The Light Chain Binding Domain of Expressed Smooth Muscle Heavy Meromyosin Acts as a Mechanical Lever

Structural data led to the proposal that the molecular motor myosin moves actin by a swinging of the light chain binding domain, or “neck.” To test the hypothesis that the neck functions as a mechanical lever, smooth muscle heavy meromyosin (HMM) mutants were expressed with shorter or longer necks by either deleting or adding light chain binding sites. The mutant HMMs were characterized kinetically and mechanically, with emphasis on measurements of unitary displacements and forces in the laser trap assay. Two shorter necked constructs had smaller unitary step sizes and moved actin more slowly than WT HMM in the motility assay. A longer necked construct that contained an additional essential light chain binding site exhibited a 1.4-fold increase in the unitary step size compared with its control. Kinetic changes were also observed with several of the constructs. The mutant lacking a neck produced force at a somewhat reduced level, while the force exerted by the giraffe construct was higher than control. The single molecule displacement and force data support the hypothesis that the neck functions as a rigid lever, with the fulcrum for movement and force located at a point within the motor domain. Structural data led to the proposal that the molecular motor myosin moves actin by a swinging of the light chain binding domain, or “neck.” To test the hypothesis that the neck functions as a mechanical lever, smooth muscle heavy meromyosin (HMM) mutants were expressed with shorter or longer necks by either deleting or adding light chain binding sites. The mutant HMMs were characterized kinetically and mechanically, with emphasis on measurements of unitary displacements and forces in the laser trap assay. Two shorter necked constructs had smaller unitary step sizes and moved actin more slowly than WT HMM in the motility assay. A longer necked construct that contained an additional essential light chain binding site exhibited a 1.4-fold increase in the unitary step size compared with its control. Kinetic changes were also observed with several of the constructs. The mutant lacking a neck produced force at a somewhat reduced level, while the force exerted by the giraffe construct was higher than control. The single molecule displacement and force data support the hypothesis that the neck functions as a rigid lever, with the fulcrum for movement and force located at a point within the motor domain. essential light chain regulatory light chain heavy meromyosin wild type β-cardiac actin binding loop mean-variance Muscle contracts as a result of the cyclic interaction of the molecular motor myosin with actin, powered by the hydrolysis of MgATP. A simple mechanistic model by which myosin could move actin was proposed based on the crystal structure of skeletal myosin subfragment 1 (1Rayment I. Rypniewski W.R. Schmidt-Base K. Smith R. Tomchick D.R. Benning M.M. Winkelmann D.A. Wesenberg G. Holden H.M. Science. 1993; 261: 50-58Crossref PubMed Scopus (1877) Google Scholar, 2Rayment I. Holden H.M. Whittaker M. Yohn C.B. Lorenz M. Holmes K.C. Milligan R.A. Science. 1993; 261: 58-65Crossref PubMed Scopus (1456) Google Scholar, 3Fisher A.J. Smith C.A. Thoden J. Smith R. Sutoh K. Holden H.M. Rayment I. Biophys. J. 1995; 68: 19S-26SPubMed Google Scholar). The key feature was an 8.5-nm single α-helix, stabilized by the essential and regulatory light chains (ELC and RLC),1 which formed an elongated neck region that emerged from the globular motor domain. It was suggested that a substantial portion of the myosin motor domain maintains a fixed orientation when attached to actin, while the neck region pivots about a fulcrum within the motor domain, thus generating a power stroke. Additional evidence in support of a lever arm rotation was obtained from the crystal structure of a motor domain-essential light chain complex with a transition state analog at the active site, which showed the lever arm in a second position that may represent myosin in the prepowerstroke state (4Dominguez R. Freyzon Y. Trybus K.M. Cohen C. Cell. 1998; 94: 559-571Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar). The skeletal subfragment 1 structure is likely to resemble the structure adopted at the end of the powerstroke. A comparison of the two conformations shows that smaller changes that originate at the active site are amplified into much larger movements of the lever arm. This motion could accommodate a powerstroke on the order of 10 nm, in the range of the 5–15 nm of displacement measured using single molecule techniques (5Finer J.T. Simmons R.M. Spudich J.A. Nature. 1994; 368: 113-119Crossref PubMed Scopus (1583) Google Scholar, 6Molloy J.E. Burns J.E. Kendrick-Jones J. Tregear R.T. White D.C.S. Nature. 1995; 378: 209-212Crossref PubMed Scopus (527) Google Scholar, 7Guilford W.H. Dupuis D.E. Kennedy G. Wu J. Patlak J.B. Warshaw D.M. Biophys. J. 1997; 72: 1006-1021Abstract Full Text PDF PubMed Scopus (223) Google Scholar, 8Kitamura K. Tokunaga M. Iwane A.H. Yanagida T. Nature. 1999; 397: 129-134Crossref PubMed Scopus (424) Google Scholar). The simplest mechanical model for the neck region predicts that myosin with a shorter neck (i.e. shorter lever arm) should generate smaller unitary displacements and move actin more slowly, whereas a longer neck should lead to larger displacements and more rapid actin movement (reviewed in Ref. 9Block S. Cell. 1996; 87: 151-157Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Several studies in which myosins of various neck lengths were produced either by removing light chains (10Lowey S. Waller G.S. Trybus K.M. Nature. 1993; 365: 454-456Crossref PubMed Scopus (268) Google Scholar, 11Trybus K.M. J. Biol. Chem. 1994; 269: 20819-20822Abstract Full Text PDF PubMed Google Scholar) or by genetically adding or deleting light chain binding sites (12Uyeda T.Q.P. Spudich J.A. Science. 1993; 262: 1867-1870Crossref PubMed Scopus (103) Google Scholar, 13Uyeda T.Q. Abramson P.D. Spudich J.A. Proc. Natl. Acad. Sci. 1996; 93: 4459-4464Crossref PubMed Scopus (392) Google Scholar) showed that constructs with necks shorter than wild type moved actin more slowly, while a construct with a longer neck moved actin more quickly. A chimera in which two α-actinin repeats were fused to the Dictyostelium motor domain showed a higher average velocity than a similar construct with only one α-actinin repeat, suggesting that nonnative structures can mimic some aspects of the native neck (14Anson M Geeves M.A. Kurzawa S.E. Manstein D.J. EMBO J. 1996; 15: 6069-6074Crossref PubMed Scopus (136) Google Scholar). Although these studies are consistent with a simple lever arm model, they all relied on the assumption that no kinetic changes resulted from these biochemical or genetic perturbations. Since velocity in the motility assay,v max, is dependent upon both step size (d) and the time spent attached to actin following the powerstroke (t on) (i.e. v max ≅d/t on) (15Huxley H.E. J. Biol. Chem. 1990; 265: 8347-8350Abstract Full Text PDF PubMed Google Scholar), changes in either parameter could equally well account for the observed differences in motility. Here we test the lever arm hypothesis at the single molecule level by measuring the displacement (d) and force (F) of a series of smooth muscle heavy meromyosin (HMM) mutants in which light chain binding sites were either added to or deleted from the neck. The laser trap data support the hypothesis that the neck acts as a lever and are consistent with structural data that suggest that the fulcrum for movement and force is located near the SH1 helix (4Dominguez R. Freyzon Y. Trybus K.M. Cohen C. Cell. 1998; 94: 559-571Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar). Wild type (WT) smooth muscle HMM (amino acids 1–1175) and neck length mutants of this heavy chain backbone were co-expressed with the regulatory and essential light chains using the baculovirus insect cell expression system (11Trybus K.M. J. Biol. Chem. 1994; 269: 20819-20822Abstract Full Text PDF PubMed Google Scholar). The HMM backbone was chosen as the basis for the different neck-length constructs so that monoclonal anti-rod antibody S2.2 could be used as a common means of attaching the molecules to the nitrocellulose substratum for both motility and laser trap studies. Proteins were purified by binding to actin and release with MgATP as described previously (11Trybus K.M. J. Biol. Chem. 1994; 269: 20819-20822Abstract Full Text PDF PubMed Google Scholar). Two constructs with neck lengths shorter than WT HMM were cloned (see Fig. 1). An HMM with no light chain binding region (“neckless”) had heavy chain residues 791–848 deleted. This resulted in a dimeric construct with the motor domain attached to the rod. The sequence of the region joining the motor domain to the rod in the neckless construct was ERDLGPLLQV. An HMM mutant lacking an RLC binding site (“-R site”) had amino acids 820–848 deleted. The sequence of the joining region in this construct, which contains the motor domain and ELC binding site attached to the rod, was QQQLLGPLLQV.Figure 1Schematic diagram of the constructs used in this study.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A long necked mutant (“giraffe”) in which a second ELC binding site was added between the native ELC and RLC binding sites was also cloned (Fig. 1). This mutant retains native contacts between the motor domain and the ELC and between the ELC and the RLC but introduces a foreign ELC-ELC interaction. The following sequence was introduced between Leu819 and Thr820: LGITDVIIAFQAQCRGYLARKAFAKRQQQL. This sequence is LG plus amino acids 792–819. To test if the orientation of the two ELCs with respect to each other influences the properties of the giraffe construct, a second variation was constructed (“giraffe's twisted sister”). This revised construct had one less amino acid in the added ELC site than the previous construct; thus, the two ELCs would be rotated by approximately 100°. This was accomplished by inserting the sequence of amino acids 791–819 (i.e. KITDVIIAFQAQCRGYLARKAFAKRQQQL) between Leu819 and Thr820. A phosphorylation-independent variant of giraffe HMM was also engineered. Based on earlier studies, substitution of the 50/20-kDa β-cardiac actin binding loop (CABL) for the native sequence (residues 626–653) activated smooth muscle HMM Freyzon Y. Trybus K.M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). the and twisted were also The movement of these constructs was more consistent than the giraffe construct, and thus the studies could be more with a native neck was used as the for the long necked were in 10 1 1 1 1 at was at time actin using to the PubMed Scopus Google Scholar). The of active was by to a myosin at 1 1 were obtained in and observed with a at K.M. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). was with the expressed and observed on R. Cohen C. J. Biol. PubMed Scopus Google Scholar). The motility was at in 1 as described by Trybus and K.M. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). was also at a higher in 1 anti-rod antibody S2.2 was used to a site of to the nitrocellulose K.M. J. Biol. PubMed Scopus Google Scholar). or more were used to the and of the velocity of The average velocity for a was by the by the a between for as described previously Warshaw D.M. PubMed Scopus Google Scholar). To for motility the velocity for a of and were and in a (see Fig. of laser trap techniques W.H. Dupuis D.E. Kennedy G. Wu J. Patlak J.B. Warshaw D.M. Biophys. J. 1997; 72: 1006-1021Abstract Full Text PDF PubMed Scopus (223) Google Scholar, D.E. W.H. Wu J. Warshaw D.M. J. Muscle 1996; Scopus Google Scholar, Freyzon Y. Warshaw D.M. Trybus K.M. J. Muscle 1998; PubMed Scopus Google Scholar). a of myosin were in using two laser A actin was between the two and The actin is the of a to a The acts as a on which a of HMM is so that on average only one HMM molecule with the actin at HMM was to the an antibody at K.M. J. Biol. PubMed Scopus Google Scholar) with HMM was in from and to to the antibody for HMM was to the with orientation but at a point on the were at and to the unitary To the displacements to the actin by a single HMM the image of one of the is a and position unitary displacements were To a was obtained by the trap approximately The loop contained an which was used to the laser trap and thus the forces W.H. Dupuis D.E. Kennedy G. Wu J. Patlak J.B. Warshaw D.M. Biophys. J. 1997; 72: 1006-1021Abstract Full Text PDF PubMed Scopus (223) Google Scholar). The was and as the force measurements in this may represent the unitary force to in the actin and the of the system (i.e. Although the forces measured are they are for The of J.B. Biophys. J. 1993; Full Text PDF PubMed Scopus Google Scholar) was used to of force and displacement from the laser trap with a of the time thus an of the data which of properties within the of the no about or of the and of the data are from to the is less to the introduced by and may be used to the size (i.e. and and of (t on) in the data as described previously W.H. Dupuis D.E. Kennedy G. Wu J. Patlak J.B. Warshaw D.M. Biophys. J. 1997; 72: 1006-1021Abstract Full Text PDF PubMed Scopus (223) Google Scholar, Freyzon Y. Warshaw D.M. Trybus K.M. J. Muscle 1998; PubMed Scopus Google Scholar). Two constructs with neck lengths shorter than that of wild type HMM were and expressed in the cell system (Fig. 1). construct an RLC binding site and that this construct RLC (Fig. The second shorter necked construct both an RLC and an ELC binding site showed that the two globular the rod, as would be from a neckless construct that the light chain binding domain as well as the that are to that the constructs that are in the laser trap essential and of To if the the of the measurements were Since both shorter necked constructs the these molecules for The of site and neckless was similar to that of WT HMM The mutants were also by an in motility which as a model system to the of myosin at the molecular shorter necked constructs moved actin more slowly than WT The neckless construct moved actin at of the velocity of WT HMM while site moved actin at the of WT HMM To the molecular basis for the differences in actin between the various the mechanical and kinetic properties of these constructs were characterized in the laser the single molecule level, actin velocity is the unitary displacement by myosin the and the time spent attached to actin following the powerstroke by the max ≅d/t of on were obtained for the mutants and compared with WT HMM by displacement time series data obtained in the laser trap (Fig. were characterized by of in which displacement were The is reduced upon of HMM to actin (5Finer J.T. Simmons R.M. Spudich J.A. Nature. 1994; 368: 113-119Crossref PubMed Scopus (1583) Google Scholar, 6Molloy J.E. Burns J.E. Kendrick-Jones J. Tregear R.T. White D.C.S. Nature. 1995; 378: 209-212Crossref PubMed Scopus (527) Google Scholar, 7Guilford W.H. Dupuis D.E. Kennedy G. Wu J. Patlak J.B. Warshaw D.M. Biophys. J. 1997; 72: 1006-1021Abstract Full Text PDF PubMed Scopus (223) Google Scholar), which as a means of the mean-variance (see This a for from shorter necked which generate displacement that are in and well within the of the Based on this neckless an average displacement of nm, smaller than the displacement obtained with WT The site which the neck length of WT also produced smaller displacements than WT HMM and and The obtained for WT HMM are similar to previously for smooth muscle myosin as well as expressed WT HMM similar W.H. Dupuis D.E. Kennedy G. Wu J. Patlak J.B. Warshaw D.M. Biophys. J. 1997; 72: 1006-1021Abstract Full Text PDF PubMed Scopus (223) Google Scholar, Freyzon Y. Warshaw D.M. Trybus K.M. J. Muscle 1998; PubMed Scopus Google Dupuis D.E. W.H. Patlak J.B. Waller G.S. Trybus K.M. Warshaw D.M. S. Proc. Natl. Acad. Sci. S. 1999; PubMed Scopus Google Scholar). of displacement showed that the site mutant had shorter than WT HMM of mechanical neck constructs WT necks different from WT different from WT neck constructs necks different from for v max are expressed as the with the in the of The for all laser trap are expressed as the S.E. these the of data by (see different from WT different from in a for v max are expressed as the with the in the of The for all laser trap are expressed as the S.E. these the of data by (see forces were also (Fig. The of force within the time series data was similar to that of displacement and thus by The neckless construct forces that were somewhat than that of WT HMM the lever arm model the mechanical properties of the neck a longer neck should result in larger unitary a longer necked construct with an additional ELC binding site was This type of construct is a more test of the lever arm hypothesis (Fig. that a than a in is of giraffe HMM by showed that the construct for an longer neck region (Fig. the construct actin in a suggesting that its properties were (Fig. The of was larger in the giraffe construct compared with WT HMM from two consistent within with the construct an additional ELC binding less than of the actin moved in the motility and they moved at a at than WT The of motility were for giraffe HMM and for giraffe The of ELC to the had no on motility. of the giraffe HMM were used in an to unitary displacement the data were of to the that smooth muscle to move actin is dependent on light chain we that light chain was to the giraffe HMM this is a active mutant that contains the elongated neck region as the giraffe construct should the of motility. had previously that of the loop to the sequence in myosin produced a active molecule Freyzon Y. Trybus K.M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), and thus we expressed a long necked that was active when (see with a native neck as the control. constructs were in the The of max was more than than of max (Fig. This result shows that the of the interaction with actin by the in the neck. of were compared in with were no differences in as would from a simple lever arm model (Fig. of (Fig. was also by the of an average velocity for a longer but no differences emerged (Fig. This in motility as well as at higher previously moved actin at the of WT and of had only a this to that of WT HMM Freyzon Y. Trybus K.M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). A second long necked construct, twisted (see was expressed in order to test if the orientation of the two ELCs had an on the of motility. The motility observed with this construct was of This is within the range of observed for both and the construct Fig. the that an velocity in the in motility is that its unitary displacements could be This in to be the an average displacement of nm, 1.4-fold than its which produced The at the actin binding loop step size the step of is from the unitary step size of WT The on for was longer than for its but both constructs shorter than obtained for WT forces produced by were approximately that by the higher of the is that the of an ELC site introduced a within the which could the of motility of was than its The of this was to a test for the myosin neck region acts as a mechanical lever that force and displacements within the motor domain. characterized the molecular of smooth muscle HMM mutants with shorter and longer neck by measuring in unitary and unitary the neck acts as a simple lever one is that in the laser the of should be to lever arm displacements of two necked constructs were and of the obtained with WT the long necked giraffe mutant displacements that were that of its control. The between unitary displacements and lever arm length is the hypothesis that the neck acts as a lever arm (Fig. of this is that the lever into the motor domain, neckless mutant force and was also observed for other neckless myosin genetically in Dictyostelium myosin S. T. Y. Yanagida T. T. Sutoh K. Biophys. 1993; PubMed Scopus Google Scholar) or from skeletal muscle myosin G. J. S. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). The portion of the long that in the neckless construct (residues which the more domain of the region (residues could an additional of lever arm for generating force and motion in this The crystal structures of skeletal subfragment 1 and smooth muscle that the rotation of the and the lever arm of two two that are located at either end of the SH1 helix (4Dominguez R. Freyzon Y. Trybus K.M. Cohen C. Cell. 1998; 94: 559-571Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar). with the structural the between neck length and (Fig. that the fulcrum for movement is nm within the motor domain. the of the motor domain in a fixed orientation with respect to actin, and the point at which myosin a rotation is to the region than to the The lever hypothesis also about the between lever arm length and unitary force (see Fig. but these are all described a between lever arm length and unitary displacement (Fig. data showed that neckless somewhat less force than WT while the giraffe construct more force than its control. data the simplest lever arm model, which would force to A second model, suggested by and Spudich in proposed that the neck region acts more an than a rigid lever and predicts that force (F) should be to data also this model, which was used to changes in data with changes in muscle length I. M. G. M. M.A. M. Nature. 1998; PubMed Scopus Google Scholar), the changes were to be the result of both a rotation and of the neck A mechanical model, that we previously proposed to the and of light skeletal muscle myosins (10Lowey S. Waller G.S. Trybus K.M. Nature. 1993; 365: 454-456Crossref PubMed Scopus (268) Google Scholar, Waller G.S. D.E. Trybus K.M. Warshaw D.M. S. Proc. Natl. Acad. Sci. 1994; PubMed Scopus Google Scholar), predicts that force should be to this model, a in the motor domain force at the end of the which acts as a rigid This force would an in the subfragment region Waller G.S. D.E. Trybus K.M. Warshaw D.M. S. Proc. Natl. Acad. Sci. 1994; PubMed Scopus Google Scholar). A comparison of the force data from the laser trap with the for the (see Fig. that the model may be the the displacement and the force data support the that the neck region acts as a rigid lever and that much of the myosin is to the neck. It should be that force measurements in this are the within the system (see and thus based on these data should be in light of this An of the neck by Yanagida and K. Iwane Yanagida T. Biophys. J. Scholar). Although they also obtained a reduced velocity with a similar neckless construct of Dictyostelium they showed that the unitary step size was by the in neck length and that the velocity was only to an increase in the time of following the powerstroke K. Iwane Yanagida T. Biophys. J. Scholar). This the that the neck region to the of the unitary displacement data support the that the neck acts as a mechanical lever arm. we also evidence that kinetic changes with several of the neck length both in the unitary and in (Fig. the assumption and that all changes in velocity are to changes in unitary displacement max ≅d/t which was in several previous studies with neck length mutants (12Uyeda T.Q.P. Spudich J.A. Science. 1993; 262: 1867-1870Crossref PubMed Scopus (103) Google Scholar, 13Uyeda T.Q. Abramson P.D. Spudich J.A. Proc. Natl. Acad. Sci. 1996; 93: 4459-4464Crossref PubMed Scopus (392) Google Scholar, M Geeves M.A. Kurzawa S.E. Manstein D.J. EMBO J. 1996; 15: 6069-6074Crossref PubMed Scopus (136) Google Scholar), is moved actin at the as its the step size was in to a previous which that a long necked myosin showed motility than WT myosin T.Q. Abramson P.D. Spudich J.A. Proc. Natl. Acad. Sci. 1996; 93: 4459-4464Crossref PubMed Scopus (392) Google Scholar). A kinetic to changes in velocity of neck also from several previous studies. in the to the reduced velocity and force of myosin Waller G.S. D.E. Trybus K.M. Warshaw D.M. S. Proc. Natl. Acad. Sci. 1994; PubMed Scopus Google Scholar). in the RLC to in motility J. Sci. 1999; Google Scholar). a RLC point with the of and Biophys. J. 1996; Full Text PDF PubMed Scopus Google Scholar). that the unitary step size was by this point these data also for the of velocity kinetic as a result of structural to the neck. that changes in the length of the light chain binding domain the unitary displacement and force of both shorter and longer necked constructs in a that is consistent with the neck as a lever arm. The fulcrum for the rotation is located nm to the end of the motor domain, from the to the neck region also to the of several as by changes in state and by changes in the unitary displacement It is that kinetic changes are in these smooth muscle myosin myosin is by light chain the neck region acts as a lever arm but may also to mechanical to the site within the motor domain. and for for in data on in the for and Kennedy for that were to the trap this