Redox-dependent gating of VDAC by mitoNEET.

MitoNEET is an outer mitochondrial membrane protein essential for sensing and regulation of iron and reactive oxygen species (ROS) homeostasis. It is a key player in multiple human maladies including diabetes, cancer, neurodegeneration, and Parkinson's diseases. In healthy cells, mitoNEET receives its clusters from the mitochondrion and transfers them to acceptor proteins in a process that could be altered by drugs or during illness. Here, we report that mitoNEET regulates the outer-mitochondrial membrane (OMM) protein voltage-dependent anion channel 1 (VDAC1). VDAC1 is a crucial player in the cross talk between the mitochondria and the cytosol. VDAC proteins function to regulate metabolites, ions, ROS, and fatty acid transport, as well as function as a "governator" sentry for the transport of metabolites and ions between the cytosol and the mitochondria. We find that the redox-sensitive [2Fe-2S] cluster protein mitoNEET gates VDAC1 when mitoNEET is oxidized. Addition of the VDAC inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) prevents both mitoNEET binding in vitro and mitoNEET-dependent mitochondrial iron accumulation in situ. We find that the DIDS inhibitor does not alter the redox state of MitoNEET. Taken together, our data indicate that mitoNEET regulates VDAC in a redox-dependent manner in cells, closing the pore and likely disrupting VDAC's flow of metabolites

Optimization of a designed protein-protein interface

Includes bibliographical references (p. 52)Protein-protein interactions play a role in practically every biological process. The design of novel protein-protein interactions will improve our understanding of the biophysical parameters that drive molecular recognition and self-assembly in these biologically critical processes. Additionally, increased knowledge in the design and prediction of protein-protein interactions will potentially improve the discovery and design of new therapeutics. In a previous project, the monomeric ??1 domain of streptococcal protein G (G??1) was computationally docked to itself in an antiparallel orientation. With the goal of producing a heterodimer in the docked orientation, the amino acid sequence at the docked interface was optimized by computational mutagenesis to produce a pair of proteins that form a heterodimer in the docked orientation. This computational design process resulted in a pair of proteins that are referred to as Monomer A and Monomer B. These two proteins were expressed, purified and characterized. The two computationally designed proteins were found to interact with each other as designed, however with a relatively low affinity. The objective of this project is to redesign the interface between Monomer A and Monomer B to obtain a pair of proteins that form a heterodimer with increased binding affinity. To this end we have taken two approaches. First, oppositely charged amino acids were added to the N- and C-termini of each monomer with the goal of strengthening the interaction by the addition of favorable electrostatic interactions. Next, metal coordination sites were engineered into the two monomers with the goal of increasing the affinity by cross-monomer coordination with metal ion(s) such as Zn(II) or Ni(II). The designed variants were expressed, purified and analyzed by size exclusion chromatography and an in vivo screening method (GFP fragment reassembly screen). While this project did not achieve the initial goal of producing a heterodimer complex with increased affinity over the computationally designed MonomerA/MonomerB complex, we did find that three of the Monomer A variants that were designed with metal coordination sites form homo-complexes in the presence of Zn(II), yet remain monomers in the absence of Zn(II). 2D [??H, ?????N] HSQC NMR spectra were collected for each of these three proteins in the presence and absence of Zn(II). Peak perturbation analysis of the spectra provided further evidence for zinc-induced complex formation

Cancer-Related NEET Proteins Transfer 2Fe-2S Clusters to Anamorsin, a Protein Required for Cytosolic Iron-Sulfur Cluster Biogenesis

Iron-sulfur cluster biogenesis is executed by distinct protein assembly systems. Mammals have two systems, the mitochondrial Fe-S cluster assembly system (ISC) and the cytosolic assembly system (CIA), that are connected by an unknown mechanism. The human members of the NEET family of 2Fe-2S proteins, nutrient-deprivation autophagy factor-1 (NAF-1) and mitoNEET (mNT), are located at the interface between the mitochondria and the cytosol. These proteins have been implicated in cancer cell proliferation, and they can transfer their 2Fe-2S clusters to a standard apo-acceptor protein. Here we report the first physiological 2Fe-2S cluster acceptor for both NEET proteins as human Anamorsin (also known as cytokine induced apoptosis inhibitor-1; CIAPIN-1). Anamorsin is an electron transfer protein containing two iron-sulfur cluster-binding sites that is required for cytosolic Fe-S cluster assembly. We show, using UV-Vis spectroscopy, that both NAF-1 and mNT can transfer their 2Fe-2S clusters to apo-Anamorsin with second order rate constants similar to those of other known human 2Fe-2S transfer proteins. A direct protein-protein interaction of the NEET proteins with apo-Anamorsin was detected using biolayer interferometry. Furthermore, electrospray mass spectrometry of holo-Anamorsin prepared by cluster transfer shows that it receives both of its 2Fe-2S clusters from the NEETs. We propose that mNT and NAF-1 can provide parallel routes connecting the mitochondrial ISC system and the CIA. 2Fe-2S clusters assembled in the mitochondria are received by NEET proteins and when needed transferred to Anamorsin, activating the CIA

Redox-dependent gating of VDAC by mitoNEET.

MitoNEET is an outer mitochondrial membrane protein essential for sensing and regulation of iron and reactive oxygen species (ROS) homeostasis. It is a key player in multiple human maladies including diabetes, cancer, neurodegeneration, and Parkinson's diseases. In healthy cells, mitoNEET receives its clusters from the mitochondrion and transfers them to acceptor proteins in a process that could be altered by drugs or during illness. Here, we report that mitoNEET regulates the outer-mitochondrial membrane (OMM) protein voltage-dependent anion channel 1 (VDAC1). VDAC1 is a crucial player in the cross talk between the mitochondria and the cytosol. VDAC proteins function to regulate metabolites, ions, ROS, and fatty acid transport, as well as function as a "governator" sentry for the transport of metabolites and ions between the cytosol and the mitochondria. We find that the redox-sensitive [2Fe-2S] cluster protein mitoNEET gates VDAC1 when mitoNEET is oxidized. Addition of the VDAC inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) prevents both mitoNEET binding in vitro and mitoNEET-dependent mitochondrial iron accumulation in situ. We find that the DIDS inhibitor does not alter the redox state of MitoNEET. Taken together, our data indicate that mitoNEET regulates VDAC in a redox-dependent manner in cells, closing the pore and likely disrupting VDAC's flow of metabolites

Molecular Dynamics Simulations of the [2Fe-2S] Cluster-Binding Domain of NEET Proteins Reveal Key Molecular Determinants That Induce Their Cluster Transfer/Release

The NEET proteins are a novel family of iron-sulfur proteins characterized by an unusual 3 cysteine and one histidine coordinated [2Fe-2S] cluster. Aberrant cluster release, dictated by the breakage of the Fe-N bond, is implicated in a variety of human diseases, including cancer and neurodegenerative diseases. Here molecular dynamics in the multi-μs timescale, along with quantum chemical calculations, on two representative members of the family (the human NAF-1 and mitoNEET proteins) show that the loss of the cluster is associated with a dramatic decrease of secondary and tertiary structure. In addition, the calculations provide a mechanism for cluster release and clarify, for the first time, crucial differences existing between the two proteins, which are reflected in the experimentally observed difference in pH-dependent cluster reactivity. The reliability of our conclusions is established by an extensive comparison with NMR data of the proteins in solution, in part measured in this work

NEET-apo-Anamorsin association and dissociation rates determined by biolayer interferometry.

<p>NEET-apo-Anamorsin association and dissociation rates determined by biolayer interferometry.</p

Structures and UV-Vis absorption spectra of NEET proteins and Anamorsin.

<p>A. (Top) Crystal structures of mNT (PDB code: 2QH7,), NAF-1 (PDB code: 3FNV); (Middle) NEET 2Fe-2S cluster with 3-Cys:1His coordination; (Bottom) Absorption spectra of 25 μM mNT and NAF-1. B. (Top) Crystal structure of the N-terminal domain of Anamorsin (PDB code: 2YUI) with an added schematic of the unstructured 2Fe-2S cluster binding domain; (Middle) Representative Anamorsin 2Fe-2S cluster with 4-Cys coordination (from ferredoxin, PDB code: 1RFK); (Bottom) Absorption spectrum of 43 μM Anamorsin isolated from <i>E</i>. <i>coli</i>.</p

NEET transfer of 2Fe-2S clusters to apo-Anamorsin is second order.

<p>NAF-1 (A) and mNT (B) transfer to apo-Anamorsin was monitored by UV-Vis absorption spectroscopy for a series of NEET concentrations. For each NEET concentration the ratio 1 NEET dimer per 2 apo-anamorisn was maintained. The rate constant, k_obs, is determined from the fit of the data to an exponential rise and is plotted versus concentration for NAF-1 (C) or mNT (D). The slope of the best line fit was used to determine apparent second order rate constants (<i>k</i>₂) for NAF-1 and mNT, which are 600 ± 90 M^-1 min^-1 and 460 ± 60 M^-1 min^-1 respectively.</p

Biolayer interferiometry shows a direct protein-protein interaction of NAF-1 and mNT with apo-Anamorsin.

<p>Sensorgrams for the binding of holo-NAF-1 (A) and holo-mNT (B) to biotinylated apo-Anamorsin immobilized to streptavidin-coated biosensors are shown. The association was followed for 900 seconds (rising signal) followed by 1800 seconds of dissociation (decaying signal). The data was fit to a one-to-one model (black curves). On- and off-rates were determined from the fits for each NEET-Anamorsin concentration (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139699#pone.0139699.t002" target="_blank">Table 2</a>).</p