An Atomic Model of the Unregulated Thin Filament Obtained by X-ray Fiber Diffraction on Oriented Actin-Tropomyosin Gels

Actin can exist in multiple conformations necessary for normal function. Actin isoforms, although highly conserved in sequence, exhibit different biochemical properties and cellular roles. We used amide proton hydrogen/deuterium (HD) exchange detected by mass spectrometry to analyze conformational differences between Saccharomyces cerevisiae and muscle actins in the G and F forms to gain insight into these differences. We also utilized HD exchange to study interdomain and allosteric communication in yeast-muscle hybrid actins to better understand the conformational dynamics of actin. Areas showing differences in HD exchange between G-and F-actins are areas of intermonomer contacts, consistent with the current filament models. Our results showed greater exchange for yeast G-actin compared with muscle actin in the barbed end pivot region and areas in subdomains 1 and 2 and for F-actin in monomer-monomer contact areas. These results suggest greater flexibility of the yeast actin monomer and filament compared with muscle actin. For hybrid G-actins, the muscle-like and yeastlike parts of the molecule generally showed exchange characteristics resembling their parent actins. A few exceptions were a peptide on top of subdomain 2 and the pivot region between subdomains 1 and 3 with muscle actin-like exchange characteristics although the areas were yeastlike. These results demonstrate that there is cross-talk between subdomains 1 and 2 and the large and small domains. Hybrid F-actin data showing greater exchange compared with both yeast and muscle actins are consistent with mismatched yeast-muscle interfaces resulting in decreased stability of the hybrid filament contacts.The ability of actin to engage in a wide range of physiological functions requires that it be subject to complex spatial and temporal control by a large array of actin-binding proteins (1-3). Such regulation demands that both the monomeric and filamentous forms of actin be able to exist in a number of different conformations. Some of these may be differentially recognized by different actin-binding proteins, and some might actually be induced by the interaction of actin with these proteins.Actin is highly conserved from yeast to humans. There is 87% sequence identity between yeast and muscle actin and 91% sequence identity between yeast and nonmuscle actins. Even with this high sequence similarity and minor differences in crystal structures, there are some major differences in yeast and muscle actin behavior. Yeast, compared with muscle actin, polymerizes faster and exchanges its bound nucleotide faster. In contrast to muscle actin, the yeast actin filament releases its P i almost immediately after ATP hydrolysis and the filament fragments more readily (4). Yeast cells cannot survive with muscle actin as the sole actin, and with ␤-nonmuscle actin as the only actin, yeast cells are very sick (5). A set of biochemical data indicates that the muscle filament is less flexible than yeast (6, 7). However, conformational and structural differences that could ex...

Section: Resultsmentioning

confidence: 99%

Actin Isoform-specific Conformational Differences Observed with Hydrogen/Deuterium Exchange and Mass Spectrometry

Stokasimov

Rubenstein

2009

“…We have interpreted the data on the Tn inhibition of S1 binding rates in terms of the McKillop and Geeves three-state model (15,53) of the thin filament regulatory states (15,53 (37,60,61). The models derived from solution studies are most directly relevant to the work here.…”

Section: Discussionmentioning

confidence: 99%

Tropomyosin Exon 6b Is Troponin-specific and Required for Correct Acto-myosin Regulation

Maytum

Bathe²,

Konrad

et al. 2004

The specificity of tropomyosin (Tm) exon 6b for interaction with and functioning of troponin (Tn) has been studied using recombinant fibroblast Tm isoforms 5a and 5b. These isoforms differ internally by exons 6a/6b and possess non-muscle exons 1b/9d at the termini, hence they lack the primary TnT 1 -tropomyosin interaction, allowing study of exon 6 exchange in isolation from this. Using kinetic techniques to measure regulation of myosin S1 binding to actin and fluorescently labeled Tm to directly measure Tn binding, we show that binding of Tn to both isoforms is similar (0.1-0.5 M) and both produce well regulated systems. Calcium has little effect on Tn binding to the actin⅐Tm complex and both exons produce a 3-fold reduction in the S1 binding rate to actin⅐Tm⅐Tn in its absence. This confirms previous results that show exon 6 has little influence on Tn affinity to actin⅐Tm or its ability to fully inhibit the acto-myosin interaction. Thin filaments reconstituted with Tn and Tm5a or skeletal Tm (containing exon 6b) show nearly identical calcium dependence of acto-myosin regulation. However, Tm5b produces a dramatic increase in calcium sensitivity, shifting the activation mid-point by almost an order of magnitude. This shows that exon 6 sequence and, hence, Tm structure in this region have a significant effect upon the calcium regulation of Tn. This finding supports evidence that familial hypertrophic cardiomyopathy mutations occurring adjacent to this region can effect calcium regulation.

“…Phalloidin Does Not Alter Cooperative Interactions between Troponin Molecules-The polymerization ability of the troponin⅐tropomyosin complex (18 -20) and atomic models of actin⅐actin contacts in F-actin (4,21) suggest that longitudinal contacts along the thin filament are the most likely source of cooperativity. However, this does not exclude the possibility that cooperativity occurs across rather than along the actin filament.…”

Section: Relationship Between Ca 2ϩ Binding To the Thin Filament Regumentioning

confidence: 99%

Cooperative Effect of Calcium Binding to Adjacent Troponin Molecules on the Thin Filament-Myosin Subfragment 1 MgATPase Rate

Butters

Tobacman

1997

The myosin subfragment 1 (S1) MgATPase rate was measured using thin filaments with known extents of Ca 2؉ binding controlled by varying the ratio of native cardiac troponin versus an inhibitory troponin with a mutation in the sole regulatory Ca 2؉ binding site of troponin C. Fractional MgATPase activation was less than the fraction of troponins that bound Ca 2؉ , implying a cooperative effect of bound Ca 2؉ on cross-bridge cycling. Addition of phalloidin did not alter cooperative effects between bound Ca 2؉ molecules in the presence or absence of myosin S1. When the myosin S1 concentration was raised sufficiently to introduce cooperative myosin-myosin effects, lower Ca 2؉ concentrations were needed to activate the MgATPase rate. MgATPase activation remained less than Ca 2؉ binding, implying a true, not just an apparent, increase in Ca 2؉ affinity. MgATPase activation by Ca 2؉ was more cooperative than could be explained by cooperativeness of overall Ca 2؉ binding, the discrepancy between Ca 2؉ binding and MgATPase activation, or interactions between myosins. The results suggest the thin filament-myosin S1 MgATPase cycle requires calcium binding to adjacent troponin molecules and that this binding is cooperatively promoted by a single cycling cross-bridge. This mechanism is a potential explanation for Ca 2؉ -mediated regulation of crossbridge kinetics in muscle fibers.Just as isometric tension is cooperatively activated by Ca 2ϩ , so is the cardiac thin filament-myosin S1 1 MgATPase rate, even under conditions where there is no cooperativity in myosin S1 binding (1, 2). A possible explanation for this behavior is that ATPase activation is proportional to Ca 2ϩ binding to the many TnCs on each thin filament and that this calcium binding is cooperative (3). We tested the idea that Ca 2ϩ binding and MgATPase activation are proportional and found to the contrary that they are not. Instead, fractional MgATPase activation was considerably less than fractional Ca 2ϩ binding and more closely paralleled the number of pairs of adjacent troponins with Ca 2ϩ bound to both.To accomplish the above experiment, we employ a constitutively inhibitory form of cardiac troponin containing an inactivating mutation of the sole regulatory site of TnC, site II (4). This troponin, designated CBMII-Tn, results in a low thin filament-myosin S1 MgATPase rate that is not increased by the addition of Ca 2ϩ , analogous to previous results in which a similar TnC mutant was exchanged into myofibrils or muscle fibers (5-7). CBMII-Tn binds to actin-tropomyosin with an affinity identical to that of normal troponin in the absence of Ca 2ϩ . This binding, which is very tight for both normal troponin and for 8,9), permits the present report in which thin filaments are assembled with defined mixtures of normal troponin and CBMII-Tn. In the presence of saturating Ca 2ϩ concentrations, such thin filaments exhibit a fractional saturation of the TnC regulatory sites that is experimentally controllable by varying the relative concentrations of the two forms ...