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Norma J Greenfield

Publications and source records attributed to Norma J Greenfield.

11 recordsLinked to original sources
PubMed2006

Solution NMR structure of the junction between tropomyosin molecules: implications for actin binding and regulation.

Tropomyosin is a coiled-coil protein that binds head-to-tail along the length of actin filaments in eukaryotic cells, stabilizing them and providing protection from severing proteins. Tropomyosin cooperatively regulates actin's interaction with myosin and mediates the Ca2+ -dependent regulation of contraction by troponin in striated muscles. The N-terminal and C-terminal ends are critical functional determinants that form an "overlap complex". Here we report the solution NMR structure of an overlap complex formed of model peptides. In the complex, the chains of the C-terminal coiled coil spread apart to allow insertion of 11 residues of the N-terminal coiled coil into the resulting cleft. The plane of the N-terminal coiled coil is rotated 90 degrees relative to the plane of the C terminus. A consequence of the geometry is that the orientation of postulated periodic actin binding sites on the coiled-coil surface is retained from one molecule to the next along the actin filament when the overlap complex is modeled into the X-ray structure of tropomyosin determined at 7 Angstroms. Nuclear relaxation NMR data reveal flexibility of the junction, which may function to optimize binding along the helical actin filament and to allow mobility of tropomyosin on the filament surface as it switches between regulatory states.

Actins↗
PubMed2005

Structural requirements of tropomodulin for tropomyosin binding and actin filament capping.

Regulation of actin filament dynamics underlies many cellular functions. Tropomodulin together with tropomyosin can cap the pointed, slowly polymerizing, filament end, inhibiting addition or loss of actin monomers. Tropomodulin has an unstructured N-terminal region that binds tropomyosin and a folded C-terminal domain with six leucine-rich repeats. Of tropomodulin 1's 359 amino acids, an N-terminal fragment (Tmod1(1)(-)(92)) suffices for in vitro function, even though the C-terminal domain can weakly cap filaments independent of tropomyosin. Except for one short alpha-helix with coiled coil propensity (residues 24-35), the Tmod1(1)(-)(92) solution structure shows that the fragment is disordered and highly flexible. On the basis of the solution structure and predicted secondary structure, we have introduced a series of mutations to determine the structural requirements for tropomyosin binding (using native gels and CD) and filament capping (by measuring actin polymerization using pyrene fluorescence). Tmod1(1)(-)(92) fragments with mutations of an interface hydrophobic residue, L27G and L27E, designed to destroy the alpha-helix or coiled coil propensity, lost binding ability to tropomyosin but retained partial capping function in the presence of tropomyosin. Replacement of a flexible region with alpha-helical residues (residues 59-61 mutated to Ala) had no effect on tropomyosin binding but inhibited the capping function. A mutation in a region predicted to be an amphipathic helix (residues 65-75), L71D, destroyed the capping function. The results suggest that molecular flexibility and binding to actin via an amphipathic helix are both required for tropomyosin-dependent capping of the pointed end of the actin filament.

Actin Depolymerizing Factors↗
PubMed2004

Structure and tropomyosin binding properties of the N-terminal capping domain of tropomodulin 1.

Two families of actin regulatory proteins are the tropomodulins and tropomyosins. Tropomodulin binds to tropomyosin (TM) and to the pointed end of actin filaments and "caps" the pointed end (i.e., inhibits its polymerization and depolymerization). Tropomodulin 1 has two distinct actin-capping regions: a folded C-terminal domain (residues 160-359), which does not bind to TM, and a conserved, N-terminal region, within residues 1-92 that binds TM and requires TM for capping activity. NMR and circular dichroism were used to determine the structure of a peptide containing residues 1-92 of tropomodulin (Tmod1(1-92)) and to define its TM binding site. Tmod1(1-92) is mainly disordered with only one helical region, residues 24-35. This helix forms part of the TM binding domain, residues 1-35, which become more ordered upon binding a peptide containing the N-terminus of an alpha-TM. Mutation of L27 to E or G in the Tmod helix reduces TM affinity. Residues 49-92 are required for capping but do not bind TM. Of these, residues 67-75 have the sequence of an amphipathic helix, but are not helical. Residues 55-62 and 76-92 display negative 1H-15N heteronuclear Overhauser enhancements showing they are flexible. The conformational dynamics of these residues may be important for actin capping activity.

Actins↗
PubMed2004

Circular dichroism analysis for protein-protein interactions.

Circular dichroism (CD) spectroscopy is a useful technique for studying protein-protein interactions in solution. CD in the far ultraviolet region (178-260 nm) arises from the amides of the protein backbone and is sensitive to the conformation of the protein. Thus CD can determine whether there are changes in the conformation of proteins when they interact. CD bands in the near ultraviolet (350-260 nm) and visible regions arise from aromatic and prosthetic groups. There are also changes in these regions when proteins bind to each other. Because CD is a quantitative technique, changes in CD spectra are directly proportional to the amount of the protein-protein complexes formed, and these changes can be used to estimate binding constants. Changes in the stability of the protein complexes as a function of temperature or added denaturants, compared to the isolated proteins, can also be used to determine binding constants.

Animals↗
PubMed2003

Tropomodulin contains two actin filament pointed end-capping domains.

Tropomodulin 1 (Tmod1) is a approximately 40-kDa tropomyosin binding and actin filament pointed end-capping protein that regulates pointed end dynamics and controls thin filament length in striated muscle. In vitro, the capping affinity of Tmod1 for tropomyosin-actin filaments (Kd approximately 50 pm) is several thousand-fold greater than for capping of pure actin filaments (Kd approximately 0.1 microM). The tropomyosin-binding region of Tmod1 has been localized to the amino-terminal portion between residues 1 and 130, but the location of the actin-capping domain is not known. We have now identified two distinct actin-capping regions on Tmod1 by testing a series of recombinant Tmod1 fragments for their ability to inhibit actin elongation from gelsolin-actin seeds using pyrene-actin polymerization assays. The carboxyl-terminal portion of Tmod1 (residues 160-359) contains the principal actin-capping activity (Kd approximately 0.4 microM), requiring residues between 323 and 359 for full activity, whereas the amino-terminal portion of Tmod1 (residues 1-130) contains a second, weaker actin-capping activity (Kd approximately 1.8 microM). Interestingly, 160-359 but not 1-130 enhances spontaneous actin nucleation, suggesting that the carboxyl-terminal domain may bind to two actin subunits across the actin helix at the pointed end, whereas the amino-terminal domain may bind to only one actin subunit. On the other hand, the actin-capping activity of the amino-terminal but not the carboxyl-terminal portion of Tmod1 is enhanced several thousand-fold in the presence of skeletal muscle tropomyosin. We conclude that the carboxyl-terminal capping domain of Tmod1 contains a TM-independent actin pointed end-capping activity, whereas the amino-terminal domain contains a TM-regulated pointed end actin-capping activity.

Actins↗
PubMed2003

The structure of the carboxyl terminus of striated alpha-tropomyosin in solution reveals an unusual parallel arrangement of interacting alpha-helices.

Coiled coils are well-known as oligomerization domains, but they are also important sites of protein-protein interactions. We determined the NMR solution structure and backbone (15)N relaxation rates of a disulfide cross-linked, two-chain, 37-residue polypeptide containing the 34 C-terminal residues of striated muscle alpha-tropomyosin, TM9a(251-284). The peptide binds to the N-terminal region of TM and to the tropomyosin-binding domain of the regulatory protein, troponin T. Comparison of the NMR solution structure of TM9a(251-284) with the X-ray structure of a related peptide [Li, Y., Mui, S., Brown, J. H., Strand, J., Reshetnikova, L., Tobacman, L. S., and Cohen, C. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 7378-7383] reveals significant differences. In solution, residues 253-269 (like most of the tropomyosin molecule) form a canonical coiled coil. Residues 270-279, however, are parallel, linear helices, novel for tropomyosin. The packing between the parallel helices results from unusual interface residues that are atypical for coiled coils. Y267 has poor packing at the coiled-coil interface and a lower R(2) relaxation rate than neighboring residues, suggesting there is conformational flexibility around this residue. The last five residues are nonhelical and flexible. The exposed surface presented by the parallel helices, and the flexibility around Y267 and the ends, may facilitate binding to troponin T and formation of complexes with the N-terminus of tropomyosin and actin. We propose that unusual packing and flexibility are general features of coiled-coil domains in proteins that are involved in intermolecular interactions.

Amino Acid Sequence↗
PubMed2003

Tropomyosin ends determine the stability and functionality of overlap and troponin T complexes.

Tropomyosin binds end to end along the actin filament. Tropomyosin ends, and the complex they form, are required for actin binding, cooperative regulation of actin filaments by myosin, and binding to the regulatory protein, troponin T. The aim of the work was to understand the isoform and structural specificity of the end-to-end association of tropomyosin. The ability of N-terminal and C-terminal model peptides with sequences of alternate alpha-tropomyosin isoforms, and a troponin T fragment that binds to the tropomyosin overlap, to form complexes was analyzed using circular dichroism spectroscopy. Analysis of N-terminal extensions (N-acetylation, Gly, AlaSer) showed that to form an overlap complex between the N-terminus and the C-terminus requires that the N-terminus be able to form a coiled coil. Formation of a ternary complex with the troponin T fragment, however, effectively takes place only when the overlap complex sequences are those found in striated muscle tropomyosins. Striated muscle tropomyosins with N-terminal modifications formed ternary complexes with troponin T that varied in affinity in the order: N-acetylated > Gly > AlaSer > unacetylated. The circular dichroism results were corroborated by native gel electrophoresis, and the ability of the troponin T fragment to promote binding of full-length tropomyosins to filamentous actin.

Amino Acid Sequence↗
PubMed2002

Functions of tropomyosin's periodic repeats.

Tropomyosin binds along actin filaments and regulates actin-myosin interaction in muscle and nonmuscle cells. Seven periodic amino acid repeats are proposed to correspond to actin binding sites, and the middle periods are important for cooperative activation of actin by myosin. The functional contributions of individual periods were studied in mutants in which periods 2-6 were individually deleted from rat striated muscle alphaalpha-tropomyosin or replaced with a leucine zipper sequence. Unacetylated recombinant tropomyosins were assayed for actin binding, regulation of the actomyosin ATPase with troponin, cooperative myosin S1-induced binding to actin, and thermal stability. Tropomyosin function is relatively insensitive to deletion of period 2, but loss increases as the deletion is shifted toward the C-terminus. Retention of function upon deletion of the periodic repeats is in the order of 2 > 3 approximately 4 approximately 6 >> 5. Internal periods are important for specific functions and are not quasiequivalent. Deletion of period 5 (residues 166-207), and especially deletion or replacement of residues 166-188, a constitutively expressed region encoded by exon 5, had severe consequences on actin affinity and cooperative myosin S1-induced binding to actin. Period 6, residues 208-242, part of the troponin binding site, is required for full inhibition of the actomyosin ATPase in the absence of calcium. The effect of the deletion can depend on its context, suggesting that sequence alone is not the only factor important for function. We propose that the local structure and stability, and consequent flexibility, of the coiled coil are major determinants of actin affinity.

Actins↗
PubMed2002

Structure and interactions of the carboxyl terminus of striated muscle alpha-tropomyosin: it is important to be flexible.

Tropomyosin (TM) binds to and regulates the actin filament. We used circular dichroism and heteronuclear NMR to investigate the secondary structure and interactions of the C terminus of striated muscle alpha-TM, a major functional determinant, using a model peptide, TM9a(251-284). The (1)H(alpha) and (13)C(alpha) chemical shift displacements show that residues 252 to 277 are alpha-helical but residues 278 to 284 are nonhelical and mobile. The (1)H(N) and (13)C' displacements suggest that residues 257 to 269 form a coiled coil. Formation of an "overlap" binary complex with a 33-residue N-terminal chimeric peptide containing residues 1 to 14 of alpha-TM perturbs the (1)H(N) and (15)N resonances of residues 274 to 284. Addition of a fragment of troponin T, TnT(70-170), to the binary complex perturbs most of the (1)H(N)-(15)N cross-peaks. In addition, there are many new cross-peaks, showing that the binding is asymmetric. Q263, in a proposed troponin T binding site, shows two sets of side-chain (15)N-(1)H cross-peaks, indicating conformational flexibility. The conformational equilibrium of the side chain changes upon formation of the binary and ternary complexes. Replacing Q263 with leucine greatly increases the stability of TM9a(251-284) and reduces its ability to form the binary and ternary complexes, showing that conformational flexibility is crucial for the binding functions of the C terminus.

Amino Acid Sequence↗
PubMed2002

Tropomyosin requires an intact N-terminal coiled coil to interact with tropomodulin.

Tropomodulins (Tmods) are tropomyosin (TM) binding proteins that bind to the pointed end of actin filaments and modulate thin filament dynamics. They bind to the N termini of both "long" TMs (with the N terminus encoded by exon 1a of the alpha-TM gene) and "short" nonmuscle TMs (with the N terminus encoded by exon 1b). In this present study, circular dichroism was used to study the interaction of two designed chimeric proteins, AcTM1aZip and AcTM1bZip, containing the N terminus of a long or a short TM, respectively, with protein fragments containing residues 1 to 130 of erythrocyte or skeletal muscle Tmod. The binding of either TMZip causes similar conformational changes in both Tmod fragments promoting increases in both alpha-helix and beta-structure, although they differ in binding affinity. The circular dichroism changes in the Tmod upon binding and modeling of the Tmod sequences suggest that the interface between TM and Tmod includes a three- or four-stranded coiled coil. An intact coiled coil at the N terminus of the TMs is essential for Tmod binding, as modifications that disrupt the N-terminal helix, such as removal of the N-terminal acetyl group from AcTM1aZip or striated muscle alpha-TM, or introduction of a mutation that causes nemaline myopathy, Met-8-Arg, into AcTM1aZip destroyed Tmod binding.

Acetylation↗