PMC

SAXS-restrained model of laforin CBM bound to α-cD. A, ab initio SAXS reconstructions of laf-CBM (in gray) calculated from experimental scattering values at 3.2 mg ml⁻¹. Overlaid to the SAXS envelope is a homology model of human laf-CBM. B, agreement of P(r) functions calculated from experimental SAXS data (blue) and data calculated from the laf-CBM model (red). Inset shows experimental scattering data (blue) overlaid to the scattering curve calculated from the laf-CBM model (red).

Catalytic activity of dimeric laforin toward amylopectin and OMFP. A, dephosphorylation of 100 μg of amylopectin by various point mutants of laforin and laf-DSP. Error bars are based on three independent repeats. B, Michaelis-Menten saturation kinetics of dimeric laforin for OMFP. Initial rates of OMFP hydrolysis by various laforin mutants are plotted against OMFP concentration, which ranges between 0 and 1600 μm. Kinetic parameters measured in this experiment are as follows: WT laforin (●): K_m = 520.0 ± 35.0 μm, V_max = 0.012, k_cat = 99,830 s⁻¹, k_cat/K_m = 1.90 × 10⁸ m⁻¹ s⁻¹; C169S (▿): K_m = 580.0 ± 10.0 μm, V_max = 0.01, k_cat = 88,641 s⁻¹, k_cat/K_m = 1.53 × 10⁸ m⁻¹ s⁻¹; R171A (▵): K_m = 400.0 ± 20.0 μm, V_max = 0.013, k_cat = 109,425 s⁻¹, k_cat/K_m = 2.70 × 10⁸ m⁻¹ s⁻¹; D235A (■): K_m = 60.0 ± 2.0 μm, V_max = 0.006, k_cat = 11,144 s⁻¹, k_cat/K_m = 1.85 × 10⁸ m⁻¹ s⁻¹. Mutants D197A, R272A, C266S, and laf-DSP did not show detectable activity.

Crystal structure of laforin DSP. A, schematic diagram of laforin domain organization. B, thermal stability of apo-laforin (red line) and in the presence of α-cD (blue line) or maltose (green line). The T_{m, app} is 60 °C for apo-laforin, 62 °C in the presence of 5 mm α-cD, and 68 °C in the presence of 10 mm maltose. C, ribbon representation of laf-DSP with catalytic triad, second triad, and active site sulfate ion shown as sticks. D, top views of the laf-DSP (residues 150–331), plant Sex4-DSP (residues 96–249) (), and human PTEN-DSP (residues 23–185) () highlighting critical loops surrounding the active site. E, surface representation of laf-DSP is as follows: catalytic and second triads are circled with dashed lines. Cys-266/Cys-169, Arg-272/Arg-171, and Asp-235/Asp-197 are colored in green, blue, and red, respectively.

Quaternary structure of human laforin. A, sedimentation velocity profiles of human laforin at 20 °C. Top panel, raw absorbance at 280 nm plotted as a function of the radial position. Data at intervals of 60 min are shown as open circles for sedimentation at 35,000 rpm. Bottom panel, fitted distribution of s*. B, ab initio SAXS reconstruction of dimeric laforin (light gray) displayed in side and top views overlaid to a pseudo-atomic model of the enzyme obtained by docking ribbon models of laf-DSP and laf-CBM inside the SAXS envelope. C, agreement of P(r) functions calculated from experimental SAXS data (blue) and data calculated from the pseudo-model (red) of dimeric laforin. Inset, experimental scattering data (blue) overlaid to the scattering curve calculated from the laforin model (red). D, a model of dimeric laforin (gray mesh) bound to α-cD (green): carbohydrate-binding residues projecting from laf-CBM (in yellow) and laf-DSP (cyan) (shown only for one active site), as well as Cys-266/Cys-169, and sulfate ions are shown as thick sticks. Underlined are residues mutated in LD ().