Thermodynamic characterization of the binding interaction between the histone demethylase LSD1/KDM1 and CoREST

Abstract

Flavin-dependent histone demethylases catalyze the posttranslational oxidative demethylation of mono- and dimethylated lysine residues, producing formaldehyde and hydrogen peroxide in addition to the corresponding demethylated protein. In vivo, histone demethylase LSD1 (KDM1; BCH110) is a component of the multiprotein complex that includes histone deacetylases (HDAC 1 and 2) and the scaffolding protein CoREST. Although little is known about the affinities of or the structural basis for the interaction between CoREST and HDACs, the structure of CoREST(286-482) bound to an α-helical coiled-coil tower domain within LSD1 has recently been reported. Given the significance of CoREST in directing demethylation to specific nucleosomal substrates, insight into the molecular basis of the interaction between CoREST and LSD1 may suggest a new means of inhibiting LSD1 activity by misdirecting the enzyme away from nucleosomal substrates. Toward this end, isothermal titration calorimetry studies were conducted to determine the affinity and thermodynamic parameters characterizing the binding interaction between LSD1 and CoREST(286-482). The proteins tightly interact in a 1:1 stoichiometry with a dissociation constant (K(d)) of 15.9 ± 2.07 nM, and their binding interaction is characterized by a favorable enthalpic contribution near room temperature with a smaller entropic penalty at pH 7.4. Additionally, one proton is transferred from the buffer to the heterodimeric complex at pH 7.4. From the temperature dependence of the enthalpy change of interaction, a constant-pressure heat capacity change (ΔC(p)) of the interaction was determined to be -0.80 ± 0.01 kcal mol(-1) K(-1). Notably, structure-driven truncation of CoREST revealed that the central binding determinant lies within the segment of residues 293-380, also known as the CoREST "linker" region, which is a central isolated helix that interacts with the LSD1 coiled-coil tower domain to create a triple-helical bundle. Thermodynamic parameters obtained from the binding between LSD1 and the linker region of CoREST are similar to those obtained from the interaction between LSD1 and CoREST(286-482). These results provide a framework for understanding the molecular basis of protein-protein interactions that govern nucleosomal demethylation.

Figure 1

Crystal structures of (a) LSD1 in complex with CoREST^286–482, (b) the functional region of CoREST (CoREST^286–482) including the linker region^293–380 and the SANT2 domain^381–450, (c) the linker region^293–380 of CoREST, and (d) their domain structures. The three dimensional structural data file was obtained from Protein Data Bank (PDB entry code 2IW5, (4)). The catalytic domain of LSD1, amine oxidase domain, is shown in green, the SWIRM domain of LSD1 is in red, the tower region of LSD1 is in blue, FAD indicated by a black arrow is in yellow, the SANT2 domain^381–450 of CoREST is in purple, and the linker region^293–380 of CoREST is in orange. All structural figures were generated using PyMoL.

Figure 2

15 % SDS-PAGE gels of (a) the purified LSD1 and (b) the purified CoREST^286–482. 4–20 % gradient SDS-PAGE gels of (c) the linker region^293–380 and (d) the SANT2 domain^381–450 of CoREST. Kaleidoscope prestained standards were used (BioRad).

Figure 3

Initial velocity curves of the catalytic activity of LSD1 using the first 21 amino acid residues of H3 with dimethylated K4 as a substrate. The assay was performed in 50 mM Tris buffer, pH 7.85 at 25 °C, and the final concentration of both proteins was 0.5 µM. Data were fitted to the Michaelis-Menten. The black curve represents the activity of LSD1, whereas the green curve represents the activity of LSD1 incubated with CoREST^286–482.

Figure 4

(a) A representative calorimetric titration of 3 µM of LSD1 with 30 µM of CoREST^286–482 in 50 mM sodium phosphate and 1 mM DTT, pH 7.4. Thermogram (top panel) represents the heat released after each injection of CoREST^286–482 into LSD1 solution. Binding isotherm (bottom panel) shows the integrated peak area plotted as a function of molar ratio (CoREST^286–482/LSD1). The following parameters were observed: n = 1.14 ± 0.00, ΔH = −21.3 ± 0.19 kcal/mol, K_a = 6.29e7 ± 8.18e6 M⁻¹. The red line indicates the best fit of the ITC data to a one site binding model. (b) Surface plasmon resonance measurement curves obtained during and after injection of LSD1 on chip surfaces with immobilized CoREST^286–482. Sensograms show the association of LSD1 to and dissociation from CoREST^286–482.

Figure 5

(a) Plot of the calorimetric enthalpy (ΔH_cal) obtained from binding of LSD1 to CoREST^286–482 as a function of the buffer ionization enthalpy (ΔH_ion) at pH 7.4. The slope of the plot indicates the number of protons transferred between the complex and the buffer upon binding, and the y-intercept indicates the intrinsic ΔH without a protonation effect. At pH 7.4, 0.83 ± 0.09 protons are absorbed to the complex from the buffer. (b) Temperature dependence of the binding enthalpy change for the interaction between LSD1 and CoREST^286–482. The slope of the plot yields the binding heat capacity change (ΔC_p), which is equal to −0.80 ± 0.01 kcal/mol·K.

Figure 6

(a) Surface representation of the binding interface between the LSD1 tower region^418–522 and the linker region^293–380 of CoREST. The blue and orange surfaces represent the LSD1 tower domain^418–522 and the linker region^293–380 of CoREST, respectively. Based on information from the three dimensional structure of the complex, pale blue and orange surfaces indicate the surface of each protein involved in key binding interactions. (b) Nonpolar residues at the binding interface. The LSD1 tower domain^418–522 and the linker region^293–380 of CoREST are represented in a ribbon diagram, and nonpolar residues involved in the binding interface are represented by a surface diagram. (c) Stereo view of packing in the trimeric coiled coil interaction between the LSD1 tower region^418–522 (blue) and the linker region^293–380 of CoREST (orange). The side chains at a and d position of a heptad repeat model are represented as sticks. (d) Stereo top view of the binding interface. All residues involved in the binding interaction are represented by lines.

Figure 7

Detailed molecular contacts between the LSD1 tower region^418–522 (blue ribbon) and the linker region^293–380 of CoREST (orange ribbon). Residues are labeled in black for LSD1 and in red for CoREST. Hydrogen bonds are represented by black dashed lines. (a) Interface part I (LSD1 residues 418–432, 514–522; CoREST residues 308–344) (b) Interface part II (LSD1 residues 433–450, 496–513; CoREST residues 345–365) (c) Interface part III (LSD1 residues 451–495; CoREST residues 366–380) (d) Stereo view of the interface part II. Residues involved in hydrogen bonds in the interface part II are shown.

Scheme 1

General mechanism of lysine demethylation by LSD1