Influence of the presence of the heme cofactor on the JK-loop structure in indoleamine 2,3-dioxygenase 1

Abstract

Indoleamine 2,3-dioxygenase 1 has sparked interest as an immunotherapeutic target in cancer research. Its structure includes a loop, named the JK-loop, that controls the orientation of the substrate or inhibitor within the active site. However, little has been reported about the crystal structure of this loop. In the present work, the conformation of the JK-loop is determined for the first time in the presence of the heme cofactor in the active site through X-ray diffraction experiments (2.44 Å resolution). Molecular-dynamics trajectories were also obtained to provide dynamic information about the loop according to the presence of cofactor. This new structural and dynamic information highlights the importance of the JK-loop in confining the labile heme cofactor to the active site.

Temporary Intermediates of L-Trp Along the Reaction Pathway of Human Indoleamine 2,3-Dioxygenase 1 and Identification of an Exo Site

“…The production and purification of the enzyme was performed according to the protocol already described in the literature. 39 After purification, the protein was exchanged into crystallization buffer consisting of 5 mM HEPES/NaOH pH 7.6, 200 mM NaCl, 5 mM DTT, and concentrated at a concentration of 16 mg/mL using Amicon filters with a 10 kDa molecular-weight cut-off membrane. The hIDO1 protein is stored at −80°C.…”

“…The solvatation, the optimization, the equilibration, and the production stage of the models were performed as previously described in the literature. 39…”

“…Recently, due to a change in the crystal packing and despite its agitation, the complete JK-loop was refined for the first time in the absence of ligand. 39 The structure shows an intermediate form of hIDO1, and the heme cofactor is located inside the enzyme, involving an electrostatic interaction with the residue K377. Since the information on the conformation of the loop is recent, the folding mechanism remains still unclear.…”

One Key and Multiple Locks: Substrate Binding in Structures of Tryptophan Dioxygenases and Hydroxylases

“…The access to this site is governed by a large flexible loop (JK-loop, residues 360-382), whose distinct closed, intermediate and open conformations regulate substrate recognition and binding of inhibitors and heme cofactor to the catalytic site. [47,[55][56][57][58] A channel for the shuttling of water and oxygen molecules from/to the catalytic site is shaped by αhelices E and F, extending into the distal and proximal pockets above and below the plane of the heme group, respectively (Figure 2). [46] Molecular recognition of l-Trp to IDO1 was investigated using a wide array of studies including biochemical, spectroscopic, biophysical, crystallographic and computational approaches.…”

“…A flexible loop (DE‐loop, residues 260–268) connects the large catalytic domain and the small signaling domain, shaping the catalytic pocket above the sixth coordination site of the heme‐iron. The access to this site is governed by a large flexible loop (JK‐loop, residues 360–382), whose distinct closed, intermediate and open conformations regulate substrate recognition and binding of inhibitors and heme cofactor to the catalytic site [47,55–58] . A channel for the shuttling of water and oxygen molecules from/to the catalytic site is shaped by α‐helices E and F, extending into the distal and proximal pockets above and below the plane of the heme group, respectively (Figure 2).…”