BioSci M160 MolBio255 StructureFunction Relationships of Integral Membrane Proteins Lecture 7

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BioSci M160 / MolBio255Structure-Function
Relationships of Integral Membrane
ProteinsLecture 7
Hartmut Hudel Luecke Biochemistry, Biophysics
Computer Science Email hudel_at_uci.edu
http//bass.bio.uci.edu/hudel
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Homework (a.k.a. home education -)

• 1) Use amino acid sequence to predict
• transmembrane segments (hydropathy)
• secondary structure (helix, strand, turn)
• hydrophilic hydrophobic face of helices using
  helical wheel analysis
• 2) Use PDB file to evaluate accuracy of
  predictions (helix boundaries, hydrophobic faces
  etc.)
• Coot http//www.ysbl.york.ac.uk/emsley/coot/
• http//www.chem.gla.ac.uk/bernhar
  d/coot/wincoot.html (Windows)

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Helical Wheel
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Wimley-White Hydrophobicity Scales
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Steve White Lab UCI http//blanco.biomol.uci.edu/m
pex/
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http//blanco.biomol.uci.edu/mpex/
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http//blanco.biomol.uci.edu/mpex/
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ADP/ATP Carrier
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Mitochondria
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Mitochondrial Outer Membrane
The outer mitochondrial membrane, which encloses
the entire organelle, has a protein-to-phospholipi
d ratio similar to the eukaryotic plasma membrane
(about 11 by weight). It contains numerous
integral membrane proteins called porins, which
contain a relatively large internal channel
(about 2-3 nm) that is permeable to molecules of
5,000 daltons or less. Larger molecules, for
example most proteins, can only traverse the
outer membrane by active transport.
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Mitochondrial Inner Membrane

• The inner mitochondrial membrane contains
  proteins with four types of functions
• Those that carry out the oxidation reactions of
  the respiratory chain
• ATP synthase, which use the H gradient to make
  ATP
• Specific transport proteins that regulate the
  passage of metabolites into and out of the matrix
• Protein import machinery
• It contains more than 100 different
  polypeptides, and has a very high
  protein-to-phospholipid ratio (more than 31 by
  weight, which is about 1 protein for 15
  phospholipids). Additionally, the inner membrane
  is rich in an unusual phospholipid, cardiolipin,
  which is usually characteristic of bacterial
  plasma membranes. Unlike the outer membrane, the
  inner membrane does not contain porins, and is
  highly impermeable almost all ions and molecules
  require special membrane transporters to enter or
  exit the matrix. In addition, there is a
  membrane potential across the inner membrane.

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Cardiolipin (CDL)
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Mitochondrial Inner Membrane
The inner mitochondrial membrane is
compartmentalized into numerous cristae, which
expand the surface area of the inner
mitochondrial membrane, enhancing its ability to
generate ATP. In typical liver mitochondria, for
example, the surface area, including cristae, is
about five times that of the outer membrane.
Mitochondria of cells which have greater demand
for ATP, such as muscle cells, contain more
cristae than typical liver mitochondria.
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The Electron Transport Chain Uses a Hydrogen
Gradient to Make ATP
If glucose is totally oxidized, using glycolysis,
decarboxylation and the Krebs cycle, 36 ATPs are
generated per glucose, compared to only 2 ATPs if
glycolysis alone is used.
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Relations Between Structure and Function of the
Mitochondrial ADP/ATP Carrier H. Nury, C.
Dahout-Gonzalez, V. Trezeguet, G.J.M. Lauquin, G.
Brandolin and E. Pebay-Peyroula
Import and export of metabolites through
mitochondrial membranes are vital processes that
are highly controlled and regulated at the level
of the inner membrane. Proteins of the
mitochondrial carrier family (MCF) are embedded
in this membrane, and each member of the family
achieves the selective transport of a specific
metabolite. Among these, the ADP/ATP carrier
transports ADP into the mitochondrial matrix and
exports ATP toward the cytosol after its
synthesis. Because of its natural abundance, the
ADP/ATP carrier is the best characterized
carrier, and a high-resolution structure of one
conformation is known. The overall structure is
basket-shaped and formed by six transmembrane
helices that are not only tilted with respect to
the membrane, but three of them are also kinked
at the level of prolines. The functional
mechanisms, nucleotide recognition, and
conformational changes for the transport,
suggested from the structure, are discussed along
with the large body of biochemical and functional
results.
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Relations Between Structure and Function of the
Mitochondrial ADP/ATP Carrier H. Nury, C.
Dahout-Gonzalez, V. Trezeguet, G.J.M. Lauquin, G.
Brandolin and E. Pebay-Peyroula

• All mitochondrial carriers are encoded by nuclear
  genes.
• The primary structure of most carriers displays
  three repeated homologous regions of about 100
  amino acids each.
• The N and C termini face the intermembrane space
  (IMS) and six transmembrane (TM) segments can be
  delineated.
• A common sequence, the MCF motif, can be found in
  each repeated region with slight deviations on
  one or two signature sequences for some carriers.
• Comparison of primary structures indicates that
  mitochondrial carriers have no orthologues in
  prokaryotes their emergence seems to be the
  evolutionary consequence of the capture of an
  ancient aerobic prokaryotic cell by the primitive
  eukaryotic cell.

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Relations Between Structure and Function of the
Mitochondrial ADP/ATP Carrier H. Nury, C.
Dahout-Gonzalez, V. Trezeguet, G.J.M. Lauquin, G.
Brandolin and E. Pebay-Peyroula

• When mitochondria are actively respiring in the
  presence of phosphate and ADP, the latter is
  exchanged against intramitochondrial ATP with a
  1-to-1stoichiometry.
• The only physiological substrates are ADP and ATP
  in their free forms, i.e., Mg-ADP and Mg-ATP
  are not recognized by the carrier.
• The ADP/ATP exchange is electrogenic, which means
  one negative charge is extruded from the matrix
  to the cytosol for each cycle, and this process
  is driven by the membrane potential.
• The kinetic parameters of the carrier are
  consistent with mitochondrial ATP production and
  the cell nucleotide concentrations under
  physiological conditions.
• The carrier could be purified in detergent
  solutions, and transport activity could be
  reconstituted after reincorporation into
  liposomes.

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Relations Between Structure and Function of the
Mitochondrial ADP/ATP Carrier
from matrix
from IMS
Overall structure of the bovine ADP/ATP carrier.
The ribbon diagram, colored blue to red from the
N terminus (N) to the C terminus (C), depicts
transmembrane helices (H1H6), loops facing the
IMS (C1 and C2), and loops facing the matrix
(M1M3). Matrix loops are partially structured in
short helices (h12, h34, and h56). Three
cardiolipins (CDL800, CDL801, and CDL802) are
bound to the structure and represented as ball
and sticks in grey. The inhibitor, CATR,
complexed with the protein is depicted in yellow.
Panels a, b, c are viewed from the IMS, the side,
and the matrix, respectively.
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Relations Between Structure and Function of the
Mitochondrial ADP/ATP Carrier
Overall topology and motifs of the bovine ADP/ATP
carrier. (a) Schematic diagram of the secondary
structure. Regions containing MCF motif residues
are colored in gray, and the RRRMMM motif is in
black . Kinks in H1, H3, and H5 are induced by
the prolines. (b) Alignment of the three MCF
motifs. On top, the consensus MCF sequence boxed
in grey. The ADP/ATP carrier signature present
in the third repeat is boxed in black.
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Relations Between Structure and Function of the
Mitochondrial ADP/ATP Carrier
Surface representation of the cavity. The
longitudinal section through the cavity shows the
wide cavity present in the bovine ADP/ATP carrier
and accessible from the IMS. R234, R235, and
R236, the three arginines of the ADP/ATP carrier
signature, located on the C-terminal end of H5
are shown, as well as E264, which forms a salt
bridge with R236 (yellow).
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Relations Between Structure and Function of the
Mitochondrial ADP/ATP Carrier
Distribution of residues within the cavity. The
figure represents a two-dimensional projection of
the residues present at the surface of the
cavity. Each circle represents an atom of a
residue located within the cavity, with a size
proportional to its solvent accessibility.
Residues are colored as follows basic, K, R
(blue) acidic, D, E (red) aromatic, F, Y,W
(grey) hydrophobic, A, V, P, M, I, L, G
(yellow) and polar, S, T, H, C, N, Q (green).
The positive patches are labeled 1 to 4, and the
tyrosine ladder is marked by Y.
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Relations Between Structure and Function of the
Mitochondrial ADP/ATP Carrier
Schematic representation of a large hydrogen-bond
network. The network connects all the TM
helices, except H4. It implicates side chains of
polar, acidic, and basic residues that are highly
conserved within ADP/ATP carriers, as well as
main-chain carbonyls (labeled CO) and water
molecules. Hydrogen bonds are deduced from
atomic distances and are represented as dotted
lines.
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Relations Between Structure and Function of the
Mitochondrial ADP/ATP Carrier
The kinked conformation of odd-numbered helices.
H1, H3, and H5, represented as ribbons, are
kinked after prolines P27, P132, and P229, which
are the first residues in each MCF motif. Acidic
and basic residues also belonging to the MCF
motif form salt bridges (dotted lines) that tie
the three helices together.
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Relations Between Structure and Function of the
Mitochondrial ADP/ATP Carrier
MCF motif of the third repeat. The section
between the C terminus of H5 and the N terminus
of H6 is represented as ribbons. Side chains of
MCF motif residues and CDL801 are shown in
ball-and-stick form. A salt bridge between R236
(ADP/ATP carrier signature) and E264 (MCF motif)
is highlighted. F270 is sandwiched between P229
and CDL801.
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Relations Between Structure and Function of the
Mitochondrial ADP/ATP Carrier
Deviation from the pseudo-threefold symmetry.
The superposition of repeat 1 (blue), 2 (pale
green), and 3 (pale yellow) highlights the
similarity of helical parts. Repeats 2 and 3 were
rotated to be superimposed on repeat 1. The
figure shows also a significant difference at the
beginning of matrix loops (between the
odd-numbered and the short helices), which is
slightly longer for M1. M1 folds back toward the
center of the protein and thus allows
interactions with M2 and M3. Small ribbon
portions of M2 and M3 are depicted in green and
yellow and represent these interactions.
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Relations Between Structure and Function of the
Mitochondrial ADP/ATP Carrier
Conserved residues in the cavity. Residues
accessible within the cavity are colored
according to their conservation among ADP/ATP
carriers similarity (grey), medium or high
similarities (yellow or orange), respectively,
and identical (red).
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Relations Between Structure and Function of the
Mitochondrial ADP/ATP Carrier
Conserved residues on external surfaces.
Residues are colored according to conservation
among ADP/ATP carriers from white to red (0 to
100 of homology).
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Relations Between Structure and Function of the
Mitochondrial ADP/ATP Carrier
Protein-protein interaction mediated by CDLs. The
two monomers seen in the crystal packing interact
directly next to the matrix side and to the IMS.
The interaction also involves cardiolipins
(grey). Van der Waals surfaces of proteins and
lipids are shown superposed on the ribbons for
the protein and on the balls and sticks for the
lipids.
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Relations Between Structure and Function of the
Mitochondrial ADP/ATP Carrier Summary

• The ADP/ATP carrier structure highlights a bundle
  of six tiltedwith half of them kinkedhelices
  forming a cavity that is wide open toward the
  IMS.
• MCF members may share a common transport
  mechanism, which is based on a common scaffold
  and could rely on the kink and tilt modifications
  of the TM helices.
• Substrate specificity may be related to the
  geometry and the chemical properties of the
  residues in the cavity, illustrated for instance
  by the distribution of patches of basic residues
  as well as by a ladder of aromatic residues.
• The sequential transport mechanism might be
  induced by the simultaneous binding of ADP and
  ATP on both sides of the membrane.
• Many published results, such as cross-linking
  experiments, protein/inhibitor stoichiometries,
  chimeric dimers, analytical ultracentrifugation
  or neutron scattering, indicate that the ADP/ATP
  carrier functions as a dimer.