Past Event: Oden Institute Seminar

NEET proteins – a novel class of 2Fe-2S proteins with a unique fold and function

Rachel Nechushtai, The Hebrew University of Jerusalem

Abstract

Metalloproteins (MPs) comprise one-third of all known protein structures. Among them, a unique class of iron-sulfur (2Fe-2S) proteins, the NEET proteins, were recently discovered. In human, the NEET family consists of three members. Two of them, mitoNEET and NAF-1, encoded by the CISD1 and CISD2 genes, respectively, are homo-dimers, containing two domains; a beta-cap, and a cluster binding domain. The 2Fe-2S clusters of NEET proteins, are coordinated by a 3Cys:1His structure which allows them to be both relatively stable, as well as transferred to apo-acceptor protein(s). This unique feature of NEET proteins is likely controlled by the NEET's 2Fe-2S's coordinating His that is positioned at the protein surface and can undergo protonation that destabilizes the cluster and enables its transfer. Most of the well-studied MPs are generally viewed as being very rigid in structure, and it is widely believed that the properties of their metal centers are primarily determined by the small fraction of amino acids that make up the local center environment. To challenge that, we initially used a globular plant-type ferredoxin (Fd) to investigate the functional landscape of a single-domain 2Fe-2S protein and the effect of a distal loop on its electron-transfer 2Fe-2S cluster. We found that the global stability and structure are minimally perturbed by a deletion mutation, whereas the functional properties are altered. Specifically, truncating the Fd-L1,2 loop does not lead to large-scale changes in the structure, determined via X-ray crystallography. In addition, the overall thermal stability of the protein is only marginally perturbed by the mutation. However, even though the mutation is distal to the iron–sulfur cluster, it leads to a significant change in the redox potential of the iron–sulfur cluster (57 mV). Structure-based all-atom simulations revealed correlated dynamical changes between the surface-exposed loop and the iron–sulfur cluster-binding region, suggesting that intrinsic communication channels within the ferredoxin fold, composed of many short-range interactions, lead to the propagation of long-range signals. We also examined both theoretically and experimentally whether distal regions can influence the metal centers in the diabetes drug target mitoNEET. A loop 20 Å away from the metal centers exerts allosteric control over the cluster binding domain and regulates multiple properties of the metal centers. Mutagenesis therefore resulted in significant shifts in the redox potential of the [2Fe-2S] cluster and orders-of-magnitude effects on the rate of [2Fe-2S] cluster transfer to an apo-acceptor protein. These surprising effects occur in the absence of any significantly-observed structural changes. Our findings suggest that long-range dynamical changes in the protein backbone can have a significant effect on the functional properties of MPs. We further used an integrated approach involving peptide array, deuterium exchange mass spectrometry (DXMS), and functional studies aided by the power of sufficient constraints from direct coupling analysis (DCA) to determine the dominant docked conformation of the NAF-1–Bcl-2 complex. NAF-1 binds to both the pro- and antiapoptotic regions (BH3 and BH4) of Bcl-2, as demonstrated by a nested protein fragment analysis in a peptide array and DXMS analysis. These studies together with our recent findings of the NEET function in Cancer, Genetic neurodegenerative disease and the importance of their transmembrane domains will be discussed.