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Structure-Function Relationships of
Integral Membrane Proteins
Hartmut “Hudel” Luecke
Biochemistry, Biophysics &
Computer Science
Email: [email protected]
http://bass.bio.uci.edu/~hudel
Mitochondria
Mitochondrial Outer Membrane
The outer mitochondrial membrane, which encloses the entire
organelle, has a protein-to-phospholipid ratio similar to the eukaryotic
plasma membrane (about 1:1 by weight). It contains numerous
integral membrane proteins called porins, which feature relatively large
internal channels (about 2-3 nm) that are permeable to molecules of
~5,000 Da or less. In contrast, larger molecules, for example most
proteins, can only traverse the outer membrane by active transport.
Mitochondrial Outer Membrane: Porins
Porins are found in the outer
membrane of Gram-negative
bacteria, in mitochondria, and
in chloroplasts.
Porins control diffusion of small
metabolites like sugars, ions,
and amino acids.
Beta barrels
Mitochondrial Inner Membrane
The inner mitochondrial membrane contains proteins with four types of functions:
1.
2.
3.
4.
Those that carry out the oxidation reactions of the respiratory chain
ATP synthase, which uses the H+ gradient from 1. to make ATP from ADP and Pi
Specific transport proteins that regulate the passage of metabolites into and out of
the matrix
Protein import machinery (TIM)
The Electron Transport Chain Uses a Hydrogen
Gradient to Make ATP
Mitochondrial Inner Membrane
The inner mitochondrial membrane contains proteins with four types of functions:
1.
2.
3.
4.
Those that carry out the oxidation reactions of the respiratory chain
ATP synthase, which uses the H+ gradient from 1. to make ATP from ADP and Pi
Specific transport proteins that regulate the passage of metabolites into and out of
the matrix
Protein import machinery (TIM)
The Electron Transport Chain Uses a Hydrogen
Gradient to Make ATP
If one molecule of glucose is fully oxidized
using glycolysis, decarboxylation and the Krebs
cycle, 36 ATPs are generated per glucose,
compared to only 2 ATPs if glycolysis alone is
used.
Glycolysis
Mitochondrial Inner Membrane
The inner mitochondrial membrane contains proteins with four types of functions:
1.
2.
3.
4.
Those that carry out the oxidation reactions of the respiratory chain
ATP synthase, which uses the H+ gradient from 1. to make ATP from ADP and Pi
Specific transport proteins that regulate the passage of metabolites into and out of
the matrix
Protein import machinery (TIM)
Carrier/transporter types
The mitochondrial ADP/ATP carrier
ADP, net charge: -3
ATP, net charge: -4
Mitochondrial Inner Membrane
Contains more than 100 different polypeptides, with a very high proteinto-phospholipid ratio (more than 3:1 by weight, which is about 1 protein
for 15 phospholipids). Additionally, the inner membrane is rich in an
unusual phospholipid, cardiolipin. 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.
Cardiolipin
Cardiolipin
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.
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. 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.
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
1.
2.
3.
4.
5.
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 repeat.
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.
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.
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
1.
2.
3.
4.
5.
When mitochondria are actively respiring in the presence of phosphate and
ADP, the latter is exchanged against intramitochondrial ATP with a 1-to1stoichiometry.
The only physiological substrates are ADP and ATP in their free forms, i.e., MgADP 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 intermembrane space 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.
Relations Between Structure and Function of the
Mitochondrial ADP/ATP Carrier
from IMS
from matrix
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 (H1–H6), loops facing the intermembrane space (IMS) (C1 and C2), and loops facing the matrix (M1–M3). Matrix loops are
partially structured in short helices (h1–2, h3–4, and h5–6). Three cardiolipins (CDL800, CDL801, and CDL802) are bound to the structure and
represented as ball&stick 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.
Relations Between Structure and Function of the
Mitochondrial ADP/ATP Carrier
Intermembrane space (IMS)
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).
Matrix
Relations Between Structure and Function of the
Mitochondrial ADP/ATP Carrier
Distribution of residues within the cavity: 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 Ys.
Relations Between Structure and Function of the Mitochondrial
ADP/ATP Carrier
Atractyloside (ATR) can be
extracted from the thistle
Atractylis gummifera
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.
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 is the first residue 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.
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 a 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.
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).
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% homology).
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.
Relations Between Structure and Function of the Mitochondrial
ADP/ATP Carrier
Summary
1.
2.
3.
4.
5.
The ADP/ATP carrier structure highlights a bundle of six tilted (with half of
them kinked) helices 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 might 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 and neutron
scattering indicate that the ADP/ATP carrier functions as a dimer but more
recent evidence supports functional monomers as well.