- Email: [email protected]
The myosin crossbridge problem
Cell, Vol. 48, 909-910,
March
27, 1987, Copyright
0 1987 by Cell Press
The Myosin Crossbridge
Thomas D. Pollard Department of Cell Biology and Anatomy Johns Hopkins University School of Medicine Baltimore, Maryland 21205
For twenty-five years the myosin crossbridge has been the focus of efforts to understand how ATP hydrolysis produces force and motion during muscle contraction. Those who believe the lovely textbook drawings that depict tilting crossbridges pulling actin filaments past myosin thick filaments may even think that the problem has been solved. Much has been learned, but the secrets of crossbridge motion have resolutely evaded the efforts of a generation of biophysicists and biochemists. A new insight is provided by the work of Hynes, Block, White, and Spudich, reported in this issue of Cell. Using a novel in vitro assay, these authors show that most of the tail of muscle myosin is not required for myosin to move along actin filaments. The value of this work will be apparent if we first consider why actin-myosin crossbridges have presented such a challenging experimental problem. Why Is the Crossbridge Problem So Difficult? The main problem is that crossbridges act asynchronously, like a completely disorganized and uncoordinated crew trying to row a racing shell (Huxley and Brown, JMB 30, 383-434, 1967). This ensures that a fraction of the crossbridges produces force at every moment, but to a biophysicist it also means that averaging techniques such as spectroscopy will record a blurred signal from a large population of crossbridges. With X-ray diffraction, for example, one can show a shift in the location of the population of crossbridges when a muscle is stimulated to contract, but the details of the individual events-including most of the information about the motion of the myosin molecule relative to the actin filaments-is averaged out. Additional problems are the speed of the molecular events and the very large size of the molecular components. Active crossbridges bind to and dissociate from the actin filaments more than 50 times per second and the actual force-producing step may be even faster. Synchrotron radiation has improved the time resolution of X-ray diffraction into the millisecond range (Huxley and Faruqi, Ann. Rev. Siophys. Bioeng. 72, 381-417, 1983) but the speed of the motion-producing step remains a problem. The myosin molecule is enormous, consisting of six polypeptide chains with a total molecular weight ap1
Long Heavy
Meromyosin
proaching 500,000. The tail is an a-helical coiled-coil and the heads are elongated globular domains (Figure 1). Most attention has been focused on the heads, because each has an actin-binding site and an active site for ATP hydrolysis. Although promising crystals of the heads are available (Winkelmann et al., JMB 787,487-501,1985), we still know little about their structure beyond the gross shape and the general locations of the active site and the light chains. Furthermore, the structure of the actin molecule and the actin filament are solved only at low resolution (reviewed by Pollard and Cooper, Ann. Rev. Biochem. 55, 987-1035, 1988). Crossbridge Chemistry Given the obstacles to structural studies, most of the recent progress has been in defining the kinetics and thermodynamics of the chemical reactions involving myosin, actin, and ATP A simplified reaction mechanism is shown in Figure 2 (see Hibberd and Trentham, Ann. Rev. Biophys. Biophys. Chem. 75, 119-161, 1986). Although this scheme appears formidable at first glance, it is straightforward once the following important features are understood. l Myosin heads exist in three different states: myosin with an empty active site (M), or with either ATP (M-ATP) or the products of ATP hydrolysis (M-ADP-Pi) bound to the active site. *All three of these myosin species can either be bound to actin (A-M) or exist freely (M), giving a total of six numbered intermediates in this minimal scheme. (Note that 1 and 4 are shown twice, but are the same. A better representation of this scheme would have these six intermediates on the surface of a cylinder.) l Myosin with bound nucleotides has a very low affinity for actin compared to free myosin (K,,~10-~ M for A-M-ATP and A-M-ADP-Pi versus K@O-10 M for A-M). This is due mainly to a large difference in the dissociation rate constants. Consequently, the nucleotide-containing intermediates are in rapid equilibrium with actin filaments, binding and dissociating over 50 times per second. *Step 3-1 is 200 times faster than step 6-+4, so actin stimulates the cycle of ATP hydrolysis. *There are two main pathways through the actin-myosin-ATPase cycle. If the actin concentration is low, the cycle will be largely l-+2+5-6+3-1 etc., with dissociation of the weakly bound intermediates and rebinding prior to product release. If the actin concentration is high, mass action will shift the rapid equilibria (steps 3-5 and 4-6) towards the bound states and the cycle will be largely strong
1
I
o@
Short
HMM
1 Hinge
i
I20nm
1
Minireview
Problem
1
weak
AM -*AM-ATP
strong
z3AMsADPePi
*AM
1
4
I 17nm I Figure
1.
150nm
t I
4
M _
Figure
2
5
M’ATP
__
M’ADP.Pi 6
-,’
M A
Cdl 910
A. Tilting
crossbridge
Actin filament
6. Bending crossbridge
C. Hinge contraction
Figure 3.
l-2*3+1 etc. This is obviously an oversimplification, since a fraction of the crossbridges will dissociate and reassociate one or more times in either case. *The quantitatively important changes in free energy occur when ATP binds to myosin (steps l-2 and 4-5) and when the products of ATP hydrolysis dissociate from the myosin (steps 3+1 and 6+4). The equilibrium constant for ATP hydrolysis on myosin is only 10. Therefore, the energy required for the production of force and motion is provided by ATP binding, stored as a conformational change in the M-ATP and M-ADP-Pi intermediates, and released when the products (particularly Pi) dissociate from the A-M-ADP-Pi complex. Where Is the Motion Produced in the Actin-Myosin Complex? This analysis of the reaction pathway pinpoints step 3+1 as the time and place where motion is produced and raises two central questions: what is the physical nature of the motion and where in the actin-myosin complex is motion produced? The paper by Hynes et al. (pp. 953-963) provides new evidence that the motion is produced within the head end of the myosin. These authors attached a proteolytic fragment of myosin with two heads and 40 nm of tail (called short heavy meromyosin) to the surface of formalin-fixed bacteria via an antibody to the short tail. When supplied with ATP, the heavy meromyosin propelled the bacteria along actin filaments like a train on a cog railway. These experiments confirm and extend in a convincing and quantitative way earlier observations of motion in model systems with purified actin and muscle heavy meromyosin by Yano et al. (J. Biochem. 84, 277-283, 1978; Nature 299, 557-559, 1982). Complementary work with myosin-I from Acanthamoeba showed that this single-headed myosin lacking an a-helical tail can also move plastic beads (Albanesi et al., JBC 260,8649-8852,1985) and membra-
nous organelles (Adams and Pollard, Nature 322, 754756, 1986) along actin filaments. To many it will come as no surprise that the head end of myosin produces the motion, since the heads have the active sites. However, those in the field are aware of an impressive body of evidence gathered by William Harrington and his colleagues (Harrington and Rodgers, Ann. Rev. Biochem. 53,35-73, 1984) that a”hinge” region in the myosin tail, 40 to 60 nm from the heads, may be involved in force production. Harrington argues that a reversible melting of the a-helix in the hinge region could produce both motion and force (Figure 3C). The fact that a myosin molecule without a hinge region can move a bacterium argues against that region’s necessity, although Hynes et al. point out that their experiment with bacteria is an essentially unloaded production of motion. Development of maximum force occurs when the velocity is zero (as when trying to lift a heavy weight) and this may yet involve the hinge region of the myosin tail. However, classical physiological evidence for an inverse relationship between force and motion argues that the two are the result of the same mechanism. The question of how the myosin heads and actin produce force and motion is still wide open. Spectroscopic experiments (Cooke, CRC Crit. Rev. Biochem. 27,53-118, 1986) seem to rule out simple tilting of the myosin heads on actin (Figure 3A) as depicted in most textbooks, so many investigators would speculate that the movement occurs in the distal part of the heads (Figure 38) at the head-tail junction or even in the proximal 40 nm of the tail included in the experiment of Hynes et al. Given that the 3-l transition most likely produces the motion, it would be helpful to have a clearer structural picture of the weakly bound intermediates (2 and 3) for comparison with the well-characterized rigor complex (1) (Amos, Ann. Rev. Biophys. Biophys. Chem. 74, 291-313, 1985). This comparison has not been possible because the weakly bound complexes appear to be more disordered than the rigor complexes (Craig et al., PNAS 82, 3247-3251, 1985). Research Opportunities The solution to the myosin crossbridge problem depends on progress in several areas. First and foremost, we need the three-dimensional structures of myosin and actin at atomic resolution. Second, new methods are needed to establish how conformational changes in the actin-myosin complex produce force and motion. Electron microscopic methods that afford views of individual molecules may be the best place to start, although the resolution will be limited. New spectroscopic methods will also be valuable. Third, we need modified myosin and actin molecules in order to test proposed mechanisms. Cloning and expression of myosin cDNAs and genomic fragments have been achieved in several laboratories; thus, novel molecules produced by in vitro mutagenesis will soon be available. This approach will provide insights in the near future, but will probably be most useful several years from now, after the first two goals have been achieved.
March
27, 1987, Copyright
0 1987 by Cell Press
The Myosin Crossbridge
Thomas D. Pollard Department of Cell Biology and Anatomy Johns Hopkins University School of Medicine Baltimore, Maryland 21205
For twenty-five years the myosin crossbridge has been the focus of efforts to understand how ATP hydrolysis produces force and motion during muscle contraction. Those who believe the lovely textbook drawings that depict tilting crossbridges pulling actin filaments past myosin thick filaments may even think that the problem has been solved. Much has been learned, but the secrets of crossbridge motion have resolutely evaded the efforts of a generation of biophysicists and biochemists. A new insight is provided by the work of Hynes, Block, White, and Spudich, reported in this issue of Cell. Using a novel in vitro assay, these authors show that most of the tail of muscle myosin is not required for myosin to move along actin filaments. The value of this work will be apparent if we first consider why actin-myosin crossbridges have presented such a challenging experimental problem. Why Is the Crossbridge Problem So Difficult? The main problem is that crossbridges act asynchronously, like a completely disorganized and uncoordinated crew trying to row a racing shell (Huxley and Brown, JMB 30, 383-434, 1967). This ensures that a fraction of the crossbridges produces force at every moment, but to a biophysicist it also means that averaging techniques such as spectroscopy will record a blurred signal from a large population of crossbridges. With X-ray diffraction, for example, one can show a shift in the location of the population of crossbridges when a muscle is stimulated to contract, but the details of the individual events-including most of the information about the motion of the myosin molecule relative to the actin filaments-is averaged out. Additional problems are the speed of the molecular events and the very large size of the molecular components. Active crossbridges bind to and dissociate from the actin filaments more than 50 times per second and the actual force-producing step may be even faster. Synchrotron radiation has improved the time resolution of X-ray diffraction into the millisecond range (Huxley and Faruqi, Ann. Rev. Siophys. Bioeng. 72, 381-417, 1983) but the speed of the motion-producing step remains a problem. The myosin molecule is enormous, consisting of six polypeptide chains with a total molecular weight ap1
Long Heavy
Meromyosin
proaching 500,000. The tail is an a-helical coiled-coil and the heads are elongated globular domains (Figure 1). Most attention has been focused on the heads, because each has an actin-binding site and an active site for ATP hydrolysis. Although promising crystals of the heads are available (Winkelmann et al., JMB 787,487-501,1985), we still know little about their structure beyond the gross shape and the general locations of the active site and the light chains. Furthermore, the structure of the actin molecule and the actin filament are solved only at low resolution (reviewed by Pollard and Cooper, Ann. Rev. Biochem. 55, 987-1035, 1988). Crossbridge Chemistry Given the obstacles to structural studies, most of the recent progress has been in defining the kinetics and thermodynamics of the chemical reactions involving myosin, actin, and ATP A simplified reaction mechanism is shown in Figure 2 (see Hibberd and Trentham, Ann. Rev. Biophys. Biophys. Chem. 75, 119-161, 1986). Although this scheme appears formidable at first glance, it is straightforward once the following important features are understood. l Myosin heads exist in three different states: myosin with an empty active site (M), or with either ATP (M-ATP) or the products of ATP hydrolysis (M-ADP-Pi) bound to the active site. *All three of these myosin species can either be bound to actin (A-M) or exist freely (M), giving a total of six numbered intermediates in this minimal scheme. (Note that 1 and 4 are shown twice, but are the same. A better representation of this scheme would have these six intermediates on the surface of a cylinder.) l Myosin with bound nucleotides has a very low affinity for actin compared to free myosin (K,,~10-~ M for A-M-ATP and A-M-ADP-Pi versus K@O-10 M for A-M). This is due mainly to a large difference in the dissociation rate constants. Consequently, the nucleotide-containing intermediates are in rapid equilibrium with actin filaments, binding and dissociating over 50 times per second. *Step 3-1 is 200 times faster than step 6-+4, so actin stimulates the cycle of ATP hydrolysis. *There are two main pathways through the actin-myosin-ATPase cycle. If the actin concentration is low, the cycle will be largely l-+2+5-6+3-1 etc., with dissociation of the weakly bound intermediates and rebinding prior to product release. If the actin concentration is high, mass action will shift the rapid equilibria (steps 3-5 and 4-6) towards the bound states and the cycle will be largely strong
1
I
o@
Short
HMM
1 Hinge
i
I20nm
1
Minireview
Problem
1
weak
AM -*AM-ATP
strong
z3AMsADPePi
*AM
1
4
I 17nm I Figure
1.
150nm
t I
4
M _
Figure
2
5
M’ATP
__
M’ADP.Pi 6
-,’
M A
Cdl 910
A. Tilting
crossbridge
Actin filament
6. Bending crossbridge
C. Hinge contraction
Figure 3.
l-2*3+1 etc. This is obviously an oversimplification, since a fraction of the crossbridges will dissociate and reassociate one or more times in either case. *The quantitatively important changes in free energy occur when ATP binds to myosin (steps l-2 and 4-5) and when the products of ATP hydrolysis dissociate from the myosin (steps 3+1 and 6+4). The equilibrium constant for ATP hydrolysis on myosin is only 10. Therefore, the energy required for the production of force and motion is provided by ATP binding, stored as a conformational change in the M-ATP and M-ADP-Pi intermediates, and released when the products (particularly Pi) dissociate from the A-M-ADP-Pi complex. Where Is the Motion Produced in the Actin-Myosin Complex? This analysis of the reaction pathway pinpoints step 3+1 as the time and place where motion is produced and raises two central questions: what is the physical nature of the motion and where in the actin-myosin complex is motion produced? The paper by Hynes et al. (pp. 953-963) provides new evidence that the motion is produced within the head end of the myosin. These authors attached a proteolytic fragment of myosin with two heads and 40 nm of tail (called short heavy meromyosin) to the surface of formalin-fixed bacteria via an antibody to the short tail. When supplied with ATP, the heavy meromyosin propelled the bacteria along actin filaments like a train on a cog railway. These experiments confirm and extend in a convincing and quantitative way earlier observations of motion in model systems with purified actin and muscle heavy meromyosin by Yano et al. (J. Biochem. 84, 277-283, 1978; Nature 299, 557-559, 1982). Complementary work with myosin-I from Acanthamoeba showed that this single-headed myosin lacking an a-helical tail can also move plastic beads (Albanesi et al., JBC 260,8649-8852,1985) and membra-
nous organelles (Adams and Pollard, Nature 322, 754756, 1986) along actin filaments. To many it will come as no surprise that the head end of myosin produces the motion, since the heads have the active sites. However, those in the field are aware of an impressive body of evidence gathered by William Harrington and his colleagues (Harrington and Rodgers, Ann. Rev. Biochem. 53,35-73, 1984) that a”hinge” region in the myosin tail, 40 to 60 nm from the heads, may be involved in force production. Harrington argues that a reversible melting of the a-helix in the hinge region could produce both motion and force (Figure 3C). The fact that a myosin molecule without a hinge region can move a bacterium argues against that region’s necessity, although Hynes et al. point out that their experiment with bacteria is an essentially unloaded production of motion. Development of maximum force occurs when the velocity is zero (as when trying to lift a heavy weight) and this may yet involve the hinge region of the myosin tail. However, classical physiological evidence for an inverse relationship between force and motion argues that the two are the result of the same mechanism. The question of how the myosin heads and actin produce force and motion is still wide open. Spectroscopic experiments (Cooke, CRC Crit. Rev. Biochem. 27,53-118, 1986) seem to rule out simple tilting of the myosin heads on actin (Figure 3A) as depicted in most textbooks, so many investigators would speculate that the movement occurs in the distal part of the heads (Figure 38) at the head-tail junction or even in the proximal 40 nm of the tail included in the experiment of Hynes et al. Given that the 3-l transition most likely produces the motion, it would be helpful to have a clearer structural picture of the weakly bound intermediates (2 and 3) for comparison with the well-characterized rigor complex (1) (Amos, Ann. Rev. Biophys. Biophys. Chem. 74, 291-313, 1985). This comparison has not been possible because the weakly bound complexes appear to be more disordered than the rigor complexes (Craig et al., PNAS 82, 3247-3251, 1985). Research Opportunities The solution to the myosin crossbridge problem depends on progress in several areas. First and foremost, we need the three-dimensional structures of myosin and actin at atomic resolution. Second, new methods are needed to establish how conformational changes in the actin-myosin complex produce force and motion. Electron microscopic methods that afford views of individual molecules may be the best place to start, although the resolution will be limited. New spectroscopic methods will also be valuable. Third, we need modified myosin and actin molecules in order to test proposed mechanisms. Cloning and expression of myosin cDNAs and genomic fragments have been achieved in several laboratories; thus, novel molecules produced by in vitro mutagenesis will soon be available. This approach will provide insights in the near future, but will probably be most useful several years from now, after the first two goals have been achieved.