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...

Interleukin-1␤ (IL-1␤) is a cytokine within the ␤-trefoil family. Our data indicate that the folding/unfolding routes are geometrically frustrated. Follow-up theoretical studies predicted backtracking events that could contribute to the broad transition barrier and the experimentally observed long-lived intermediate. The backtracking route is attributed to the topological frustration introduced by the packing of the functional loop (the ␤-bulge, residues 47-53) to the nascent barrel. We used real-time refolding NMR experiments to test for the presence of backtracking events predicted from our theoretical studies. Structural variants of IL-1␤, a ␤-bulge deletion, and a circular permutation that opens the protein in the middle of the experimentally observed kinetic intermediate, were also refolded and studied to determine the affects on the observed folding reactions. The functional loop deletion variant demonstrated less backtracking than in WT protein whereas the permutation still maintains backtracking in agreement with theoretical predictions. Taken together, these findings indicate that the backtracking results from geometric frustration introduced into the fold for functional purposes.protein folding ͉ real-time NMR

Topologically complex proteins fold by multiple routes as a result of hard-to-fold regions of the proteins. Oftentimes these regions are introduced into the protein scaffold for function and increase frustration in the otherwise smooth-funneled landscape. Interestingly, while functional regions add complexity to folding landscapes, they may also contribute to a unique behavior referred to as hysteresis. While hysteresis is predicted to be rare, it is observed in various proteins, including proteins containing a unique peptide cyclization to form a fluorescent chromophore as well as proteins containing a knotted topology in their native fold. Here, hysteresis is demonstrated to be a consequence of the decoupling of unfolding events from the isomerization or hula-twist of a chromophore in one protein and the untying of the knot in a second protein system. The question now is- can hysteresis be a marker for the interplay of landscapes where complex folding and functional regions overlap?

Proteins fold into three-dimensional structures in a funneled energy landscape. This landscape is also used for functional activity. Frustration in this landscape can arise from the competing evolutionary pressures of biological function and reliable folding. Thus, the ensemble of partially folded states can populate multiple routes on this journey to the native state. Although protein folding kinetics experiments have shown the presence of such routes for several proteins, there has been sparse information about the structural diversity of these routes. In addition, why a given protein populates a particular route more often than another protein of similar structure and sequence is not clear. Whereas multiple routes are observed in theoretical studies on the folding of interleukin-1β (IL-1β), experimental results indicate one dominant route where the central portion of the protein folds first, and is then followed by closure of the barrel in this β-trefoil fold. Here we show, using a combination of computation and experiment, that the presence of functionally important regions like the β-bulge in the signaling protein IL-1β strongly influences the choice of folding routes. By deleting the β-bulge, we directly observe the presence of routeswitching. This route-switching provides a direct link between route selection and the folding and functional landscapes of a protein.pulse-labeling | geometric frustration T he energy landscape theory and the funnel concept provide a solution to the protein folding search problem (1). The presence of multiple routes to the native state within this funnel is evident in both simulation and experimental studies (2) and is thought to add to the robustness of the landscape. The question remains, what factors control the route selection for folding? There is speculation that functional residues are a hindrance to folding and may add heterogeneity and roughness to the landscape (2-8). These residues need to be conserved for the protein to function correctly and therefore cannot be optimized for folding. However, the rest of the protein can evolve to make folding more efficient by making the energy landscape more funneled. Thus, it is possible that the most frustrated parts of the protein are the functional sites within the native structure. We hypothesize that functional regions within a protein that are geometrically frustrated drive route selection.The 12 β-stranded inflammatory cytokine, interleukin-1β (IL-1β), is an excellent system to probe the modulation of the folding landscape by perturbing functional regions. Structure-based models (where native interactions dominate the energy function) have identified the functionally important β-bulge (located between β-strands 4 and 5) as a region that is geometrically frustrated and suggest that contacts with this region direct folding route selection (9, 10). Deletion of the β-bulge maintains high affinity receptor binding but abrogates signaling activity, effectively converting the agonist into an antagonist molecule (11). To experime...

Regulation of protein function via cracking, or local unfolding and refolding of substructures, is becoming a widely recognized mechanism of functional control. Oftentimes, cracking events are localized to secondary and tertiary structure interactions between domains that control the optimal position for catalysis and/or the formation of protein complexes. Small changes in free energy associated with ligand binding, phosphorylation, etc., can tip the balance and provide a regulatory functional switch. However, understanding the factors controlling function in single-domain proteins is still a significant challenge to structural biologists. We investigated the functional landscape of a single-domain planttype ferredoxin protein and the effect of a distal loop on the electron-transfer center. We find the global stability and structure are minimally perturbed with mutation, whereas the functional properties are altered. Specifically, truncating the L1,2 loop does not lead to large-scale changes in the structure, determined via X-ray crystallography. Further, 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 (∼20 Å), it leads to a significant change in the redox potential of the ironsulfur cluster (57 mV). Structure-based all-atom simulations indicate correlated dynamical changes between the surface-exposed loop and the iron-sulfur cluster-binding region. Our results suggest intrinsic communication channels within the ferredoxin fold, composed of many short-range interactions, lead to the propagation of long-range signals. Accordingly, protein interface interactions that involve L1,2 could potentially signal functional changes in distal regions, similar to what is observed in other allosteric systems.electron transfer | functional energy landscape | iron-sulfur proteins | protein folding O ver the last several decades, our understanding of protein function has evolved from a rather static perspective, where signaling and function have been understood through surface complementarity arguments, such as the "lock and key" paradigm (1, 2), to a more dynamic view where protein conformational fluctuations are inextricably linked to function (3). As our understanding of protein dynamics expands, we are revealing many mechanisms by which proteins exploit conformational fluctuations to perform cellular function. In multidomain proteins, relative repositioning of domains is often linked to their levels of activity. For example, large-scale domain rearrangements in the four-domain Src kinase (4) and C-terminal Src kinase (5, 6) lead to these proteins being in so-called "on" or "off" states, and functional regulation may be obtained by adjusting the balance between these conformations (7). In proteins such as adenylate kinase, domain rearrangements can be rate limiting during each round of catalysis (8), where the enzyme cycles between ligandcompetent and ligand-release conformations. Because the kinetics of these tra...