Conserved Asp-137 Imparts Flexibility to Tropomyosin and Affects Function

Tropomyosin (Tm) is an α-helical coiled-coil that controls muscle contraction by sterically regulating the myosin-actin interaction. Tm moves between three states on F-actin as either a uniform or a non-uniform semi-flexible rod. Tm is stabilized by hydrophobic residues in the “a” and “d” positions of the heptad repeat. The highly conserved Asp-137 is unusual in that it introduces a negative charge on each chain in a position typically occupied by hydrophobic residues. The occurrence of two charged residues in the hydrophobic region is expected to destabilize the region and impart flexibility. To determine whether this region is unstable, we have substituted hydrophobic Leu for Asp-137 and studied changes in Tm susceptibility to limited proteolysis by trypsin and changes in regulation. We found that native and Tm controls that contain Asp-137 were readily cleaved at Arg-133 with t½ of 5 min. In contrast, the Leu-137 mutant was not cleaved under the same conditions. Actin stabilized Tm, causing a 10-fold reduction in the rate of cleavage at Arg-133. The actin-myosin subfragment S1 ATPase activity was greater for the Leu mutant compared with controls in the absence of troponin and in the presence of troponin and Ca2+. We conclude that the highly conserved Asp-137 destabilizes the middle of Tm, resulting in a more flexible region that is important for the cooperative activation of the thin filament by myosin. We thus have shown a link between the dynamic properties of Tm and its function. Tropomyosin (Tm) is an α-helical coiled-coil that controls muscle contraction by sterically regulating the myosin-actin interaction. Tm moves between three states on F-actin as either a uniform or a non-uniform semi-flexible rod. Tm is stabilized by hydrophobic residues in the “a” and “d” positions of the heptad repeat. The highly conserved Asp-137 is unusual in that it introduces a negative charge on each chain in a position typically occupied by hydrophobic residues. The occurrence of two charged residues in the hydrophobic region is expected to destabilize the region and impart flexibility. To determine whether this region is unstable, we have substituted hydrophobic Leu for Asp-137 and studied changes in Tm susceptibility to limited proteolysis by trypsin and changes in regulation. We found that native and Tm controls that contain Asp-137 were readily cleaved at Arg-133 with t½ of 5 min. In contrast, the Leu-137 mutant was not cleaved under the same conditions. Actin stabilized Tm, causing a 10-fold reduction in the rate of cleavage at Arg-133. The actin-myosin subfragment S1 ATPase activity was greater for the Leu mutant compared with controls in the absence of troponin and in the presence of troponin and Ca2+. We conclude that the highly conserved Asp-137 destabilizes the middle of Tm, resulting in a more flexible region that is important for the cooperative activation of the thin filament by myosin. We thus have shown a link between the dynamic properties of Tm and its function. Tropomyosin (Tm) 3The abbreviations used are:TmtropomyosinTntroponinS1myosin subfragment 1WTwild typeMALDI-TOFmatrix-assisted laser desorption ionization time-of-flightMOPS4-morpholinepropanesulfonic acidNTAnitrilotriacetic acidMSmass spectrometry. is a coiled-coil α-helix whose isoforms function in several ways when bound to F-actin. In skeletal and cardiac muscle, Tm controls contraction by regulating the binding of myosin to actin in conjunction with troponin (Tn) and Ca2+ (1Gordon A.M. Homsher E. Regnier M. Physiol. Rev. 2000; 80: 853-924Crossref PubMed Scopus (1363) Google Scholar). Tm is also involved in the myosin-induced cooperative activation of the thin filament (2Lehrer S.S. Geeves M.A. J. Mol. Biol. 1998; 277: 1081-1089Crossref PubMed Scopus (170) Google Scholar). In smooth muscle, Tm is involved in the cooperative activation of contraction by phosphorylated myosin (3Smiley L.B. Barany M. Biochemistry of Smooth Muscle Contraction. Academic Press, San Diego1966: 63-75Google Scholar). Non-muscle Tm stabilizes actin filaments and modulates the binding of other proteins (4Lees-Miller J.P. Helfman D.M. BioEssays. 1991; 13: 429-437Crossref PubMed Scopus (307) Google Scholar). Equilibria between three thin filament states, B (blocked), C (closed or Ca2+-induced), O or M (open or myosin-induced), are involved in the regulation of muscle contraction (5McKillop D.F. Geeves M.A. Biophys. J. 1993; 65: 693-701Abstract Full Text PDF PubMed Scopus (671) Google Scholar). Ca2+ binding to Tn facilitates the initial binding of myosin heads in the first step. In the second step, myosin heads cooperatively facilitate the movement of Tm into the O-state to allow isomerization of the heads and the generation of force. There are several single site Tm mutations that are involved in familial hypertrophic cardiomyopathy (6Gomes A.V. Barnes J.A. Harada K. Potter J.D. Mol. Cell. Biochem. 2004; 263: 115-129Crossref PubMed Scopus (56) Google Scholar, 7Tardiff J.C. Heart Fail. Rev. 2005; 10: 237-248Crossref PubMed Scopus (154) Google Scholar, 8Bottinelli R. Coviello D.A. Redwood C.S. Pellegrino M.A. Maron B.J. Spirito P. Watkins H. Reggiani C. Circ. Res. 1998; 82: 106-115Crossref PubMed Scopus (140) Google Scholar, 9Jagatheesan G. Rajan S. Petrashevskaya N. Schwartz A. Boivin G.P. Arteaga G.M. Solaro R.J. Liggett S.B. Wieczorek D.F. Am. J. Physiol. 2007; 293: H949-H958Crossref PubMed Scopus (43) Google Scholar, 10Michele D.E. Metzger J.M. J. Mol. Med. 2000; 78: 543-553Crossref PubMed Scopus (38) Google Scholar) that increase the Ca2+ sensitivity, indicating a shift into the B-state. However, the link between these mutations and the functional changes associated with familial hypertrophic cardiomyopathy is unknown. tropomyosin troponin myosin subfragment 1 wild type matrix-assisted laser desorption ionization time-of-flight 4-morpholinepropanesulfonic acid nitrilotriacetic acid mass spectrometry. Coiled-coils are primarily stabilized by inter-helical hydrophobic interactions in positions a and d in the 7-residue pseudo repeat, a-to-g. Residues at a- and d-positions consist mainly of Leu, Ala, Leu, Val, Tyr, and Met. At position 137 in both α- and β-Tm there is an Asp in a d-position instead of the typical hydrophobic residue. The remaining 24 Asp residues in Tm are outside of the hydrophobic ridge. Asp-137 is conserved in all skeletal and smooth muscle Tms and Tm from Drosophila to humans. Also, for non-muscle Tms, there is an Asp in an equivalent d-position. Asp residues with a short negatively charged side chain located at equivalent d-positions on both chains would be expected to destabilize the coiled-coil due to electrostatic repulsion. To determine the role of Asp-137 in a hydrophobic position, we substituted a Leu for the Asp in recombinant chicken skeletal α-Tm. In a preliminary study we determined that the Leu substitution increased the local and global thermal stability (11Sumida J.P. Hayes D. Langsetmo K. Lehrer S.S. Biophys. J. 2006; 90Google Scholar). Here we report that tryptic cleavage occurs rapidly at Arg-133 for the native protein and that Leu substitution for Asp-137 markedly inhibits this process, indicating a local instability in native Tm in this region. F-actin inhibits the rate of Tm cleavage 10-fold, but proteolysis occurs in the same place, Arg-133. Although Asp-137 locally destabilizes the coiled-coil, it does not affect the binding to actin. However, the actin-myosin subfragment-1 (S1) ATPase activity is greater for the Leu mutant than for its control, C190A, in the absence of troponin and in the presence of troponin and Ca2+. This study provides evidence for the importance of structural flexibility due to a local unstable region that controls the function of Tm in its regulation of muscle activity. Protein Preparation—All the Tms used in this work are recombinant Tms that have an Ala-Ser N-terminal extension, mimicking the N-terminal acetylation of native Tm (12Monteiro P.B. Lataro R.C. Ferro J.A. Reinach F.C. J. Biol. Chem. 1994; 269: 10461-10466Abstract Full Text PDF PubMed Google Scholar). To facilitate manipulation of the gene, a C190A Tm DNA construct corresponding to the TPM1 gene for chicken skeletal tropomyosin (Reinach laboratory, Ref. 12Monteiro P.B. Lataro R.C. Ferro J.A. Reinach F.C. J. Biol. Chem. 1994; 269: 10461-10466Abstract Full Text PDF PubMed Google Scholar) was inserted between a BamH1 and Nde1 restriction site in a PAED4 plasmid vector. This vector was then used as a template for the D137L/C190A clone. Because both D137L/C190A and C190A lack the native cysteine at 190, complications due to potential disulfide cross-linking were avoided. The effects of the Leu substitution in D137L/C190A were controlled for by comparing its function with C190A and WT (Cys-190), which retains the native Cys at position 190. Point mutations for these controls and the D137L/C190A Tm were performed by modifying the C190A template using the QuikChange procedure (Stratagene) and confirmed by sequence analysis. BL21 Escherichia coli cells were transformed using the PAED4 plasmid. Bacterial colonies were grown on ampicillin plates, and individual colonies were selected and used to inoculate a starter culture, which was grown overnight. Plasmid DNA was isolated and purified (Qiagen Miniprep) from samples of the overnight culture. BamH1 and Nd11 restriction nucleases were used to excise the gene insert and visualized on an agarose gel to check the condition of the transformed plasmid. The overnight cultures were then used to inoculate 1 liter of Luria Bertani medium, and these preparations were then induced with 0.5 mm isopropyl-1-thio-β-d-galactopyranoside when the O.D. of the growth medium reached 0.6. Induction of the TPM1 gene expression was confirmed by SDS-PAGE analysis of the growth medium at various stages during the growth. Cells were grown for an additional 3 h after inoculation, at which time they were harvested by centrifuging the medium at 4000 rpm for 30 min, discarding the supernatant and placing the pelleted cells in a –80 °C freezer for overnight storage. Cells were lysed using freeze-thaw by adding 50 ml of lysis buffer, (50 mm Tris, 5 mm EDTA, 100 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, and 25% sucrose) to the frozen pellet. Cellular debris was separated from the solution by centrifugation at 15,000 rpm for 20 min. The resulting supernatant was placed into an 80 °C water bath and incubated for an additional 10 min after the appearance of a precipitate. The precipitate was removed by centrifugation at 15,000 rpm for 40 min. The pH of the resulting solution was lowered to 4.6 to precipitate tropomyosin at its isoelectric point. The precipitate from the isoelectric precipitation was resuspended in HEPES buffer mm 5 mm 50 mm NaCl, pH and was purified using a using a from 100 mm to 1 N-terminal sequence and mass analysis were used to the of The presence of of the purified Tm solution was determined by of the Leu mutant that it not to actin. However, an E. coli K. A. S. G. R. J. Muscle Res. PubMed Scopus Google Scholar, H. K. A. K. S. A. PubMed Scopus Google Scholar, D.M. D.M. J. Biochem. PubMed Scopus Google Scholar) that from the the of DNA was removed by removed the and cleaved using a with a in 100 mm NaCl, pH Tm, and were determined using both and using for Tm and for was from and purified by of S.S. G. PubMed Scopus Google Scholar, J. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). S1 was by S. S. PubMed Scopus Google Scholar). The S1 and were at –80 °C after in in the presence of was a from the of D. and J. S1 was removed by of a of S1 and F-actin in the presence of 5 mm and the of and Tm was performed in a of N-terminal sequence analysis was performed to determine N-terminal sequence of all recombinant Tms and confirmed by mass and that all recombinant Tms were Tm to F-actin binding was confirmed by and by of ATPase with Tm S.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). ATPase were on a 50 using the that the of from to an a in at S. A. PubMed Scopus Google Scholar). Tm binding of ATPase was during of Tm S.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M.A. M. Lehrer S.S. 2000; PubMed Scopus Google Scholar). The Ca2+ of ATPase for the in the presence of troponin was performed using a 1 mm buffer 20 mm 5 mm mm NaCl, pH the was to be 80 or 100 mm for performed in the absence and presence of Ca2+ Ca2+ were and 1 Scholar) to the D.M. Am. J. Physiol. PubMed Google Scholar) and and was used to the Ca2+ The was determined by the ATPase on the Ca2+ to the using were from the of and of in HEPES buffer in NaCl, 5 mm 10 mm 10 mm HEPES buffer, pH was with trypsin in a 30 °C water At various were removed and with trypsin to the was to that trypsin the The samples were then for gel and mass Tm bound to Tm at was in the absence and presence of F-actin for in The samples were with acid and to mass using a N-terminal acid SDS-PAGE protein of samples were and with using an The was used to the first 10 N-terminal residues. was performed on using the of the with the at and with the hydrophobic was compared at Asp-137 and in the Tm H. C. S. A. 2005; PubMed Scopus Google Scholar) using of Leu mutant a and a wild type Tm were used in the tryptic of the controls C190A and WT two each the of the Tm two an N-terminal and a due to initial tryptic cleavage of skeletal at Arg-133 L.B. J. Biol. Chem. Full Text PDF PubMed Google Scholar). with native muscle Tm that also was cleaved at Arg-133 not The same site of cleavage was for these recombinant chicken We also that at the of the Tms as by a in were used to the of the Tm and and to determine the of initial cleavage at Arg-133. both the WT and C190A the of the with a t½ of min and were and were cleaved into with In contrast, the Leu mutant D137L/C190A was not cleaved at Arg-133 during a However, the Leu mutant was by trypsin as by the appearance of The mass and the of cleavage and were determined by mass and N-terminal of C190A and D137L/C190A with trypsin for the of the are in the The are not at min the presence of the presence of and and the presence of the presence of at min and 3 and at min. cleaved were during the at sequence of from gel D137L/C190A and and C190A cleaved and The the the determined N-terminal the mass determined by and the of Tm by mass of Tm a of the single chain at and a of the at of the laser ionization procedure not the were for each time of Here we of C190A at 3 and min. At 3 min, in to the and the trypsin 20 and are At min, remaining Tm chains are 1 as as Tm to be the presence of chains of indicating that as a in in with L.B. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of also be but of and and with it was not The mass the when compared with N-terminal The of D137L/C190A with trypsin for short and a At min, of Tm chains and chains are The in mass is which to acid residues for of min of two other of mass cleavage to and is in with the gel The Leu mutant trypsin the Tm are not by the presence of a of mass The and initial cleavage were determined by and the from and the to N-terminal Tm the expected N-terminal sequence of chicken with the Ala-Ser The sequence of from the proteins C190A and WT to cleavage at which a whose sequence with The mass of chicken from to from mass with that from the that of residues The determined mass of the with that for residues with the Ala-Ser N-terminal The N-terminal acid sequence at that the first site of was after the at This is the same site of for the in of The second site is at and the site to be in the region determined from the of Tm to determine the of F-actin on the trypsin proteolysis of Tm, we compared the of trypsin on the with Tm and F-actin 1 h of with Tm was cleaved rapidly but the presence of actin the rate of cleavage of Tm by a of 10 F-actin was not by the 1 h of with that Tm on and there was at This that F-actin Tm from in with the acid of the and C P. L.B. PubMed Scopus Google Scholar) and the of the region A. A. J. Mol. Biol. 2006; PubMed Scopus Google which would cleavage at The rate of cleavage bound to F-actin that actin stabilizes However, the of and that cleavage at Arg-133 Tm is bound to actin. The Leu mutant was not cleaved or bound to actin during the same time as the as of binding properties of the Tms were studied by with F-actin and also by of F-actin S1 ATPase activity with Tm S.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar) using the F-actin that for the Leu mutant and controls Tm was found in the supernatant when 1 Tm was with 10 actin mm HEPES buffer, pH 5 mm 30 mm and to the actin. Tm of the ATPase at that the Leu mutant D137L/C190A to actin with binding not To determine the of the more Leu mutant on the ATPase of the thin filament actin we used the S. A. PubMed Scopus Google Scholar). At the ATPase was more for the Leu mutant than the The S1 of the ATPase that the thin filament the Leu mutant a greater activity than the C190A both in the thin filament in the presence of Tn and in the absence of In with in the absence of Ca2+ the were the is that Asp-137 destabilizes a region in the middle of Tm, resulting in a more flexible that is important for the cooperative activation of the thin filament by myosin. We this unstable region in native Tm as a which the or when to a Tm to be a non-uniform flexible as to a uniform flexible rod. This study that dynamic instability flexibility in Tm and that flexibility and function are have shown that thermal and local of acid residues in the middle of the Tm α-helix to the global Lehrer S.S. J. Mol. Biol. PubMed Scopus Google Scholar, S.S. J. Mol. Biol. PubMed Scopus Google Scholar, H. PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar, A. 2006; Full Text Full Text PDF PubMed Scopus Google In a preliminary study we that Leu substituted for Asp-137 in C190A control, the by and increased the of the local (11Sumida J.P. Hayes D. Langsetmo K. Lehrer S.S. Biophys. J. 2006; 90Google Scholar). are to allow for cleavage Protein Chem. PubMed Scopus Google Scholar, Biol. PubMed Scopus Google Scholar, M. Rev. Biochem. PubMed Scopus Google Scholar). work with skeletal Tm that Arg-133 is the site of initial cleavage by trypsin and that it was to the Asp in the hydrophobic of the coiled-coil heptad L.B. J. Biol. Chem. Full Text PDF PubMed Google Scholar). We that when Asp-137 is with Leu, the susceptibility of Tm to tryptic cleavage is study that Tm at indicating that the unstable region to position The site of trypsin cleavage in proteins is the to and There are and residues the residues of Tm, with located between positions and 190. The lack of limited proteolysis at other in the middle of Tm that the does not a single cooperative the region Asp-137 other unstable in the middle of cleavage at Arg-133 also occurs at °C L.B. J. Biol. Chem. Full Text PDF PubMed Google from the indicating that the Asp-137 region is more unstable than the of the At other N-terminal to to trypsin and the rate of with and with the of the in the between and 40 °C H. PubMed Scopus Google Scholar). of instability from structural that are or that resulting in local in proteins have studied using C. S. J. Mol. Biol. 2004; PubMed Scopus Google and cooperative in proteins have with 269: PubMed Scopus Google Scholar). these C. S. J. Mol. Biol. 2004; PubMed Scopus Google Scholar, J. Am. 2006; PubMed Scopus Google the cleavage is limited by the of time the is in the a a of at the cleavage The that Tm is cleaved primarily at Arg-133 and that Tm is from cleavage when Asp-137 is by Leu is with both an of in the region of Arg-133 compared with other and with the that Asp-137 is for this both the Leu mutant and its at resulting in a occurs at the same rate as cleavage at Arg-133. This was shown by of and were at short of trypsin This Tm is also on by a In with work L.B. J. Biol. Chem. Full Text PDF PubMed Google a tryptic at is also at at the for the Leu mutant occurs cleavage in the middle of the at the same rate as cleavage for the This that to of cleavage for the is to that the cleavage at in the region not at of other located to the C Tm cleavage at Arg-133 is also when Tm is bound to actin. However, the rate of cleavage was actin stabilizes the of Tm or the of Because the N-terminal at by trypsin was also the were also stabilized on actin. This is in with the due to P. L.B. PubMed Scopus Google Scholar, A. A. J. Mol. Biol. 2006; PubMed Scopus Google Scholar). The Leu substitution not the binding of Tm to actin. However, the Leu substitution increase the filament ATPase and the by Tm on the ATPase We this in of the of thin filament regulation (5McKillop D.F. Geeves M.A. Biophys. J. 1993; 65: 693-701Abstract Full Text PDF PubMed Scopus (671) Google Scholar). Because the Ca2+ is the does not to be In contrast, the increased activity at Ca2+ and the greater activity in the absence of Tn a shift in the or an increase in the cooperative M.A. Lehrer S.S. Biophys. J. 1994; Full Text PDF PubMed Scopus Google which is with a in flexibility. In it is to that an increase in activation by Tm compared with skeletal Tm associated with an increase in due to which increased between Tms S.S. Geeves M.A. PubMed Scopus Google Scholar). structural have that conserved in the hydrophobic also destabilize the S. K. N. Med. Biol. 2007; PubMed Scopus Google there is a in this region. However, of trypsin cleavage is in the absence or presence of there are several and Because the rate of proteolysis of a region is to the of time that it is cleavage is a for local the in susceptibility a in the local at these two H. C. S. A. 2005; PubMed Scopus Google Scholar) determined the of a of Tm in the hydrophobic was between the two heptad by and Because in the between the was for this this is due to a of the side chains of the hydrophobic of the in the region with the Asp-137 region provides an into the would a hydrophobic side chain were at the 137 position the Leu side chains the hydrophobic in with the charged side chains that are of the hydrophobic The of a of side chain interactions by the is with stability and increased of flexibility in Tm at Asp-137 is by a by R. A. S. 2007; PubMed Scopus Google after this work was that that are in the myosin coiled-coil Asp residues are into its hydrophobic ridge. Asp-137 in muscle tropomyosin is highly conserved a of from to humans. its role in the middle of tropomyosin to a flexible region is to its function. would a more Tm be that flexibility is for actin binding A. PubMed Scopus Google Scholar, A. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). that function be by increased flexibility actin at the local myosin the be Tm is the flexibility at this position not the to shift associated with We that the of flexibility is to the There are several single site mutations that are associated with familial hypertrophic cardiomyopathy (6Gomes A.V. Barnes J.A. Harada K. Potter J.D. Mol. Cell. Biochem. 2004; 263: 115-129Crossref PubMed Scopus (56) Google Scholar, 7Tardiff J.C. Heart Fail. Rev. 2005; 10: 237-248Crossref PubMed Scopus (154) Google Scholar, 8Bottinelli R. Coviello D.A. Redwood C.S. Pellegrino M.A. Maron B.J. Spirito P. Watkins H. Reggiani C. Circ. Res. 1998; 82: 106-115Crossref PubMed Scopus (140) Google Scholar, 9Jagatheesan G. Rajan S. Petrashevskaya N. Schwartz A. Boivin G.P. Arteaga G.M. Solaro R.J. Liggett S.B. Wieczorek D.F. Am. J. Physiol. 2007; 293: H949-H958Crossref PubMed Scopus (43) Google Scholar, 10Michele D.E. Metzger J.M. J. Mol. Med. 2000; 78: 543-553Crossref PubMed Scopus (38) Google Scholar). the Leu is not associated with familial hypertrophic this study provides a to Tm mutations that are associated with skeletal and cardiac Tm dynamic properties to affect function. We and for in the expression and of Tm, for in the ATPase and for the for N-terminal sequence and Langsetmo and for