Iron-sulfur (Fe-S) proteins are key players in vital processes involving energy homeostasis and metabolism from the simplest to most complex organisms. We report a 1.5 Å x-ray crystal structure of the first identified outer mitochondrial membrane Fe-S protein, mitoNEET. Two protomers intertwine to form a unique dimeric structure that constitutes a new fold to not only the Ϸ650 reported Fe-S protein structures but also to all known proteins. We name this motif the NEET fold. The protomers form a two-domain structure: a -cap domain and a cluster-binding domain that coordinates two acid-labile 2Fe-2S clusters. Binding of pioglitazone, an insulin-sensitizing thiazolidinedione used in the treatment of type 2 diabetes, stabilizes the protein against 2Fe-2S cluster release. The biophysical properties of mitoNEET suggest that it may participate in a redox-sensitive signaling and/or in Fe-S cluster transfer. diabetes ͉ FeS cluster ͉ iron homeostasis ͉ thiazolidinedione ͉ oxidative stress I ron (Fe) is a vital trace element for virtually all organisms. Incorporation of this transition metal into iron-sulfur (Fe-S) clusters forms cofactors integral to diverse biological pathways involved in the capture and metabolism of light and chemical energy (1, 2). Because free iron can be highly toxic, an elaborate array of proteins has evolved to facilitate the transfer of iron through cell compartments, to insert iron into Fe-S clusters, and to incorporate Fe-S clusters into proteins. Fe-S cluster assembly takes place primarily, although not exclusively, within the mitochondrial matrix of eukaryotic cells, and defects in mitochondrial cluster assembly and export have profound consequences for rates of growth, iron accumulation, oxidative stress, and heme biosynthesis (1, 2).Mitochondrial dysfunction is associated with insulin resistance and the development of type 2 diabetes (3). Recent studies suggest that disease pathogenesis involves diminished mitochondrial oxidative capacity in insulin-sensitive tissues. Pharmacologic agents extensively used to treat insulin resistance such as the thiazolidinedione (TZD) pioglitazone are known to enhance oxidative capacity and normalize lipid metabolism (4, 5). Although TZDs are conventionally thought to operate through binding to peroxisome proliferator-activated receptors, a recent study by Colca and colleagues (6) identified an additional binding target within mitochondrial membranes that was named mitoNEET, on the basis of the subcellular localization (mito) and the presence of the amino acid sequence Asn-Glu-Glu-Thr (NEET).MitoNEET was determined to be an integral protein of the outer mitochondrial membrane (OMM) by a series of studies, including immuno-electron microscopy and detailed fractionation studies of highly purified rat liver mitochondria. An amino-terminal signal sequence within the first 32 residues, containing a predicted transmembrane domain, targets mitoNEET to the outer membrane. The orientation of this protein toward the cytoplasm was established by proteolytic digestion...
BackgroundThe Acel_2062 protein from Acidothermus cellulolyticus is a protein of unknown function. Initial sequence analysis predicted that it was a metallopeptidase from the presence of a motif conserved amongst the Asp-zincins, which are peptidases that contain a single, catalytic zinc ion ligated by the histidines and aspartic acid within the motif (HEXXHXXGXXD). The Acel_2062 protein was chosen by the Joint Center for Structural Genomics for crystal structure determination to explore novel protein sequence space and structure-based function annotation.ResultsThe crystal structure confirmed that the Acel_2062 protein consisted of a single, zincin-like metallopeptidase-like domain. The Met-turn, a structural feature thought to be important for a Met-zincin because it stabilizes the active site, is absent, and its stabilizing role may have been conferred to the C-terminal Tyr113. In our crystallographic model there are two molecules in the asymmetric unit and from size-exclusion chromatography, the protein dimerizes in solution. A water molecule is present in the putative zinc-binding site in one monomer, which is replaced by one of two observed conformations of His95 in the other.ConclusionsThe Acel_2062 protein is structurally related to the zincins. It contains the minimum structural features of a member of this protein superfamily, and can be described as a “mini- zincin”. There is a striking parallel with the structure of a mini-Glu-zincin, which represents the minimum structure of a Glu-zincin (a metallopeptidase in which the third zinc ligand is a glutamic acid). Rather than being an ancestral state, phylogenetic analysis suggests that the mini-zincins are derived from larger proteins.
With over 60,000 protein structures available in the Protein Data Bank, it is frequently possible use one of them to obtain starting phase information and to solve new crystal structures. Molecular replacement1–4 procedures, which search for placements of a starting model within the crystallographic unit cell that best account for the measured diffraction amplitudes, followed by automatic chain tracing methods5–8, have allowed the rapid solution of large numbers of protein structures. Despite extensive work9–14, molecular replacement or the subsequent rebuilding usually fail with more divergent starting models based on remote homologues with less than 30% sequence identity. Here we show that this limitation can be substantially reduced by combining algorithms for protein structure modeling with those developed for crystallographic structure determination. An approach integrating Rosetta structure modeling with Autobuild chain tracing yielded high-resolution structures for 8 of 13 X-ray diffraction datasets that could not be solved in the laboratories of expert crystallographers and that remained unsolved after application of an extensive array of alternative approaches. We estimate the new method should allow rapid structure determination without experimental phase information for over half the cases where current methods fail, given diffraction datasets of better than 3.2Å resolution, four or fewer copies in the asymmetric unit, and the availability of structures of homologous proteins with >20% sequence identity.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.