) fructose+ADP (in sticks) bound VcFRK. Shift of large lid loop upon sugar binding is evident. (e) Sequence alignment of VcFRK with other fructokinases overlaid with secondary structures (top). Important loops are indicated (bottom) with same color scheme as (a). Conserved residues involved in sugar binding (green), anion hole formation (cyan), divalent cation binding (violet) and negatively charged patch formation near ATP (red) are indicated.

Figure 2

Fructose binding and specificity of VcFRK. (a) Movement of the large lid loop upon fructose (green sticks) binding (apo:cadbury, fructose bound:yellow) is shown in red arrow. (b) VcFRK residues involved in sequestering fructose through H-bonds. Position of these residues in apo form is also indicated (Cadbury). (c) Polar interactions that tether the large lid loop with the αβα sandwich domain. Important residues and water molecules bridging large lid loop and αβα domain are labeled. T261 and H108 are indicated. (d) F_o − F_c omit electron density map, contoured at 4σ (green mesh), around fructose(grey sticks). (e) Representative ITC titration curve of VcFRK with fructose (FRU), Fructose-6-phosphate (F6P), Glucose (GLU) and Ribose (RIB).

Figure 3

Enzymatic activity of VcFRK. Saturation curve was fit with the Michaelis–Menten equation to obtain estimates of K_m. Error bars correspond to the standard deviation of three independent measurements. Variation of enzymatic activity of VcFRK with (a) fructose, (b) ATP (WT and VcFRK-DM in various forms as indicated), (c) monovalent cations K⁺ and Cs⁺ and (d) divalent cations Mg²⁺ and Ca²⁺, (e) Crystal structure of VcFRK showing a disulphide bond between C108 and C261; fructose and part of ADP is also shown. 2F_o − F_c electron density map (blue mesh; 1σ) around the disulphide bond is shown.

Figure 4

Mono/di valent cations in activation and catalysis of VcFRK. (a) 2F_o − F_c electron density map (slate mesh; 1.5σ) around K⁺ (pink ball) binding residues overlaid with F_o-F_c map (green mesh; 15σ) confirming the position of K⁺. (b) Coordination of K⁺ (blue dash) with large ATP loop residues and metal binding loop. (c) Na⁺ ion (violet ball) coordination (blue dash) with metal binding loop and W1 and W2 (red balls). W1 connects large ATP loop with Na⁺ (red dash). (d) Comparison of activated (yellow) and non-activated VcFRK (cyan); K⁺ (violet sphere), ADP, Ca²⁺ and fructose are shown for clarity. Main-chain of ²⁶¹TTGAGD²⁶⁶ is shown as sticks, flipping of T262 carbonyl oxygen towards metal is indicated (green arrow). Point of deviation of large ATP loop in apo structure (red arrow) and its occlusion of ATP binding site is evident. (e) Divalent metal binding seen in fructose-ADP-Ca²⁺ (grey) and fructose-ADP-BeF₃⁻-Ca²⁺ bound VcFRK structure. Residues involved in metal binding (through water), ADP, fructose and BeF₃⁻ are labeled.

Figure 5

Change in surface grooves and electrostatic charge upon activation and sugar/ADP-BeF₃^- binding of VcFRK. (a) Monovalent cation bound VcFRK where binding sites for fructose and ATP are indicated. (b) Superposition of monovalent cation bound (in surface) and apo (in yellow ribbon) VcFRK showing large ATP loop of apo VcFRK encroaches ATP binding site (black arrow). (c) Fructose bound VcFRK where all but O6 atom of fructose is buried. Negative charge redistribution around sugar is evident when compared with (a). (d) Fructose-ADP-BeF₃^- bound structure where β phosphate of ADP, BeF₃^- and divalent metal are shielded from solvent.

Figure 6

Active site design to bind fructose in a preferred orientation. (a) O6′ atom of fructose is poised to attack ATP in VcFRK, crucial residues stabilizing this orientation are labeled. (b) O1′ atom of fructose is orientated to attack ATP in KHK (PDB code 2HW1) residues crucial to bind fructose in this preferred orientation are labeled and (c) phosphorylation of fructose at O6′ atom in ROK-FRK (PDB code 3LM9). Residues crucial to bind in this orientation and 2His-2Cys-Zn cluster close to the sugar binding site are shown. D266, D258 and D103 that deprotonate the pertinent sugar hydroxyl group in VcFRK, KHK and ROK-FRK respectively occupy identical position. Stabilization of O2′ in binding sugar in a preferred orientation is evident; site of phosphorylation is shown in black arrow.

Figure 7

Redirection of phosphate group by negative charge in other sugar kinases. (a) Adenosine kinase (PDB code 1LII) and (b) tagatose 6-phosphate kinase (PDB code 2JG1) where residues from the same monomer engaged to form the negative patch. In dimeric (c) KDGK (PDB code 1V1A) and (d) ribokinase (PDB code 4XDA) β-clasp mode of dimerization where negative charge(s) at the lid loop reinforce with the negative charge around ATP of the other protomer.