Contraction in striated and cardiac muscles is regulated by the motions of a Ca 2؉ -sensitive tropomyosin͞troponin switch. In contrast, troponin is absent in other muscle types and in nonmuscle cells, and actomyosin regulation is myosin-linked. Here we report an unusual crystal structure at 2.7 Å of the C-terminal 31 residues of rat striated-muscle ␣-tropomyosin (preceded by a fragment of the GCN4 leucine zipper). The C-terminal 22 residues (263-284) of the structure do not form a two-stranded ␣-helical coiled coil as does the rest of the molecule, but here the ␣-helices splay apart and are stabilized by the formation of a tail-to-tail dimer with a symmetry-related molecule. The site of splaying involves a small group of destabilizing core residues that is present only in striated muscle tropomyosin isoforms. These results reveal a specific recognition site for troponin T and clarify the physical basis for the unique regulatory mechanism of striated muscles.
This study shows that besides the residues in the flap and residues 79-81 in the S1 substrate-binding pocket which undergo conformational changes upon inhibitor binding, residues 29 and 30 can also adapt their conformation to fit certain inhibitors. Conformational flexibility of the HIV protease plays an important role in inhibitor binding.
In regulated myosins, motor and enzymatic activity are toggled between on- and off-states by a switch located on its lever arm, or regulatory domain (RD). This region consists of a long alpha-helical "heavy chain" stabilized by a "regulatory" and an "essential" light chain. The on-state is activated by phosphorylation of the regulatory light chain of smooth muscle RD, or by direct binding of Ca2+ to the essential light chain of molluscan RD. Crystal structures are available only for the molluscan RD. To understand the pathway between the on and off states in more detail, we have now also determined the crystal structure of a molluscan (scallop) RD in the absence of Ca2+. Our results indicate that loss of Ca2+ abolishes most of the interactions between the light chains and may increase flexibility of the RD heavy chain. We propose that disruption of critical links with the C-lobe of the regulatory light chain is the key event initiating the off-state in both smooth muscle and molluscan myosins.
Two crystal forms of a complex between trypsin-modified elongation factor Tu-MgGDP from Escherichia coli and the antibiotic tetracycline have been solved by X-ray diffraction analysis to resolutions of 2.8 and 2.1 A, respectively. In the P2(1) form, cocrystals were grown from a solution mixture of the protein and tetracycline. Six copies of the trypsin-modified EF-Tu-MgGDP-tetracycline complex are arranged as three sets of dimers in the asymmetric unit. In the second crystal form, tetracycline was diffused into P4(3)2(1)2 crystals, resulting in a monomeric complex in the asymmetric unit. Atomic coordinates have been refined to crystallographic R factors of 18.0% for the P2(1) form and 20.0% for the P4(3)2(1)2 form. In both complexes, tetracycline makes significant interactions with the GTPase active site of EF-Tu. The phenoldiketone moiety of tetracycline interacts directly with the Mg(2+), the alpha-phosphate group of GDP and two amino acids, Thr25 and Asp80, which are conserved in the GX(4)GKS/T and DX(2)G sequence motifs found in all GTPases and many ATPases. The molecular complementarity, previously unrecognized between invariant groups present in all GTPase/ATPases and the active moiety of tetracycline, may have wide-ranging implications for all drugs containing the phenoldiketone moiety as well as for the design of new compounds targeted against a broad range of GTPases or ATPases.
Detergent-soluble DNA is the fraction (2-4%) of DNA that is released into the supernate upon mild detergent lysis. It is nonmitochondrial in origin. It labels efficiently with deoxy[3H]ribonucleosides and the labeling is prevented by inhibitors of polymerase a and ribonucleotide reductase. In previous publications we have characterized detergent-soluble DNA from splenocytes of immunologically activated mice. In this publication we show that incorporation of [3H]thymidine into detergent-soluble DNA is prevented by pretreatment with novobiocin, 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), and teniposide (VM26), three inhibitors of type II topoisomerases. Camptothecin, an inhibitor of type I topoisomerases, also reduces incorporation of [3H]thymidine but only to 50% of control levels. In addition to affecting incorporation of [3lHlthymidine, preincubation with the topoisomerase II inhibitors m-AMSA and VM26 alters the amount of DNA recovered in the detergent-soluble fraction. At low concentrations of m-AMSA the amount of detergent-soluble DNA increases somewhat, whereas at higher drug concentrations a marked decrease is observed. Treatment with VM26 results in diminished amounts of DNA being released into the detergent-soluble fraction as well. However, maximal inhibition of detergent-soluble DNA release by VM26 requires the presence of camptothecin. Therefore, we suggest that topoisomerases play an important role in making a small part of lymphocyte chromatin detergent labile. Furthermore, these results are consistent with recent studies demonstrating a role for topoisomerases in yeast replication. Thus, the newly synthesized portion of detergent-soluble DNA may arise as DNA replication intermediates not yet stabilized into mature chromatin.Topoisomerases are a group of enzymes that interconvert various topological isomers of DNA without altering the nucleotide sequence (1-3). Type II topoisomerases result in 2n changes in linking number through transient doublestranded breaks and covalent insertion of the enzyme at the 5' end of DNA. The energy for rejoining the broken strand(s) comes from an enzyme-associated ATPase activity (4). Type I topoisomerases produce unit changes in linking number by means of single-stranded breaks and covalent insertion of the enzyme at the 3' end of DNA (5). Because of the topological constraints of DNA replication, some sort of topoisomerase involvement is likely (6). It is now accepted that resolution of intertwined pairs of newly replicated DNA molecules involves type II DNA topoisomerases (7,8)
We reported recently that splenocytes from concanavalin A-stimulated mice rapidly incorporated [3HJthy- In experiments testing the effects of aphidicolin (5 or 10 ,ug/ml) or hydroxyurea (2 or 5 mM), the inhibitor was added 15-60 min before addition of [3H]dThd and its presence was maintained throughout the pulse-chase period.Isolation and Analysis of DNA from the DS and DetergentInsoluble (DI) Fractions. DS DNA was purified from NP-40 supernate as described (9), except that cesium chloride density centrifugation and dialysis were omitted. Each isolate from 5 x 107 cells was dissolved in 100 ,ul of 10 mM Tris-HCl (pH 8.0). Aliquots were resolved by electrophoresis using native 1.2% agarose gels. After visualization of the DNA with ethidium bromide, the gel was cut into 2.0-mm slices.Each slice was dissolved as described (9)
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