Marek Orzechowski

Flexible Fitting of High-Resolution X-Ray Structures into Cryoelectron Microscopy Maps Using Biased Molecular Dynamics Simulations☆

A methodology for flexible fitting of all-atom high-resolution structures into low-resolution cryoelectron microscopy (cryo-EM) maps is presented. Flexibility of the modeled structure is simulated by classical molecular dynamics and an additional effective potential is introduced to enhance the fitting process. The additional potential is proportional to the correlation coefficient between the experimental cryo-EM map and a synthetic map generated for an all-atom structure being fitted to the map. The additional forces are calculated as a gradient of the correlation coefficient. During the molecular dynamics simulations under the additional forces, the molecule undergoes a conformational transition that maximizes the correlation coefficient, which results in a high-accuracy fit of all-atom structure into a cryo-EM map. Using five test proteins that exhibit structural rearrangement during their biological activity, we demonstrate performance of our method. We also test our method on the experimental cryo-EM of elongation factor G and show that the model obtained is comparable to previous studies. In addition, we show that overfitting can be avoided by assessing the quality of the fitted model in terms of correlation coefficient and secondary structure preservation.

Biased coarse-grained molecular dynamics simulation approach for flexible fitting of X-ray structure into cryo electron microscopy maps.

Several approaches have been introduced to interpret, in terms of high-resolution structure, low-resolution structural data as obtained from cryo-EM. As conformational changes are often observed in biological molecules, these techniques need to take into account the flexibility of proteins. Flexibility has been described in terms of movement between rigid domains and between rigid secondary structure elements, which present some limitations for studying dynamical properties. Normal mode analysis has also been used, but is limited to medium resolution data. All-atom molecular dynamics fitting techniques are more appropriate to fit structures into higher-resolution data as full protein flexibility is considered, but are cumbersome in terms of computational time. Here, we introduce a coarse-grained approach; a Go-model was used to represent biological molecules, combined with biased molecular dynamics to reproduce accurately conformational transitions. Illustrative examples on simulated data are shown. Accurate fittings can be obtained for resolution ranging from 5 to 20A. The approach was also tested on experimental data of Elongation Factor G and Escherichia coli RNA polymerase, where its validity is compared to previous models obtained from different techniques. This comparison demonstrates that quantitative flexible techniques, as opposed to manual docking, need to be considered to interpret low-resolution data.

Gestalt-binding of tropomyosin on actin during thin filament activation.

Our thesis is that thin filament function can only be fully understood and muscle regulation then elucidated if atomic structures of the thin filament are available to reveal the positions of tropomyosin on actin in all physiological states. After all, it is tropomyosin influenced by troponin that regulates myosin-crossbridge cycling on actin and therefore controls contraction in all muscles. In addition, we maintain that a complete appreciation of thin filament activation also requires that the mechanical properties of tropomyosin itself are recognized and then related to the effect of myosin-association on actin. Taking the Gestalt-binding of tropomyosin into account, coupled with our electron microscopy structures and computational chemistry, we propose a comprehensive mechanism for tropomyosin regulatory movement over the actin filament surface that explains the cooperative muscle activation process. In fact, well-known point mutations of critical amino acids on the actin–tropomyosin binding interface disrupt Gestalt-binding and are associated with a number of inherited myopathies. Moreover, dysregulation of tropomyosin may also be a factor that interferes with the gatekeeping operation of non-muscle tropomyosin in the controlling interactions of a wide variety of cellular actin-binding proteins. The clinical relevance of Gestalt-binding is discussed in articles by the Marston and the Gunning groups in this special journal issue devoted to the impact of tropomyosin on biological systems.

Energy landscapes reveal the myopathic effects of tropomyosin mutations.

Striated muscle contraction is regulated by an interaction network connecting the effects of troponin, Ca(2+), and myosin-heads to the azimuthal positioning of tropomyosin along thin filaments. Many missense mutations, located at the actin-tropomyosin interface, however, reset the regulatory switching mechanism either by weakening or strengthening residue-specific interactions, leading to hyper- or hypo-contractile pathologies. Here, we compute energy landscapes for the actin-tropomyosin interface and quantify contributions of single amino acid residues to actin-tropomyosin binding. The method is a useful tool to assess effects of actin and tropomyosin mutations, potentially relating initial stages of myopathy to alterations in thin filament stability and regulation. Landscapes for mutant filaments linked to hyper-contractility provide a simple picture that describes a decrease in actin-tropomyosin interaction energy. Destabilizing the blocked (relaxed)-state parallels previously noted enhanced Ca(2+)-sensitivity conferred by these mutants. Energy landscapes also identify post-translational modifications that can rescue regulatory imbalances. For example, cardiomyopathy-associated E62Q tropomyosin mutation weakens actin-tropomyosin interaction, but phosphorylation of neighboring S61 rescues the binding-deficit, results confirmed experimentally by in vitro motility assays. Unlike results on hyper-contractility-related mutants, landscapes for tropomyosin mutants tied to hypo-contractility do not present a straightforward picture. These mutations may affect other components of the regulatory network, e.g., troponin-tropomyosin signaling.

Structure and flexibility of the tropomyosin overlap junction.

To be effective as a gatekeeper regulating the access of binding proteins to the actin filament, adjacent tropomyosin molecules associate head-to-tail to form a continuous super-helical cable running along the filament surface. Chimeric head-to-tail structures have been solved by NMR and X-ray crystallography for N- and C-terminal segments of smooth and striated muscle tropomyosin spliced onto non-native coiled-coil forming peptides. The resulting 4-helix complexes have a tight coiled-coil N-terminus inserted into a separated pair of C-terminal helices, with some helical unfolding of the terminal chains in the striated muscle peptides. These overlap complexes are distinctly curved, much more so than elsewhere along the superhelical tropomyosin cable. To verify whether the non-native protein adducts (needed to stabilize the coiled-coil chimeras) perturb the overlap, we carried out Molecular Dynamics simulations of head-to-tail structures having only native tropomyosin sequences. We observe that the splayed chains all refold and become helical. Significantly, the curvature of both the smooth and the striated muscle overlap domain is reduced and becomes comparable to that of the rest of the tropomyosin cable. Moreover, the measured flexibility across the junction is small. This and the reduced curvature ensure that the super-helical cable matches the contours of F-actin without manifesting localized kinking and excessive flexibility, thus enabling the high degree of cooperativity in the regulation of myosin accessibility to actin filaments.

An atomic model of the tropomyosin cable on F-actin.

Tropomyosin regulates a wide variety of actin filament functions and is best known for the role that it plays together with troponin in controlling muscle activity. For effective performance on actin filaments, adjacent 42-nm-long tropomyosin molecules are joined together by a 9- to 10-residue head-to-tail overlapping domain to form a continuous cable that wraps around the F-actin helix. Yet, despite the apparent simplicity of tropomyosins coiled-coil structure and its well-known periodic association with successive actin subunits along F-actin, the structure of the tropomyosin cable on actin is uncertain. This is because the conformation of the overlap region that joins neighboring molecules is poorly understood, thus leaving a significant gap in our understanding of thin-filament structure and regulation. However, recent molecular-dynamics simulations of overlap segments defined their overall shape and provided unique and sufficient cues to model the whole actin-tropomyosin filament assembly in atomic detail. In this study, we show that these MD structures merge seamlessly onto the ends of tropomyosin coiled-coils. Adjacent tropomyosin molecules can then be joined together to provide a comprehensive model of the tropomyosin cable running continuously on F-actin. The resulting complete model presented here describes for the first time (to our knowledge) an atomic-level structure of αα-striated muscle tropomyosin bound to an actin filament that includes the critical overlap domain. Thus, the model provides a structural correlate to evaluate thin-filament mechanics, self-assembly mechanisms, and the effect of disease-causing mutations.

HCM and DCM cardiomyopathy-linked α-tropomyosin mutations influence off-state stability and crossbridge interaction on thin filaments

Calcium regulation of cardiac muscle contraction is controlled by the thin-filament proteins troponin and tropomyosin bound to actin. In the absence of calcium, troponin-tropomyosin inhibits myosin-interactions on actin and induces muscle relaxation, whereas the addition of calcium relieves the inhibitory constraint to initiate contraction. Many mutations in thin filament proteins linked to cardiomyopathy appear to disrupt this regulatory switching. Here, we tested perturbations caused by mutant tropomyosins (E40K, DCM; and E62Q, HCM) on intra-filament interactions affecting acto-myosin interactions including those induced further by myosin association. Comparison of wild-type and mutant human α-tropomyosin (Tpm1.1) behavior was carried out using in vitro motility assays and molecular dynamics simulations. Our results show that E62Q tropomyosin destabilizes thin filament off-state function by increasing calcium-sensitivity, but without apparent affect on global tropomyosin structure by modifying coiled-coil rigidity. In contrast, the E40K mutant tropomyosin appears to stabilize the off-state, demonstrates increased tropomyosin flexibility, while also decreasing calcium-sensitivity. In addition, the E40K mutation reduces thin filament velocity at low myosin concentration while the E62Q mutant tropomyosin increases velocity. Corresponding molecular dynamics simulations indicate specific residue interactions that are likely to redefine underlying molecular regulatory mechanisms, which we propose explain the altered contractility evoked by the disease-causing mutations.

Energy Landscapes Reveal the Myopathic Effects of Tropomyosin Mutations

Striated muscle contraction is regulated by a complex interaction network connecting the effects of troponin, Ca2+, and myosin-heads to the azimuthal positioning of tropomyosin along thin filaments. In relaxed muscle, tropomyosin sterically blocks crossbridge cycling on actin to inhibit contraction, while in stimulated muscle, tropomyosin repositions over actin, and crossbridge cycling and contraction result. Many missense mutations, located at the actin-tropomyosin interface, however, reset the regulatory switching mechanism either by weakening or strengthening residue-specific interactions, leading to hyper- or hypo-contractile pathologies. We have computed energy landscapes for the actin-tropomyosin interface and have quantified contributions of single amino acid residues to actin-tropomyosin binding. The information acquired can prove to be a useful diagnostic tool to assess effects of actin and tropomyosin mutations by relating the critical initial stages of disease development to alterations in thin filament stability and regulation. We find that the landscapes for mutant filaments associated with hyper-contractility, for example those linked to hypertrophic cardiomyopathy (HCM), skeletal muscle arthrogryposis and congenital fiber-type disproportion (CFTD), provide a simple picture. In these cases, most of the mutations examined are associated with a decrease in actin-tropomyosin interaction energy that will destabilize the blocked (relaxed)-state. Our measurements parallel previously noted enhanced Ca2+-sensitivity conferred by these mutants. We show, in addition, that energy landscape computation, in combination with known actin-tropomyosin sequence and structural information, can be used prospectively to identify potential effects of post-translational modifications to rescue regulatory imbalances. However, unlike results on hyper-contractility-related mutants, landscapes for tropomyosin mutants tied to hypo-contractility do not present a straightforward picture. These mutations may affect other components of the regulatory network, e.g., troponin-tropomyosin signaling or tropomyosin-myosin interaction.

Mechanical Unfolding of the Beet Western Yellow Virus −1 Frameshift Signal

Using unfolding by optical tweezers and steered molecular dynamics (SMD) simulations we have demonstrated the critical importance of Mg2+ ions for the mechanical stability of the BWYV RNA pseudoknot. The optical tweezers experiments pointed to a critical role of stem 1 of the pseudoknot, a finding that was confirmed using the SMD simulations. These simulations supported the notion that the stability of stem 1 is critical for −1 frameshifting, a translational recoding event essential for replication of the BWYV. Furthermore, they clarified the precise role of two Mg2+ ions, Mg45 and Mg52, in −1 frameshifting. The ions were shown to play a critical role in stabilizing stem 1 by two possible mechanisms depending upon the hydration of the Mg2+ ions. Mg2+ ions were either directly forming a salt bridge between the strands of stem 1, or they stabilized parallel orientation of the strands in stem 1. Interestingly, these findings explain the drop in frameshifting efficiency, down to null levels, of the C8U mutant. The large effect of this mutant upon the frameshift efficiency seemed surprising as only a single hydrogen bond appeared to have been sacrificed. However, the SMD simulations clarify how the C8U mutation affects the Mg2+ coordination and destabilizes stem 1 of the pseudoknot.