Herbert C. Cheung

Conformation of the Regulatory Domain of Cardiac Muscle Troponin C in Its Complex with Cardiac Troponin I

Calcium activation of fast striated muscle results from an opening of the regulatory N-terminal domain of fast skeletal troponin C (fsTnC), and a substantial exposure of a hydrophobic patch, essential for Ca2+-dependent interaction with fast skeletal troponin I (fsTnI). This interaction is obligatory to relieve the inhibition of strong, force-generating actin-myosin interactions. We have determined intersite distances in the N-terminal domain of cardiac TnC (cTnC) by fluorescence resonance energy transfer measurements and found negligible increases in these distances when the single regulatory site is saturated with Ca2+. However, in the presence of bound cardiac TnI (cTnI), activator Ca2+induces significant increases in the distances and a substantial opening of the N-domain. This open conformation within the cTnC·cTnI complex has properties favorable for the Ca2+-induced interaction with an additional segment of cTnI. Thus, the binding of cTnI to cTnC is a prerequisite to achieve a Ca2+-induced open N-domain similar to that previously observed in fsTnC with no bound fsTnI. This role of cardiac TnI has not been previously recognized. Our results also indicate that structural information derived from a single protein may not be sufficient for inference of a structure/function relationship.

NMR ANALYSIS OF CARDIAC TROPONIN C-TROPONIN I COMPLEXES: EFFECTS OF PHOSPHORYLATION

Phosphorylation of the cardiac specific amino‐terminus of troponin I has been demonstrated to reduce the Ca2+ affinity of the cardiac troponin C regulatory site. Recombinant N‐terminal cardiac troponin I proteins, cardiac troponin I(33–80), cardiac troponin I(1–80), cardiac troponin I(1–80)DD and cardiac troponin I(1–80)pp, phosphorylated by protein kinase A, were used to form stable binary complexes with recombinant cardiac troponin C. Cardiac troponin I(1–80)DD, having phosphorylated Ser residues mutated to Asp, provided a stable mimetic of the phosphorylated state. In all complexes, the N‐terminal domain of cardiac troponin I primarily makes contact with the C‐terminal domain of cardiac troponin C. The non‐phosphorylated cardiac specific amino‐terminus, cardiac troponin I(1–80), was found to make additional interactions with the N‐terminal domain of cardiac troponin C.

Kinetic Studies of Calcium Binding to the Regulatory Site of Troponin C from Cardiac Muscle

We have studied the kinetics of the structural transitions induced by calcium binding to the single, regulatory site of cardiac troponin C by measuring the rates of calcium-mediated fluorescence changes with a monocysteine mutant of the protein (C35S) specifically labeled at Cys-84 with the fluorescent probe 2-[4′-(iodoacetamido)anilino]naphthalene-6-sulfonic acid. At 4°C, the binding kinetics determined in the presence of Mg2+ was resolved into two phases with positive amplitude, which were completed in less than 100 ms. The rate of the fast phase increased linearly with [Ca2+] reaching a maximum of ∼590 s−1, and that of the slow phase was approximately 100 s−1 and did not depend on Ca2+ concentration. Dissociation of bound Ca2+ from the regulatory site occurred with a rate of 102 s−1, whereas the dissociation from the two high affinity sites was about two orders of magnitude slower. These results are consistent with the following scheme for the binding of Ca2+ to the regulatory site: where the asterisks denote states with enhanced fluorescence. The apparent second-order rate constant for calcium binding is Kok1 = 1.4 × 108M−1 s−1. The two first-order transitions occur with observed rates of k1 + k−1 ≈ 590 s−1 and k2 + k−2 ≈ 100 s−1, and the binding of Ca2+ to the regulatory site is not a simple diffusion-controlled reaction. These transitions provide the first information on the rates of Ca2+-induced conformational changes involving helix movements in the regulatory domain.

Kinetic and spectroscopic evidence for three actomyosin:ADP states in smooth muscle.

Smooth muscle myosin II undergoes an additional movement of the regulatory domain with ADP release that is not seen with fast skeletal muscle myosin II. In this study, we have examined the interactions of smooth muscle myosin subfragment 1 with ADP to see if this additional movement corresponds to an identifiable state change. These studies indicate that for this myosin:ADP, both the catalytic site and the actin-binding site can each assume one of two conformations. Relatively loose coupling between these two binding sites leads to three discrete actin-associated ADP states. Following an initial, weakly bound state, binding of myosin:ADP to actin shifts the equilibrium toward a mixture of two states that each bind actin strongly but differ in the conformation of their catalytic sites. By contrast, fast myosins, including Dictyosteliummyosin II, have reciprocal coupling between the actin- and ADP-binding sites, so that either actin or nucleotide, but not both, can be tightly bound. This uncoupling, which generates a second strongly bound actomyosin ADP state in smooth muscle, would prolong the fraction of the ATPase cycle time that this actomyosin spends in a force-generating conformation and may be central to explaining the physiologic differences between this and other myosins.

Structural Kinetics of Cardiac Troponin C Mutants Linked to Familial Hypertrophic and Dilated Cardiomyopathy in Troponin Complexes

The key events in regulating cardiac muscle contraction involve Ca2+ binding to and release from cTnC (troponin C) and structural changes in cTnC and other thin filament proteins triggered by Ca2+ movement. Single mutations L29Q and G159D in human cTnC have been reported to associate with familial hypertrophic and dilated cardiomyopathy, respectively. We have examined the effects of these individual mutations on structural transitions in the regulatory N-domain of cTnC triggered by Ca2+ binding and dissociation. This study was carried out with a double mutant or triple mutants of cTnC, reconstituted into troponin with tryptophanless cTnI and cTnT. The double mutant, cTnC(L12W/N51C) labeled with 1,5-IAEDANS at Cys-51, served as a control to monitor Ca2+-induced opening and closing of the N-domain by Förster resonance energy transfer (FRET). The triple mutants contained both L12W and N51C labeled with 1,5-IAEDANS, and either L29Q or G159D. Both mutations had minimal effects on the equilibrium distance between Trp-12 and Cys-51-AEDANS in the absence or presence of bound Ca2+. L29Q had no effect on the closing rate of the N-domain triggered by release of Ca2+, but reduced the Ca2+-induced opening rate. G159D reduced both the closing and opening rates. Previous results showed that the closing rate of cTnC N-domain triggered by Ca2+ dissociation was substantially enhanced by PKA phosphorylation of cTnI. This rate enhancement was abolished by L29Q or G159D. These mutations alter the kinetics of structural transitions in the regulatory N-domain of cTnC that are involved in either activation (L29Q) or deactivation (G159D). Both mutations appear to be antagonistic toward phosphorylation signaling between cTnI and cTnC.

A Pathway of Structural Changes Produced by Monastrol Binding to Eg5

Monastrol is a small molecule inhibitor that is specific for Eg5, a member of the kinesin 5 family of mitotic motors. Crystallographic models of Eg5 in the presence and absence of monastrol revealed that drug binding produces a variety of structural changes in the motor, including in loop L5 and the neck linker. What is not clear from static crystallographic models, however, is the sequence of structural changes produced by drug binding. Furthermore, because crystallographic structures can be influenced by the packing forces in the crystal, it also remains unclear whether these druginduced changes occur in solution, at physiologically active concentrations of monastrol or of other drugs that target this site. We have addressed these issues by using a series of spectroscopic probes to monitor the structural consequences of drug binding. Our results demonstrated that the crystallographic model of an Eg5-ADP-monastrol ternary complex is consistent with several solution-based spectroscopic probes. Furthermore, the kinetics of these spectroscopic signal changes allowed us to determine the temporal sequence of drug-induced structural transitions. These results suggested that L5 may be an element in the pathway that links the state of the nucleotide-binding site to the neck linker in kinesin motors.

A model for molecules with twisted intramolecular charge transfer characteristics: solvent polarity effect on the nonradiative rates of dyes in a series of water—ethanol mixed solvents

Abstract The model of Grabowski et al. has been modified and used to account for the absence of a fluorescence band from the twisted form of molecules with torsional mechanisms. We have measured the ground and excited state spectroscopic properties of rhodamine B dissolved in a series of water—ethanol mixed solvents, and used the data to test the model. We also applied our model to 8-anilino-1-naphthalenesulfonate (ANS) in the same solvent system, using previously published decay and quantum yield data. The results show that the variation of the nonradiative rates may be attributed to changes in solvent polarity for both systems.

A kinetic model for the binding of Ca2+ to the regulatory site of troponin from cardiac muscle.

The kinetics of the binding of Ca2+ to the single regulatory site of cardiac muscle troponin was investigated by using troponin reconstituted from the three subunits, using a monocysteine mutant of troponin C (cTnC) labeled with the fluorescent probe 2-[(4′-(iodoacetamido)anilino]naphthalene-6-sulfonic acid (IAANS) at Cys-35. The kinetic tracings of binding experiments for troponin determined at free [Ca2+] > 1 μm were resolved into two phases. The rate of the fast phase increased with increasing [Ca2+], reaching a maximum of about 35 s−1 at 4 °C, and the rate of the slow phase was approximately 5 s−1 and did not depend on [Ca2+]. Dissociation of bound Ca2+ occurred in two phases, with rates of about 23 and 4 s−1. The binding and dissociation results obtained with the binary complex formed between cardiac troponin I and the IAANS-labeled cTnC mutant were very similar to those obtained from reconstituted troponin. The kinetic data are consistent with a three-step sequential model similar to the previously reported mechanism for the binding of Ca2+ to a cTnC mutant labeled with the same probe at Cys-84 (Dong et al. (1996) J. Biol. Chem. 271, 688–694). In this model, the initial binding in the bimolecular step to form the Ca2+-troponin complex is assumed to be a rapid equilibrium, followed by two sequential first-order transitions. The apparent bimolecular rate constant is 5.1 × 107 m −1 s−1, a factor of 3 smaller than that for cTnC. The rates of the first-order transitions are an order of magnitude smaller for troponin than for cTnC. These kinetic differences form a basis for the enhanced Ca2+ affinity of troponin relative to the Ca2+ affinity of isolated cTnC. Phosphorylation of the monocysteine mutant of troponin I by protein kinase A resulted in a 3-fold decrease in the bimolecular rate constant but a 2-fold increase in the two observed Ca2+ dissociation rates. These changes in the kinetic parameters are responsible for a 5-fold reduction in Ca2+ affinity of phosphorylated troponin for the specific site.

Kinesin Has Three Nucleotide-dependent Conformations IMPLICATIONS FOR STRAIN-DEPENDENT RELEASE

Although crystallographic information is available on several nucleotide-induced states in myosin, little is known about the corresponding structural changes in kinesin, since a crystallographic model is only available for the kinesin:ADP complex. This makes it difficult to characterize at a molecular level the structural changes that occur in this motor through the course of its ATPase cycle. In this study, we report on the production of a series of single tryptophan mutants of a monomeric human kinesin motor domain, which demonstrate nucleotide-dependent changes in microtubule affinity that are similar to wild type. We have used these mutations to measure intramolecular distances in both strong and weak binding states, using florescence resonance energy transfer. This work provides direct evidence that movement of the switch II loop and helix are essential to mediate communication between the catalytic and microtubule binding sites, evidence that is supported as well by molecular modeling. Kinetic studies of fluorescent nucleotide binding to these mutants are consistent with these distance changes, and demonstrate as well that binding of ADP produces two structural transitions, neither of which are identical to that produced by the binding of ATP. This study provides a basis for understanding current structural models of the kinesin mechanochemical cycle.

Activation of striated muscle: nearest-neighbor regulatory-unit and cross-bridge influence on myofilament kinetics.

We have formulated a three-compartment model of muscle activation that includes both strong cross-bridge (XB) and Ca(2+)-activated regulatory-unit (RU) mediated nearest-neighbor cooperative influences. The model is based on the tight coupling premise--that XB retain activating Ca(2+) on the thin filament. Using global non-linear least-squares, the model produced excellent fits to experimental steady-state force-pCa and ATPase-pCa data from skinned rat soleus fibers. In terms of the model, nearest-neighbor influences over the range of Ca(2+) required for activation cause the Ca(2+) dissociation rate from regulatory-units (k(off)) to decrease and the cross-bridge association rate (f) to increase each more than ten-fold. Moreover, the rate variations occur in separate Ca(2+) regimes. The energy of activation governing f is strongly influenced by both neighboring RU and XB. In contrast, the energy of activation governing k(off) is less affected by neighboring XB than by neighboring RU. Nearest-neighbor cooperative influences provide both an overall sensitization to Ca(2+) and the well-known steep response of force to free Ca(2+). The apparent sensitivity for Ca(2+)-activation of force and ATPase is a function of cross-bridge kinetic rates. The model and derived parameter set produce simulated behavior in qualitative agreement with steady-state experiments reported in the literature for partial TnC replacement, increased [P(i)], increased [ADP], and MalNEt-S1 addition. The model is an initial attempt to construct a general theory of striated muscle activation-one that can be consistently used to interpret data from various types of muscle manipulation experiments.