The rise in obesity in the United States parallels a dramatic increase in
obesity-associated diseases, most notably type-2 diabetes. This disease is
predicted to reach epidemic proportions in the next several decades (Zimmet
et al 2001, Urek et al 2007). Thus, understanding the
biochemical processes
underlying type-2 diabetes and identifying new targets for therapeutic
intervention are critical for national and world health. A drug of choice to
treat type-II diabetes is pioglitazone, a thiazolidinedione (TZD) derivative
originally thought to exert its effect through activation of the nuclear
transcription factor PPARg. Recently, a novel
protein target for pioglitazone
was discovered and was called mitoNEET (Colca et al 2004).
This protein is
anchored to the outer mitochondrial membrane (OMM) (Wiley et al 2007).
Contrary to predictions that this was a zinc-finger transcription factor we
discovered that mitoNEET is a novel 2Fe-2S protein.
|  |
|
Figure 1. Overall structural organization and domain topology of dimeric
mitoNEET. (A) The backbone tracing of each protomer colored in green and
magenta, respectively, together with the observed 2Fo-Fc electron density
(grey) map contoured at 1.5s. A 2Fe-2S cluster is
present in each protomer,
and is depicted in CPK rendition as yellow (sulfur) and red (iron) spheres.
The protomers pack in a parallel fashion. The N-termini and C-termini are
indicated. (B) Ribbon diagram highlighting the two domains and protomer
interactions within the mitoNEET dimer: a six stranded beta sandwich
forms the intertwined beta cap domain and a larger cluster binding domain
carries two 2Fe-2S clusters (one per protomer).
| |
In an effort to understand the structural properties of this protein, a soluble
form of recombinant human mitoNEET was crystallized in an orthorhombic space
group P212121, with unit-cell parameters a =
46.81 Å, b = 49.62 Å, c = 59.01 Å
(Paddock et al 2007). The structure was determined by x-ray diffraction
from 1.5 Å resolution data collected from SSRL Beamline 9-2. Initial phasing
was obtained from Fe-MAD (multiwavelength anomalous dispersion) datasets
collected at wavelengths corresponding to the inflection, high energy remote,
and absorption peak of Fe. Data was processed using automated MAD script
developed at SSRL. The structural model was refined to an R-factor = 18.2 %.
The crystal structure reveals that mitoNEET folds into a unique homodimeric
structure with one 2Fe-2S cluster bound to each monomer within the dimmer
(Figs. 1 & 2). A structural similarity search revealed that this fold is novel
when compared with the >650 known Fe-S proteins, and it is also unique when
compared with the >44,200 known members of the structural databases. The
protein is folded into two spatially distinct subregions: a b-rich or "b-cap"
domain and a helical 2Fe-2S binding or "cluster-binding" domain (Fig. 1B). The
b-rich domain contains a strand swap from opposite
ends of the primary sequence to form the b-cap structure (Fig. 1B).
|
 |
|
Figure 2. The overall distribution of charges within mitoNEET create a
macro-dipole separated by hydrophobic belt. (A) The ribbon diagram of
mitoNEET is displayed in two orientations. The bottom view is
rotated 90º from the top along the vertical axis shown in the center. The protomers of the
unit are colored in green and purple, respectively. Each 2Fe-2S cluster is
shown with yellow (sulfur) and red (iron) spheres. (B) The ribbons of
each protomer are colored grey and the packing of the ten aromatic residues
(five from each protomer) are emphasized by yellow dots. Apolar residues are
also localized to this region, but are not shown. (C) The separation
of charged residues in mitoNEET indicates segregation of the aromatic and
apolar regions of the protein. The negatively charged residues are labeled in
red and the positively charged residues in blue. The upper panel emphasizes
both the asymmetry of charges within the interior of the molecule and the
separation of these charges by the nonpolar residues (panel B).
|
A prominent feature of the structure is the presence of two 2Fe-2S clusters
that are separated by  16 Å from each other within
the larger helical cluster-binding domain (30 Å across) (Fig. 2). The mitoNEET dimer has an
unusual distribution of aromatics (Fig. 2B) forming a ring around the central
b-sheet region and charges (Fig. 2C) that create an internal macro-dipole.
Three Cys residues and one histidine residue on each monomer are ligands to the
2Fe-2S clusters. Interestingly, mitoNEET shares this unusual 3Cys cluster
coordination with the structurally unrelated cluster scaffold protein IscU
(Ramelot et al 2004).
Complementary biophysical investigations showed that the cluster is redox
active and labile below pH 8.0 (Wiley et al 2007), characteristics
possibly related to its function. Optical and NMR experiments demonstrated
that the presence of the diabetes drug pioglitazone increased the stability by
10-fold.
The unusual lability was associated with the coordinating ligand His-87 (Wiley
et al 2007), which cannot serve as a stabilizing ligand for the
2Fe-2S when
protonated. This unusual characteristic of the protein raises the interesting
possibility that mitoNEET participates in Fe-S cluster assembly or
storage. We predict that the cluster-binding domain is situated near the OMM
in vivo placing mitoNEET in a unique position to receive and
transfer clusters that have crossed the outer mitochondrial membrane, a
process that is not presently fully understood.
Although TZDs activate peroxisome proliferator-activating receptors
(PPARg),
data suggesting alternative modes of action involving mitochondria has
accumulated. Our structural results may have important implications for both
mechanisms of drug action and future optimization of TZDs, especially in light
of the fact that the structures of PPARg and
mitoNEET are nearly completely
dissimilar. Whether the beneficial effects of TZDs on mitochondria including
biogenesis and normalization of lipid oxidation are mediated through
mitoNEET is unknown. However, these data, combined with those of Colca
et al. (1994), suggest that pioglitazone can bind and alter the
properties of mitoNEET that is
expressed in many insulin-responsive tissues. Although further biological and
biophysical experiments are needed to relate in vitro binding to
in vivo effects, mitoNEET may prove to be an alternative target
for drug actions.
Acknowledgments:
This work was supported by that National Institutes of Health (GM 41637,
GM18024, GM 18849, GM54038, and DK54441) and the Zevi Hermann Shipira
Foundation. Portions of this research were carried out at the SSRL, a national
user facility operated by Stanford University on behalf of the U.S. Department
of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular
Biology Program is supported by the Department of Energy, Office of Biological
and Environmental Research, and by the National Institutes of Health, National
Center for Research Resources, Biomedical Technology Program, and the National
Institute of General Medical Sciences.
Primary Citation:
Paddock, M. L., Wiley, S. E., Axelrod, H. L., Cohen, A. E., Roy, M., Abresch,
E. C., Capraro, D., Murphy, A. N., Nechushtai, R., Dixon, J. E. and Jennings,
P. A. (2007) MitoNEET is a uniquely folded 2Fe-2S outer mitochondrial membrane
protein stabilized by pioglitazone. Proc Natl Acad Sci USA 104,
14342-14347.
References:
Colca, J. R., McDonald, W. G., Waldon, D. J., Leone, J. W., Lull, J. M.,
Bannow, C. A., Lund, E. T. & Mathews, W. R. (2004) Am J Physiol Endocrinol
Metab 286, E252-260.
Ramelot, T.A., Cort, J. R., Goldsmith-Fischman, S., Kornhaber, G. J., Xiao,
R., Shastry, R., Acton, T. B., Honig, B., Montelione, G. T. & Kennedy, M. A.
(2004) J Mol Biol 344, 567-583.
Urek, R., Crncevic-Urek, M. & Cubrilo-Turek, M. (2007) Acta Med
Croatica 61, 161-164.
Wiley, S. E., Paddock, M. L., Abresch, E. C., Gross, L., van der Geer, P.,
Nechushtai, R., Murphy, A. N., Jennings, P. A. & Dixon, J. E. (2007)
J Biol Chem. 282, 23745 - 23749.
Zimmet, P., Alberti, K. G. and Shaw, J. (2001) Nature 414, 782-787.
|
| PDF
Version | | Lay Summary | |
Highlights Archive
|
| SSRL is supported
by the Department of Energy, Office of Basic Energy Sciences. The SSRL
Structural Molecular Biology Program is supported by the Department of Energy,
Office of Biological and Environmental Research, and by the National Institutes
of Health, National Center for Research Resources, Biomedical Technology
Program, and the National Institute of General Medical Sciences. |
|
