GAPDH heme delivery to Indoleamine 2,3-dioxygenase 1 involves their complex formation and complementary charge pairing at the protein-protein interface

TL;DR

This study shows how the protein GAPDH directly transfers heme to IDO1 through a specific charge interaction, helping IDO1 become functional in immune responses and cancer.

Contribution

The study reveals a direct heme transfer mechanism between GAPDH and IDO1 via a novel Lys-Asp charge pairing at their interface.

Findings

GAPDH transfers heme to IDO1 directly through a previously unknown protein interface.

A Lys-Asp charge pairing is essential for the heme transfer between GAPDH and IDO1.

The interaction was confirmed in purified proteins and in human cells.

Abstract

In eukaryotes, the last steps of heme biosynthesis occur in mitochondria and so heme must be transported to reach many heme-dependent proteins that mature and function outside this organelle. Although the enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) has emerged as a key intracellular heme chaperone, how it performs heme deliveries to its numerous clients is poorly understood. It is unknown if handoffs of the GAPDH-bound heme require that it make direct contact with its clients or instead involve GAPDH passing its heme to middlemen proteins to execute the final heme transfers. To address this question, we studied GAPDH heme transfer to the client protein indoleamine 2,3-dioxygenase 1 (IDO1), whose enzyme activity is heme-dependent and regulates mammalian immune responses and cancer progression. A chemical crosslinking-mass spectrometry approach identified two Lys residues that formed an inter-protein crosslink across a previously uncharacterized GAPDH-IDO1 interface. This guided our building a model of the GAPDH-IDO1 complex so we could interrogate by point mutagenesis the role of the GAPDH-IDO1 contact in enabling delivery of GAPDH heme to IDO1. We characterized behaviors of the GAPDH and IDO1 variants in their purified form and when they were expressed in the HEK293T human cell line. This revealed GAPDH heme transfer to IDO1 in cells requires that they make a direct contact which relies on a specific Lys-Asp charge pairing interaction forming across the complex interface. These findings illuminate a key step in the maturation of functional IDO1 and improve our understanding of how GAPDH may perform its heme trafficking function in mammals.

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