Structural Basis for Catalytic Activity and Enzyme Polymerization of Phospholipid Hydroperoxide Glutathione Peroxidase-4 (GPx4)^,,

Abstract

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a moonlighting selenoprotein, which has been implicated in anti-oxidative defense, sperm development, and cerebral embryogenesis. Among GPx-isoforms, GPx4 is unique because of its capability to reduce complex lipid hydroperoxides and its tendency toward polymerization, but the structural basis for these properties remained unclear. To address this, we solved the crystal structure of the catalytically active U46C mutant of human GPx4 to 1.55 A resolution. X-ray data indicated a monomeric protein consisting of four alpha-helices and seven beta-strands. GPx4 lacks a surface exposed loop domain, which appears to limit the accessibility of the active site of other GPx-isoforms, and these data may explain the broad substrate specificity of GPx4. The catalytic triad (C46, Q81, and W136) is localized at a flat impression of the protein surface extending into a surface exposed patch of basic amino acids (K48, K135, and R152) that also contains polar T139. Multiple mutations of the catalytic triad indicated its functional importance. Like the wild-type enzyme, the U46C mutant exhibits a strong tendency toward protein polymerization, which was prevented by reductants. Site-directed mutagenesis suggested involvement of the catalytic C46 and surface exposed C10 and C66 in polymer formation. In GPx4 crystals, these residues contact adjacent protein monomers.

Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis

“…Ideally, a cocrystal structure of RSL3-bound GPX4 would be solved to obtain structural information on the binding mechanism and to initiate a search for drug-like GPX4 inhibitors. The apo-GPX4 structure is available (Protein Data Bank ID code 2OBI) (20). However, no cocrystal structure containing GPX4 is available, implying technical difficulties in achieving this goal, consistent with our experience.…”

“…The GPX4 U46C protein contains Cys in place of Sec in the active site, which allows for efficient bacterial protein production; GPX4 U46C protein contains reduced peroxidase activity compared with WT GPX4 (20). Despite the lack of binding of (1S, 3R)-RSL3-Fcn to U46C GPX4 in the pull-down assay, we suspected that an in vitro reaction with greater protein abundance and sensitivity might allow for detection of the less efficient covalent interaction between these two species.…”

“…Although Escherichia coli does have a native selenoprotein, formate dehydrogenase (51), the selenocysteine incorporation mechanism is sufficiently different to impose a strict species barrier in direct heterologous synthesis of recombinant selenoproteins in E.coli (52). Indeed, the apo-GPX4 crystal structure available is the Sec-to-Cys mutant GPX4 protein, not WT protein (20). Engineering of a bacterial expression system has reportedly produced rat thioredoxin reductase, another mammalian selenoprotein (53).…”

Molecular biology of glutathione peroxidase 4: from genomic structure to developmental expression and neural function

“…During the past 12 months, five crystal structures for seleno-GPx isoforms and selenium-free enzymes have been deposited in the PDB database (http://www.rcsb.org/pdb/home/home.do) including catalytically inactive U-to-G mutants of human GPx1 (PDB entry 2F8A) and GPx4 (PDB entry 2GS3), a U-to-C mutant of GPx2 (PDB entry 2HE3) and GPx5 (PDB entry 2I3Y). Recently we have crystallized the catalytically active U46C mutant of GPx4 at resolution of 1.55 Å (PDB entry 2OBI; Scheerer et al, 2007). The two X-ray data sets for GPx4 (2GS3, 2OBI) confirm that the enzyme is a monomeric protein, in contrast to GPx1, GPx2 and GPx5.…”

“…Although structural alignments of GPx4 and GPx1, GPx2 and GPx5 revealed high similarities, GPx4 shows two peculiarities. GPx4 lacks two surface-exposed loop structures (loops 1 and 2) that appear to increase the accessibility of the active site, suggesting a molecular explanation for broad substrate specificity in contrast to other GPx enzymes (Scheerer et al, 2007). Second, surface loop 2 is lacking, which can be considered the structural basis for the monomeric character of GPx4.…”