Mycobacterium tuberculosis CYP130: crystal structure, biophysical characterization, and interactions with antifungal azole drugs

Abstract

CYP130 is one of the 20 Mycobacterium tuberculosis cytochrome P450 enzymes, only two of which, CYP51 and CYP121, have so far been studied as individually expressed proteins. Here we characterize a third heterologously expressed M. tuberculosis cytochrome P450, CYP130, by UV-visible spectroscopy, isothermal titration calorimetry, and x-ray crystallography, including determination of the crystal structures of ligand-free and econazole-bound CYP130 at a resolution of 1.46 and 3.0A(,) respectively. Ligand-free CYP130 crystallizes in an "open" conformation as a monomer, whereas the econazole-bound form crystallizes in a "closed" conformation as a dimer. Conformational changes enabling the "open-closed" transition involve repositioning of the BC-loop and the F and G helices that envelop the inhibitor in the binding site and reshape the protein surface. Crystal structure analysis shows that the portion of the BC-loop relocates as much as 18A between the open and closed conformations. Binding of econazole to CYP130 involves a conformational change and is mediated by both a set of hydrophobic interactions with amino acid residues in the active site and coordination of the heme iron. CYP130 also binds miconazole with virtually the same binding affinity as econazole and clotrimazole and ketoconazole with somewhat lower affinities, which makes it a plausible target for this class of therapeutic drugs. Overall, binding of the azole inhibitors is a sequential two-step, entropy-driven endothermic process. Binding of econazole and clotrimazole exhibits positive cooperativity that may reflect a propensity of CYP130 to associate into a dimeric structure.

Fig. 1. Binding of antifungal azole drugs to CYP130

The concentration dependence of econazole (A), clotrimazole (B), miconazole (C) and ketoconazole (D) binding deduced from the difference absorption changes obtained from the titration of CYP130 (2.5 µM) with increasing concentrations of the inhibitor. The structure of the inhibitor is shown in each panel.

Fig. 2. Stoichiometry of CYP130-inhibitor binding

The bell-shaped Job plots at a total protein and inhibitor concentration of 15 µM display a maximum close to a mole fraction of 0.55, the value that corresponds to a 1:1 ratio for the binding to CYP130 of econazole (open circles) and miconazole (closed circles).

Fig. 3. Stereo views of ligand-free and econazole-bound CYP130

A, the ligand-free CYP130 is in a ribbon representation colored according to the secondary structure elements: helices are in blue, β-sheets in green, and loops and turns in grey. The BC-loop region (highlighted in pink) is well-structured having two short helices αB' and αB". A hydrogen-bonding network of water molecules linking the stability of the distal water ligand to the I-helix N-terminus is marked by the red spheres. In orange are shown water molecules having contacts with the bulk solvent. Residue T239 in the N-terminal portion of the I-helix is shown in sticks. B, superimposition of the ligand-free (gray) and econazole-bound (lime green) forms. The BC-loop region containing residues 80–91 which relocates up to 18 Å to a position where they interact with the econazole is shown in pink in the ligand-free and in yellow-green in the econazole-bound forms. The F and G helices are shown as pink cylinders in the ligand-free and yellow-green cylinders in econazole bound forms. The G helix is on top. Econazole is in cyan and heme is in yellow. Images were generated using VMD software (51) unless specified otherwise.

Fig. 4. CYP130 active site

A, major conformational differences between the ligand-free (gray) and econazole–bound (green) states. The BC-region is in pink, heme is in yellow and econazole is in cyan. Gaps in the protein chain between the F and G helices due to the missing electron density are marked with the open circles. Relocation distances for selected structural elements are given in Angstroms. B, H-bonding network. The fragment of the ligand-free crystal structure showing the water molecules (red spheres) that stabilize the distal water in CYP130. Water molecules having contacts with the bulk solvent are colored in orange. Distances between oxygen atom centers are in Angstroms. T239 is shown in sticks. The iron axial water ligand is labeled with a capital L.

Fig. 5. Dimerization interface

The dimerization interfaces of (A) CYP130 (2000 ų) and (B) CYP154C1 (610 ų) formed largely via interactions between the G helices in anti-parallel orientations, overlapping N-termini of the I helices, and multiple contacts in the BC-loop regions are shown. The monomers are colored in green and blue, heme is in yellow and econazole in (A) is in cyan.

Fig. 6. Glutaraldehyde cross-linking

Analysis of the CYP130 cross-linked products by native- (A, C) and SDS- (B) gel electrophoresis. A, cross-linking performed at a protein concentration of 20 µM is shown. Two soluble P450 enzymes, CYP51 from Mtb and PikC from S. venezuelae, were used as controls. B, the molecular weight of the cross-linked CYP130 products was confirmed by the SDS-gel electrophoresis. C, effect of ionic strength on stability of the CYP130 aggregates is shown. As the KCl concentration increases from 0 to 300 mM, the dimer product stabilized by glutaraldehyde cross-linking persists but the formation of tetramers and higher oligomers is suppressed.

Fig. 7. Econazole binding in the active site

A, stereo view of econazole (yellow-green) bound in the active site of CYP130 is shown. Cl atoms are colored in green, N atoms in blue and O atoms in red. Amino acid residues within 4 Å of econazole are labeled in black. Fragments of the 2F_o−F_c electron density composite omit map contoured at 1.0 σ are in blue. To avoid excessive cluttering, heme was excluded from the map calculation and T242 was excluded from the view as projecting on top of econazole. The image was generated using the SETOR program (52). B, a kink of the I-helix introduced by the binding of econazole is shown. The ligand-free (gray) and econazole-bound (green) CYP130 structures were superimposed with an r.m.s.d. of 0.93 Ų for all the protein residues. Iron (ochre) and oxygen (red) atoms are shown as spheres. The iron axial water ligand is labeled with a capital L. The arrows show the directions of movements upon transition from the ligand-free to the econazole-bound state. C, alignment of the BC-loop fragment (85–91) (grey) in the groove formed between the mono- and double-chlorinated phenyl rings of econazole is shown. The additional chlorination site in miconazole is indicated by an arrow. The solid surface represents the van der Waals surface of econazole (volume of 330 ų) and is colored according to the underlying atoms: oxygen in red, nitrogen in blue, chlorine in green. The mesh surface shows the accessible space in the active site (600 ų). A fragment of the I-helix (237–247) is represented by the grey ribbon. The heme is in orange. This image was generated using the program CHIMERA (53).

Fig. 8. Calorimetric binding studies of azole drugs

The isothermal calorimetric enthalpy changes (upper panel) and the resulting binding isotherms (lower panel) are shown for reverse titrations of CYP130 with econazole (A) and miconazole (B). The data were best fitted to a two-step sequential binding model. The binding parameters obtained are listed in Table 4.