Presentation is loading. Please wait.

Presentation is loading. Please wait.

ATP Synthase: What Does It Take to Make a Rotary Enzyme

Similar presentations

Presentation on theme: "ATP Synthase: What Does It Take to Make a Rotary Enzyme"— Presentation transcript:

1 ATP Synthase: What Does It Take to Make a Rotary Enzyme
ATP Synthase: What Does It Take to Make a Rotary Enzyme? Stan Dunn Department of Biochemistry Schulich School of Medicine & Dentistry 24 April 2013 Thank you Provost Deakin. I also want to thank everyone who wrote letters of support, and especially Dave Litchfield the Chair of Biochemistry, for organizing the nomination. I was so delighted when I learned last year that I had been been selected. I value the award particularly because it recognizes balanced effort and accomplishment over all three branches of the academic mission, and this is something that I have always believed in. Finally thanks to the organizers of this event for asking me to participate this year, since I was unable to do so last year. Today we are asked to provide a brief overview of our research. The focus of my research has been to understand the structure and mechanism of ATP synthase. Is is an enzyme of great biological importance, with a remarkable rotary mechanism, causing some people to refer to it as the world’s smallest motor. But lets begin with the biological context.

2 ATP (Adenosine TriPhosphate) Is the Primary Biological “Energy Currency”
Food Cell maintenance, protein and nucleic acid biosynthesis Active transport Muscle contraction Lets begin with the biological significance. Organisms degrade their food in order to obtain energy and to produce the building blocks for cell renewal and growth. Most of the energy that is released is captured through a process called oxidative phosphorylation; ATP synthase is the enzyme that condenses ADP and orthophosphate in this process. Then the cell can utilize the energy stored in ATP by coupling its hydrolysis to useful processes, like you see listed here. So ATP is often called the biological energy currency. You go through ATP so fast that every day you synthesize and hydrolyze an amount of ATP that is about about equivalent to your body weight. Oxidative phosphorylation CO2 and H2O

3 The Mechanism of Oxidative Phosphorylation
Respiratory complexes use the energy released during the oxidation of food to drive H+ ions across the inner mitochondrial membrane, producing an electrochemical H+ gradient containing potential energy; ATP synthase allows the H+ to return, using the released energy to convert ADP + Pi into ATP Oxxidative phosphorylation works in a surprisingly indirect way. During those final stages of food oxidation, the electrons that are extracted from it are passed though protein complexes of the respiratory chain. These complexes are all membrane-bound, and the redox reactions they catalyze result in the movement of Hydrogen ions, which we commonly call protons, across the membrane. This produces a steep ion gradient that stores potential energy. The protons can go down that steep gradient, releasing their energy, by passing through ATP synthase. ATP synthase uses that energy to drive ATP synthesis. So now we can focus on that enzyme, which here is presented simply as a pink box. The question then is how does the structure and mechanism of ATP synthase to link, or couple, ion translocation across the membrane to ATP synthesis or hydrolysis. Respiratory Complexes I, III, IV ATP synthase, aka Complex V

4 F-ATP Synthase: two motors sharing one driveshaft/rotor
peripheral a3b3gde ATP synthesis/ hydrolysis chemical rotary motor The stator: a3b3gdab2 The stator stalk: b2d ADP + Pi ATP a a b b2 g Fo integral ab2c10 H+ conduction electrical rotary motor H+ e So lets take a closer look at the enzyme. It contains a membrane integral Fo sector, composed mostly of a ring of c subunits and a single a subunit, which provide the pathway for ion translocation. Translocation causes the c-ring to rotate, much like a turbine. So you could call Fo an ion-driven, or electrical, rotary motor. The enzyme also has a membrane-peripheral sector called F1, and F1 is composed largely of 3 alpha and 3 beta subunits. Each beta contains a catalytic site where ATP can be made or hydrolyzed, and the shape or conformation of beta changes during these reactions. You can also see that there are two subunits called gamma and epsilon that link the c ring to alpha3 beta3. What you can’t see is a narrow, curved section of gamma that sticks up into the core of the alpha/beta hexamer. The irregular shape of this part of gamma also causes the beta subunits to adopt different conformations. During oxidative phosphorylation, when ions flow through Fo, gamma/epsilon/c10 rotates as a unit. Rotation of the irregular gamma inside alpha3/beta three drives conformational changes in the beta subunits, that result in the binding of ADP + phosphate, their condensation, and finally release of the product ATP. In this rotational mechanism, some device is required to prevent alpha3beta3 from simply spinning with the rotor. This function is fulfilled by the stator stalk that links alpha3beta3 with the a subunit of Fo. This stalk consists of 2 b subunits, and the delta subunit at the top of the complex. Today I want to tell you about some of our work on the structure and function of this b dimer. The rotor: gec10 A common driveshaft C10 Image courtesy of Achim Weber a H+

5 Analysis of E. coli bsol bsol Properties of bsol Dimer
Membrane Tether Dimerization F1-Binding bsol Properties of bsol Dimer Highly extended, not globular Binds to F1 sector, through d subunit Highly a-helical Sequence shows heptad repeat pattern So we expected bsol to form a left-handed coiled coil E. coli b has 156 lamino acid residues. The first 23 span the membrane while the remainder are cytoplasmic. Introduce b. 156 residues. N-terminal transmembrane helix. Remainder cytoplasmic portion call bsol. These can be expressed without the membrane domain in forms that we call bsol. Initially characterized as an elongated, highly helical dimer. In subsequent studies dimerization was shown to be reversible with a Kd on microM range, determined by sed equilibrium. C-terminus involved in binding delta and indeed other parts of F1. Both this and the dimerization domain were defined by deletion analysis. CD spectra show indicate a coiled coil arrangment of helices in the dimerization domain. Left-handed coiled coil

6 Arrangement of helices in dimerization domain Disulfide formation between introduced cysteine residues Ala to Cys mutation changes the side chain from –CH3 to –CH2SH Heptad: b c d e f g a b c d Residue: WT res: A S A T D Q L K K A In a standard LHCC, the heptad d positions are very well oriented for disulfide formation. Proving this was the first task I assigned to my student Derek McLachlin. His experiments did not support the LHCC model, disulfide were not formed at positions 61 and 68, d positions of the heptad Our conclusion: b2 forms ”an atypical coiled coil”

7 “Troubles are good for you.” -Efraim Racker, Cornell University

8 “Work hard, don’t have fun, and save your money for your old age
“Work hard, don’t have fun, and save your money for your old age.” -Leon Heppel, Cornell University So we worked hard, we carried out some other studies of the enzyme, we had some fun, and we explored various avenues to see how we might learn more about the structure.

9 The dimerization domain of b
Dimer in solution, but monomer in crystal (crystallization from MPD/isopropanol/water) Panel A shows hydrophobic face with alanines in orange, branched aliphatics in green. Panel B highlights alanines along the face Panel C shows a model dimerized about this face: this will produce a right-handed coiled coil In collaboration with my colleague Brian Shilton, my studen Paul Del Rizzo reported the structure of a polypeptide containing most of the dimerization domain. Under the conditions of crystallization, this polypeptide formed an isolated helix. Examination of the helix revealed a strip of alanine residues, shown in oragne, that defined a right handed strip on the helix. The hydorphobicity of the strip was reinforced by larger nonpolar residues that flanked the central alanines. This is the only obvious potential dimerization interface; such dimerization would produce a right-handed coiled coil as shown in the model in panel c. This realizatiion set me off on a flurry or literature and database searching, where I learned that RHCCs had recently become a topic of interest, and that they had a distinctive sequence pattern. RHCCs had earlier been proposed by Linus Pauling, but had not been found, the way that the LHCCs proposed by Francis Crick had.

10 Selective disulfide bond formation between a and h positions
Observation: preferential disulfide formation between an (a) position from one helix and the (h) from the adjacent helix. This result implies that (a) & (h) positions are at the helix-helix interface … but how can 79 link to 72 and 83? 14. Although a high resolution structure has so far eluded us, we have recently obtained protein chemical evidence that tells us much about the structure. We returned to a technique we used earlier, selective disulfide bond formation. In these experiments we have mostly used the expressed dimerization domain, achieving selective disulfide bond formation by dialysis vs a trace of copper in the presence of a high concentration of cysteine. This experiment focussed on the a and h positions, we have tried others but have not achieved more than traces of dimer. Individual positions had a modest to moderate tendency to form homodisulfides. As you move toward the center of the domain this tendency is decreased. Then we tested all pairwise combinations, and found strong disulfide formation in all cases between adjacent a and h positions--as you can see in the boxed lanes. For example 79(h) was linked with 83(a), but also with 72(a). On the one hand this confirms the a/h surface as the center of the dimerization interaction, demonstrating the significance of the 11-residue pattern and the right-handedness of the coiled coil. However it raises the question of how 79 can link to either of two positions, 72 and 83, that should be so far apart.

11 Offset right-handed coiled coil model of b
bC bN i 68(h) x 72(a) ii 72(a) x 79(h) iii 79(h) x 83(a) iv 83(a) x 90(h) These disulfide-linked forms had shapes and stabilities characteristic of native proteins They also interacted strongly with F1-ATPase 15. To explain this we have developed a model with the helices of the two subunits offset by about 5.5 residues, or one half of a hendecad, producing an asymmetric dimer with positions we call bC and bN. As you can see, this places posiiton 79 of bC close to positoin 83 of bN, but position 79 of bN close to 72 of bC. To obtain evidence that this is the correct explanation, we have prepared both in-register homodisulfides and offset heterodisulfides, with constructs shown here, and analyzed the properties of these proteins.

12 Structure of Thermus thermophilus EG
alanine zipper Danielle Stock and co-workers, NSMB 2010

13 Functional relevance of right-handedness of b2: structural stabilization to torque on stator
24. Here we have a schematic diagram to help me explain the significance of right-handedness. Focus on the LHCC of gamma in the central stalk, and the RHCC of b in the peripheral stalk. During rotational catalysis, proton flow through Fo drive c-ring rotation, and the energy from at least 3 protons must be stored before an ATP can be made. It is generally recognized that the c-ring rotation will tighten the left-handed coiled in the gamma rotor. But the rotational force on alpha3beta3 will tend to twist the peripheral stalk in the opposite direction. That is, it would loosen a left-handed coiled coil in this position, but will tighten a right-handed coiled coil. Thus for both stalks, as rotational forces become stronger, the structures become tighter and more able to resist them. For this reason, we think that the most critical feature of the unusual RHCC structure of b is simply that it is right-handed. close

14 Gabriele Deckers-Hebestreit
Acknowledgments Derek McLachlin Paul Del Rizzo Yumin Bi Canada Quim Madrenas Liz Meiering International Brian Cain Stephan Wilkens Wolfgang Junge Michael Bӧrsch Gabriele Deckers-Hebestreit Paola Turina Masamitsu Futai Brian Shilton Greg Gloor Graduate students Ardy Goliaei Kristi Wood Dan Cipriano Lee-Ann Briere Matt Revington Undergraduates Carla Busnello Karen Dunkerley Nancy Wang Kevin Talbott Chelsea Botsford Stahs Pripotnev Western James Choy Gary Shaw Greg Gloor Eric Ball Lindi Wahl Mike Strong I would like to present my coauthors on this work. Paul and Kristi are current graduate students in the lab, Dan recently completed his degree and is now a postdoc with Mike Forgac at Tufts. Yumin is a research technician who has contriubuted immeasurably to this work. My colleague Brian collaborates on crystallographic studies, Matt and Derek were former Ph.D. graduates, and Jennie, Pam and Joel were udnergraduates who contributed to the work. Also thanks to the groups of Karlheinz Altendorf and Rod Capaldi for the generous gifts of antibodies used in the work.

15 Does b2 have a function beyond simply holding F1 on the membrane?

16 Electron micrograph of mitochondrial membranes
What is the structure of ATP synthase and how does it utilize an H+ gradient to make ATP? So what is the structure of ATP synthase, and how does it coulpe ion translocation and ATP synthesis? The enzyme is so porminent in mitochdrial membranes that it can be readily seen in electronmicrographs as these “lollipop”-like structures. It can also be extracted from membranes, purified, and imaged by electron microscopy, revealing the part that sits in the membrane, as well as the peripheral part you can see over here. Time to here Electron micrograph of mitochondrial membranes Purified E. coli ATP synthase

17 Right-handed vs. left-handed coiled coils
Characterized by heptad pattern where a, d positions are usually hydrophobic, knobs-into-holes packing of side chains, in-register helices Expected to have a hendecad (11-residue) pattern, but such a structure had never been seen, it was only theoretical! The difference between left and right-handed coiled coils is greater than might be expected. LHCCs are common and their sequences are characterized by repetition of a heptad pattern, where sidechains in a and d positions pack in the interface by Knobs into holes packing. 7 residues encompass 2 turns of the about the interhelical axis. The potential for RHCC has been recognized since the days of Linus Pauling, yet few have been found. One type of RHCC was postulated to have 11 residues encompass 3 turns of the helix, with the a, h d, and e positions in the interface. There is more potential for steric clashes here, because the a and h positions are much closer to the interhelical axis than a and d in a heptad. However, it has been suggested that the two stranded RHCC structure mabe possible provided small residues were in these positions.. We believe that we have this kind of structure in the b dimer, but unfortunately no high resolution dimeric structures of this type are available.

18 MSA and periodicity search by Fourier transform using REPPER FTwin support 11-residue repeat
This multiple sequence alignment of the dimerization regions of b and b’ sequences shows the 11-residue hendecad repeat, with small alanine residues (highlighted in orange) quite common in a and h positions, and larger hydrophobic residue in d and e positions (green). FT analyses of the sequences in the alignment using various scoring matrices confirms the predominant periodicity of the sequences as 3.68 (11/3) as expected for a RHCC. For a left-handed coiled coil the periodicity would be 3.5 (7/2) Peak periodicity is at 3.68 (11 residues/3 turns of helix, right-handed) Left-handed periodicity would be 3.5 (7 residues/2 turns of helix)

19 Mitochondria are derived from a bacterium
subcellular organelles Escherichia coli: a bacterial model Mitochondria are the descendents of bacteria that invaded eukaryotic cells long ago in evolutionary history, so its not surprising that bacteria contain many of the same components, including ATP syntahse. Many of us who work in the field of oxidative phosphorylation use the becterial systems to study basic processes, since they are far easier to manipulate and often have additional characterisitcs that make them favourable for basic research.

20 Stalk structures in distantly related ATPases
Archaeal A-type Eukaryotic V-type Zhang et al., JBC, 2008 Kish-Trier and Wilkens, JBC 2009

21 How stiff is the stator stalk relative to the rotor
How stiff is the stator stalk relative to the rotor? (collaboration with Wolfgang Junge) Single molecule analysis -motions of parts of enzyme were constrained by engineered disulfide bonds -a short fluorescently labeled actin filament or magnetic bead was attached -torsional stiffness (k) of various sections was determined by observing thermal fluctuations of the bead or filament Normally this setup will measure the torsional stiffness of the rotor; when a disulfide link is made between the a subunit and the c ring, b2 will restrict the fluctuations

22 The Results

23 Glycolysis occurs in cytoplasm, generates a little bit of ATP
Citric acid cycle and oxidative phosphorylation occur in mitochondria, generate a lot of ATP through ATP Synthase Now a little bit of ATP can be produced through glycosysis, but the vast majority os obtained through oxidative phosphorylation, which takes place in subcellular organelles called mitochondria. During oxidative phosphorylation, the energy released in the final stages of food degradation is used to make ATP, and this is the function of ATP synthase. I should point out that a related ATP synthase present in chloroplasts is involved in using the energy from the sun to make ATP in the process of photophosphorylation. 2:56 ATP Synthase

24 Functional effects of softening the stator stalk I
Oxidative phosphorylation in vivo

Download ppt "ATP Synthase: What Does It Take to Make a Rotary Enzyme"

Similar presentations

Ads by Google