Molecular Motors. Dave Wee 24 Sept Mathematical & Theoretical Biology Seminar

Transcription

1 Molecular Motors Dave Wee 24 Sept 2003 Mathematical & Theoretical Biology Seminar

2 Overview Types of motors and their working mechanisms. Illustrate the importance of motors using the example of : ATP-Synthase Chromatin remodeling motor (ACF) Discuss the importance of molecular motor modeling in system wide modeling of biological systems. Illustrate the modeling of protein motion using Brownian motion concept.

3 Types of Motors 1. Pump - Membrane protein that transports ions and small molecules across the membrane. 2. Rotary Motor - Membrane-bound structure having a spinning shaft, while the motor is fixed on the membrane. - E.g. ATP Synthase. 3. Linear Motor - Mostly found in cytoplasm. - Need a track to walk on. Filaments and microtubules serve as tracks. - E.g. Myosin, Kinesin, Dynein.

4 Rotary Motor ATP Synthase Found on bacterial and mitochondria membrane. ATP manufacturer which is the fuel that powers many other biochemical processes. Has 2 rotary motors acting in opposition F 0 and F 1 motors. Each operates on an entirely different mechanism. F 0 motor : Localization Membrane. Energy source Transmembrane electromotive force. F 1 motor : Localization Cytoplasm. Energy source ATP hydrolysis.

5 ATP Synthase 2 nd Structure Cytoplasm Periplasm

6 F 1 Motor Structure Shaft?-subunit. Surrounded by 6 alternating a and ß subunits. ATP binding sites 6 ATP binding sites, each nestles in the cleft between the a and ß subunits. Only 3 can catalyze the hydrolysis of ATP. F 1 F 0 These catalytic sites are mostly in the ß-subunits, with a few but crucial residues in the a- subunits.

7 F 1 Motor Mechanism Power Stroke 1. An ATP molecule diffuses by Brownian motion into an open catalytic site, and is only weakly bound. 2. The catalytic site wraps around it tightly by progressively forming up to H-Bonds. 3. This binding transition from weak to tight pulls the top part of the ß subunit towards the bottom part, like a hinge to about 30 o. 4. Binding free energy -> Power stroke with nearly constant force. 5. At the end of binding transition, some elastic energy is stored in the ß-sheet.

8 F 1 Motor Mechanism Exhaust Stroke 1. ATP is hydrolyzed to ADP and P. 2. The free energy of hydrolysis and the formation of 2 product molecules weaken the binding from the catalytic site. 3. The binding force is so weak such that thermal fluctuations can knock the products out of the catalytic site. 4. The elastic energy stored in the ß-sheet is released during this stage and the top part of the ß subunit returns to its original open state.

9 F 1 Motor Mechanism Complete Stroke 1 bend of a ß-sheet rotates the shaft in a single step of 120 o, with a brief pause at 90 o. When the 3 catalytic sites hydrolyze sequentially, they drives the rotation of the shaft. (See animation) The unbending of a ß-sheet will help the bending of its neighbour ß-sheet.

10 F 0 Motor Structure Has between 10 to 14 c- subunits assembled in a cylinder (c n cylinder), attached to the shaft and?-subunit. Has a transmembrane assembly consisting of the A and B subunits. F 1 The c n cylinder interfaces with the transmembrane assembly. Rotor : Shaft, c n cylinder &?-subunit. Stator(Stationary) : a-ß and A-B subunits & d. F 0

11 F 0 Motor Driving Force Driven by ion-motive force,?µ c across a membrane.?µ c contributed by : Ion(H + /Na + ) concentration gradient,?ph. Electrical potential difference,?f. Thus,?µ c =?ph +? f

12 F 0 Motor Mechanism Ionic driving force : Concentration at periplasm is higher than at cytoplasm. Electrical driving force : A blocking +ve charge in the stator. A -ve charge array wrapped around the c n cylinder. Mechanism : 1. Rotor turns anti-clockwise due to electrical driving force. 2. Once a ve site reaches the input channel, a +ve ion from the periplasm binds to it. 3. Since potential on its left is lower than on the right (as a result of the +ve blocking charge), the rotor continues to turn anti-clockwise. 4. Once it moves out of the stator, the +ve ion quickly dissociates from the site because of its low concentration in the cytoplasm. 5. (See animation)

13 Linear Motors Background Myosin Moves on actin filaments. 18 different families. Kinesin Moves on microtubules. 10 different families. Dynein Moves on microtubules. 2 different families. Each family consists of up to several dozen members. Each member may vary very significantly in makeup and function even within the same family.

14 Linear Motors Generic Structure Motor domain ATP binding site Virtually identical in structure for all 3 types of motors, even though sequence homology is very low! Track binding site Accessory structural motifs Mechanical amplifiers Coiled-coil domains Dimerization Regulation Interactions with other molecular, eg, cargo

15 Linear Motors Generic Behavior Exist as monomers, dimers, trimers or tetramers. Can take 1 or more steps before dissociating. Processive motors move along the track for long distances without detaching. They are individualist and long distance runners. Non-processive motors lose contact to the track usually after 1 cycle. They work as a team and are optimized for brief, fast interactions, such as in the muscles. They are 100m sprinters.

16 Linear Motors Generic Mechanism 1. ATP binds to motor domain and is hydrolyzed. 2. Loss of?-phosphate group from ATP creates a space that will cause a rearrangement (movement) of the ATP binding site. This is called the 1 st level of amplification. 3. The amplification is propagated to the track binding site causing it to change its structure too. 4. The 2 nd level of amplification (more powerful and useful) involves the mechanical amplifiers. This is where myosin, kinesin and dynein differs in operation.

17 Myosin In Detail High-resolution electron micrograph Motor domain Accessory structural motifs Coiled-coil domains

18 Myosin In Detail Green arrow propagation of structural change upon ATP hydrolysis from ATP binding site to track binding site. Red arrow the 1 st level amplification is relayed to the mechanical amplifier. In myosin, the amplifier is a a- helical lever, that swings up and down up to an angle of 70 o. Due to biological requirements in muscles, most myosins are non-processive motors. Motor domain of myosin Both Myosin V (g) and Myosin I (h) uses their tail domains (part of the coiled-coil domain) to bind to their cargo.

19 Kinesin In Detail High-resolution electron micrograph Motor domain Coiled-coil domains Accessory structural motifs

20 Kinesin In Detail Green arrow same as in myosin. Red arrow same as in myosin. However, in kinesin, the amplifier is a flexible neck linker. It can be docked to the motor at times. 2 proposed models of movement : Hand-over-hand model inchworm type model

21 Kinesin In Detail a Direct interaction. b Interaction mediated by a linker protein. c Interaction mediated by a linker complex.

22 Dynein In Detail High-resolution electron micrograph Motor domain Accessory structural motifs

23 Dynein In Detail Mechanistic analysis is severely hampered by the lack of a high-resolution structure. But it is believed that the propagation of ATP hydrolysis is transmitted from the right side to the left side.

24 Linear Motors Other issues 1. Diffusion effects & forces generated 2. Directionality of movement 3. Regulation of motors behaviors 4. Cellular functions & importance of motors

25 1. Diffusion Effects & Forces Generated Diffusion affects processive motors only. Movement along the track may entail both a mechanical component and a diffusive component, with different motors using different proportion of each. A single motor can move its cargo many times its own size through viscous cytoplasm at near maximum speed.

26 2. Directionality of movement Microtubules : Polar. The +ve end is fast growing while ve end is slow growing. The ve end is the anchor point. They can anchor to other microtubules to form a network. The myth of motors moving to +ve ends only is shattered when some myosin and kinesin members can move to ve ends too! How and which part of the motor determines its direction is still unknown.

27 3. Regulation of motors behaviors 2 ways to regulate motors : Turning the motor on/off. Inhibit/promote its interaction with cargo. Possible mechanisms : Phosphorylation -ve regulator of cargo binding of several motors. Causes detachment from cargo in some cases. Intramolecular interactions Tail domain (important for binding to cargo) can inhibit the motor domain, thus prevent it from moving. Cooperative behaviors : A motor can pass its cargo to a motor of a different class when it is being inhibited or when the terrain is not its forte. Some organelles switch tracks from actin filaments to microtubules and vice versa, showing evidence of cooperating between myosin and kinesin.

28 4. Cellular functions & importance of motors Transportation (including mrnas) Ciliary movement or contraction Energy generation Cellular homeostasis Cell architecture and cytoskeletal remodelling Chromatin assembly

29 4. Cellular functions & importance of motors 1. Retrograde transport of centrosomal components 2. Anterograde and retrograde transport of intermediate filaments 3. Anterograde and retrograde transport of ribonucleoprotein (RNP) complexes 4. Myosin, kinesin and dynein motors interact with components of the microtubule plus-end complex 5. Anchorage of dynein at the actin-rich cell cortex 6. Interaction of a kinesin-like protein with actin 7. Catenin-mediated anchorage of dynein at adherens junctions

30 4. Cellular functions & importance of motors 1. Myosin myopathies muscle disorder 2. Griscelli syndrome pigmentation disorder 3. Hearing loss 4. Retinitis pigmentosa photoreceptor degeneration 5. Primary ciliary dyskinesia 6. Kartagener syndrome situs inversus 7. Polycystic kidney disease 8. Virus transport 9. Anthrax susceptibility 10. Neurodegenerative diseases

31 4. Cellular functions & importance of motors Interesting but yet unanswered questions : How does a motor finds its cargo? What directs it to the correct target destination (especially the microtubules network is so complicated)? How is its activity regulated? Can it have variable speed? When a motor has unloaded its cargo, what will happen to it? Would it be dissociated or U-turn back to carry more cargo???

32 This is just the beginning As more knowledge on motors regulatory mechanisms and their new biological roles are discovered, it will be shown that motors are important in almost all areas of biological processes. Hence, to model and study a biological system s behavior, the modeling of motor dynamics must be incorporated into gene network modeling. This could be a new direction in system modeling.

33 Chromatin Assembly Background DNA is packaged into a periodic nucleoprotein complex, known as chromatin. The basic repeating unit of chromatin is the nucleosome. Each nucleosome consists of : 8 histone proteins (H2A, H2B, H3, H4) x 2 146bp of DNA turns around these proteins

34 Chromatin Assembly Background H2A H2A H2B H3 DNA 11 nm H3 H4 H2B H4 Linker DNA (approx 50 bp) Nucleosome bead (8 histones nucleotide pairs of DNA)

35 Chromatin Assembly Chromatin assembly happens in : DNA replication in S phase of cell cycle. DNA repair, transcription and histone exchange. Factors needed for chromatin assembly : Histone-chaperone complex Chaperone deliver histones to the appropriate chromatin assembly sites. ATP-utilizing chromatin assembly and remodelling factor (ACF) A processive ATP-driven motor that tracks on DNA. (see fig 1)

36 Chromatin Assembly in vitro, ACF can assemble the nucleosomes periodically, a remarkable biological feat! 2 models are proposed : Iterative-annealing model (fig 2 & 3) Directed-deposition model (fig 4) ACF is also responsible for chromatin remodeling a very important factor controlling gene activity!

37 Stochastic Modeling of Protein Motion Protein motion is dominated by Brownian motion. Its velocity is dependent on the random thermal fluctuation of surrounding water molecules. This is important because it allows proteins to move against an opposing force by utilizing occasional large thermal fluctuation. Even if 2 proteins start at the same position at t=0, they will have different paths after some time. Hence, this is a stochastic process.

38 Stochastic Modeling of Protein Motion Formulation 1-D flow, x n = x(t) x(t) is approximated by a discrete random variable, with constant time step,?t. Assumptions Markov process At each?t, it can only take 1 step of length?x it must either move left or right Objective P(right) = P(left) = 0.5 To derive the probability density function for finding the protein s displacement at time t.

39 Stochastic Modeling of Protein Motion At any time n?t, the total # of steps taken is n = R n + L n R n : # of steps to the right L n : # of steps to the left Thus, P(R n = r) = n C r (0.5) r (0.5) n-r = n C r (0.5) n We have, x n =?x(r n - L n ) =?x(2r n - n) Making R n the subject, R n = ½(x n /?x + n) But x n /?x is the # of unit lengths the protein is located. We let it be k. So, R n = ½(k + n) Finally, P(k n, t) = n C ½(k + n) (0.5) n

40 Stochastic Modeling of Protein Motion Results: See fig 12.2 It can also be shown that : E(x n ) = 0 Var(x n ) = ((?x) 2 /?t)t

41 References 1. Manfred Schliwa & Gunther Woehlke, Molecular motors, Nature Vol 422, 2003, p Reinhard Lipowsky, Molecular motors and Stochastic Models, LNP 557, 2000, p Chapter 12, Molecular motors : Theory, p George Oster & Hongyun Wang, Chapter 8, How protein motors convert chemical energy into mechanical work, p Karl A. Haushalter & James T. Kadonaga, Chromatin assembly & DNA-translocating motors, Nature, 2003, p H. Wang & G. Oster, Ratchets, power strokes, and molecular motors, Applied Physics A, 2002, p Peter Bayley, Biophysical studies of the cytoskeleton, European Biophysics Journal, 1998, p