2 Structure-Function Relationships of Integral Membrane Proteins Hartmut “Hudel” LueckeBiochemistry, Biophysics &Computer Science
3 due Feb 3, 2011 via email (.doc file) Homework (a.k.a. home education ;-)1) Use amino acid sequence to predicttransmembrane segments (hydropathy)secondary structure (helix, strand, turn)hydrophilic & hydrophobic face of helices using helical wheel analysis2) Use PDB file to evaluate accuracy of predictions (helix boundaries, hydrophobic faces etc.)Coot:(Windows)
12 The mitochondrial ADP/ATP carrier ADP, net charge: -3ATP, net charge: -4
13 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.
15 Mitochondrial Inner Membrane The inner mitochondrial membrane contains proteins with four types of functions:Those that carry out the oxidation reactions of the respiratory chainATP synthase, which uses the H+ gradient from 1. to make ATP from ADP and PiSpecific transport proteins that regulate the passage of metabolites into and out of the matrixProtein import machinery
16 Mitochondrial Inner Membrane Contains more than 100 different polypeptides, with a very high protein-to-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
18 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.
19 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.
21 The Electron Transport Chain Uses a Hydrogen Gradient to Make ATP
22 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-PeyroulaImport 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.
23 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-PeyroulaAll 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.
24 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.
25 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-PeyroulaWhen 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 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.
26 Relations Between Structure and Function of the Mitochondrial ADP/ATP Carrier from matrixfrom IMSOverall 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.
27 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
28 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.
29 Relations Between Structure and Function of the Mitochondrial ADP/ATP Carrier Atractyloside (ATR) can be extracted from the thistle Atractylis gummiferaSchematic 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.
30 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.
31 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.
32 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.
33 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).
34 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).
35 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.
36 Relations Between Structure and Function of the Mitochondrial ADP/ATP Carrier SummaryThe 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.