Tropomyosin isoforms show unexpected differential effects on actin polymerization

Tropomyosin is a rod-like coiled-coil protein that forms a continuous filament that is weakly associated, but firmly-attached to the surface of the actin filaments in all eukaryotic cells. Simple eukaryotes such as yeasts have only one or two different tropomyosin isoforms which are known to be essential and perform roles in regulating the actin cytoskeleton. However higher eukaryotes have larger numbers of tropomyosins, the number of which appear linked to organismal complexity. Mammals have 4 genes producing over 40 different isoforms by alternative splicing. In higher organisms tropomyosin is best known and characterized in the regulation of striated muscle contraction. The role of tropomyosin outside of muscle is less well understood. It is generally thought to have a regulatory role in controlling interactions of actin-binding proteins and in providing additional stability to actin-filaments. In the latter case has been considered that tropomyosin binds to actin-filaments some time after their formation, both making them mechanically stiffer and protecting them from breakdown. We have produced a range of recombinant tropomyosins from all four mammalian genes and characterized their actin-binding affinities in a cosedimentation assay. We have then used them to systematically study the effects of different isoforms of tropomyosin on actin polymerization for the first time. We have monitored actin polymerization by the well-characterised change in fluorescence of a pyrene-label attached to actin. Actin polymerisation is monitored by measuring the significant fluorescence enhancement on polymerization. Our results characterize the actin-affinities of some of the TPM3 and TPM4 isoforms for the first time, These are in the same general range as mammalian isoforms previously characterized by our group and others. We demonstrate differential effects of the different isoforms on actin-polymerisation for the first time. The data unexpectedly show the most significant effects of the different isoforms appears to be in the early initiation / elongation stages of polymerizations. This is unexpected as tropomyosin is only considered to have significant affinity for actin filaments through itself forming a polymer along the surface of an actin filament. Different isoforms appear capable of both enhancing and inhibiting the early stages of polymerization, with examples of the shorter 6-actin spanning TPM1 gene isoforms showing a significant reduction in the lag-phase of early polymerization. These differential effects on different isoforms provides a new role for tropomyosin in not only stabilizing filaments, but also in helping catalyze their formation

The intrinsically disordered Tarp protein from chlamydia binds actin with a partially preformed helix

Tarp (translocated actin recruiting phosphoprotein) is an effector protein common to all chlamydial species that functions to remodel the host-actin cytoskeleton during the initial stage of infection. In C. trachomatis, direct binding to actin monomers has been broadly mapped to a 100-residue region (726-825) which is predicted to be predominantly disordered, with the exception of a ~10-residue α helical patch homologous to other WH2 actin-binding motifs. Biophysical investigations demonstrate that a Tarp726-825 construct behaves as a typical intrinsically disordered protein; within it, NMR relaxation measurements and chemical shift analysis identify the ten residue WH2-homologous region to exhibit partial α-helix formation. Isothermal titration calorimetry experiments on the same construct in the presence of monomeric G-actin show a well defined binding event with a 1:1 stoichiometry and Kd of 102 nM, whilst synchrotron radiation circular dichroism spectroscopy suggests the binding is concomitant with an increase in helical secondary structure. Furthermore, NMR experiments in the presence of G-actin indicate this interaction affects the proposed WH2-like α-helical region, supporting results from in silico docking calculations which suggest that, when folded, this α helix binds within the actin hydrophobic cleft as seen for other actin-associated proteins

Engineering nucleotide specificity of succinyl-CoA synthetase in blastocystis: the emerging role of gatekeeper residues

Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper residues) produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called "electrostatic gatekeeper effect". This proof-of-concept study demonstrates that the nucleotide specificity can be engineered by altering the electrostatic properties of the gatekeeper residues outside the binding site. Using Blastocystis succinyl-CoA synthetase (SCS, EC 6.2.1.5), we demonstrated that the gatekeeper mutant (ED) resulted in ATP-specific SCS to show high GTP specificity. Moreover, nucleotide binding site mutant (LF) had no effect on GTP specificity and remained ATP-specific. However, via combination of the gatekeeper mutant with the nucleotide binding site mutant (ED+LF), a complete reversal of nucleotide specificity was obtained with GTP, but no detectable activity was obtained with ATP. This striking result of the combined mutant (ED+LF) was due to two changes; negatively charged gatekeeper residues (ED) favored GTP access, and nucleotide binding site residues (LF) altered ATP binding, which was consistent with the hypothesis of the "electrostatic gatekeeper effect". These results were further supported by molecular modeling and simulation studies. Hence, it is imperative to extend the strategy of the gatekeeper effect in a different range of crucial enzymes (synthetases, kinases, and transferases) to engineer substrate specificity for various industrial applications and substrate-based drug design

Actomyosin regulatory properties of yeast tropomyosin are dependent upon N-terminal modification

The yeast tropomyosin 1 gene (TPM1) encodes the major isoform of the two tropomyosins (Tm) found in yeast. The gene has been expressed in E. coli and the protein purified. The gene product (yTm1) is a 199-amino acid protein that has a low affinity for actin compared to the native yTm1 purified from yeast. Mass spectrometry shows that the native protein is acetylated while the recombinant protein is not. A series of yTm1 N-terminal constructs were made with either an Ala-Ser dipeptide extension previously shown to restore actin binding to skeletal muscle Tm or the natural extension found in fibroblast Tm 5a/b. All constructs bound actin tightly and showed similar CD spectra and thermal stability. All constructs induced cooperativity in the equilibrium binding of myosin subfragment 1, to actin but the binding curves differed significantly between the constructs. The apparent cooperative unit size (n) and closed/open equilibrium (K-T) were determined using a fluorescence titration technique [Maytum et al. (1998) Biophys, J. 74, A347]. The data could be accounted for by changes in K-T (0.1-1) With no change in n, Values of n were approximately twice the structural unit size (5 actin sites). The presence of yTm on actin had little effect upon the overall affinity of S1 for actin despite showing an ability to regulate the acto-myosin interaction, These results show that the short yTm can aid our understanding of actomyosin regulation and that the N-terminus of Tm has a major influence upon its regulatory properties