Dynamic Transition from Î±-Helices to Î²-Sheets in Polypeptide Coiled

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Dynamic transition from #-helices to #-sheets in polypeptide coiled-coil motifs Kirill A. Minin, Artem Zhmurov, Kenneth A. Marx, Prashant K. Purohit, and Valeri Barsegov J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b06883 • Publication Date (Web): 18 Oct 2017 Downloaded from http://pubs.acs.org on October 18, 2017

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Journal of the American Chemical Society

Dynamic transition from α-helices to β-sheets in polypeptide coiled-coil motifs Kirill A. Minin,† Artem Zhmurov,† Kenneth A. Marx,‡ Prashant K. Purohit,¶ and Valeri Barsegov∗,‡,† †Moscow Institute of Physics and Technology, Dolgoprudny, 141701, Russia ‡Department of Chemistry, University of Massachusetts, Lowell, MA 01854, USA ¶Department of Mechanical Engineering and Applied Mechanics, University of Pennsylvania, Philadelphia, PA 19104, USA E-mail: Valeri [email protected] Abstract We carried out dynamic force manipulations in silico on a variety of coiled-coil protein fragments from myosin, chemotaxis receptor, vimentin, fibrin, and phenylalanine zippers that vary in size and topology of their α-helical packing. When stretched along the superhelical axis, all superhelices show elastic, plastic, and inelastic elongation regimes, and undergo a dynamic transition from the α-helices to the β-sheets, which marks the onset of plastic deformation. Using the Abeyaratne-Knowles formulation of phase transitions, we developed a new theoretical methodology to model mechanical and kinetic properties of protein coiled-coils under mechanical non-equilibrium conditions and to map out their energy landscapes. The theory was successfully validated by comparing the simulated and theoretical force-strain spectra. We derived the scaling laws for the elastic force and the force for α-to-β transition, which can be used to understand natural proteins’ properties as well as to rationally design novel biomaterials of required mechanical strength with desired balance between stiffness and plasticity.

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INTRODUCTION In 1953 coiled-coils were proposed independently by Crick and Pauling as protein motif structures comprised of supercoiled α-helical segments; at the time Crick realized these structures would be stabilized by side chain interactions from the α-helices whose axes were twisted about 20 degrees with respect to each other, thereby repeating similar interactions every seven residues along each α-helix. 1 In the intervening decades, structural biology studies have identified a wider range of coiled-coil motifs, including examples that deviate from Crick’s canonical heptad pattern. Currently, coiled-coils have been identified and characterized as ubiquitous, critically important, highly stable biomechanical structures, occuring either macroscopically at the tissue level (hair, nails, blood clots, etc.), microscopically within individual cellular structures (intracellular cytoskeleton, extracellular matrix, flagella, etc.), or as motif components of proteins involved in a variety of functions (membrane fusion, signal transduction, solute transport, etc.). Many well studied proteins performing mechanical functions in biological processes, utilize the superhelical coiled-coil architecture, including ones we have incorporated into the present study based upon their structural diversity and available atomic structures: muscle proteins (myosin), intermediate filaments (vimentin), whole blood clots and thrombi (fibrin), chemotaxis (chemotaxis receptors), cellular transport (kinesin), and bacterial adhesion (protein tetrabrachion). 1 More recently, the unique superhelical symmetry and physical properties of coiled-coils has inspired the design of new materials, 2,3 from short supercoils, 4 to long and thick fibers, 5 to nanotubes, 6 spherical cages 7 and synthetic virions. 8 In our previous work, we studied equilibrium mechanical properties and the α-to-β transition in fibrinogen coiled coils using a constant pulling force. 9 In this study, we took a step further. We have explored the physico-chemical properties of a wide range of polypeptide motifs formed by two-, three-, four- and five-stranded coiled-coil protein supehelices, involving both parallel and anti-parallel arrangements of their α-helices. Dynamic force manipulations in silico, which fully mimic single-protein forced unfolding experiments in vitro, 2


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have been combined with theoretical modeling of protein coiled-coils. Using this approach, we have found that all the coiled-coil protein fragment systems studied, ranging from twoto-five helices and forming parallel or anti-parallel supercoil architecture, are characterized by three distinctly different regimes of forced extension and that they uniformly undergo a remarkable dynamic structural transition from all α-helices to all β-sheets. Experimental biophysical force extension studies have been carried out on a number of coiled coil containing protein fiber systems, such as keratin, 10 marine snail egg case protein, 11,12 hagfish slime protein, 13 (reviewed in 14 ). These systems all show dynamic structural transition results qualitatively similar to our simulations. However, these experimental studies are carried out on far longer time scales than our simulation results can achieve, as well as resulting from far more heterogeneous structures than our short defined coiled coil motifs. In a theoretical study, two-stranded coiled coils were analyzed to determine the critical length (number of amino acids) for formation of the β-phase. 15 It is worth noting that our study continues beyond a mere characterization of the coiled coils’ dynamic transitions, to provide a theoretical basis for understanding the different coiled-coils’ stability and their nanomechanics when undergoing the pulling force induced elongation and transition from the α-helices to the β-sheets. To span the range of polypeptide coiled-coil motifs, here we used the coiled-coil fragments from the available atomic structure models of myosin, vimentin, fibrin, bacterial chemotaxis receptor and phenylalanine zippers all displayed in Figure 1; see also Supporting Information (SI) for more details on architecture of polypeptide superhelices. (i) Myosin II is a muscle protein containing a double-stranded parallel coiled-coil. 16 Upon muscle contraction, tension is transfered along the myosin tail 16 (there is a body of experimental force data available for myosin 17,18 ). (ii) Vimentin is an elementary building block of intermediate filaments in cells, 19,20 helping to determine their resistance to mechanical factors. 21 The structure of vimentin contains an α-helical rod domain, which can be divided into several double-helical parallel coiled-coil segments. 21 (iii) Fibrin forms a fibrous network in blood, 22 which acts as a

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scaffold to stem bleeding. The triple-helical parallel coiled-coils are responsible for the unique visco-elastic properties of fibrin. 9,23 (iv) Bacterial chemotaxis receptor is responsible for signal transduction across cell membranes. 24 The cytoplasmic domain contains two double-stranded anti-parallel coiled-coils wrapped around each another forming a four-stranded superhelix. Lastly, (v) Phenylalanine zippers, artificial four-to-five stranded parallel coiled-coils, are promising biomaterials with tunable materials properties. 6,25 We mechanically tested the coiled-coil motifs by applying a time-dependent pulling force, which corresponds to the dynamic force-ramp mode used in single-molecule protein stretching experiments. Direct comparison of the results of dynamic force manipulations in silico and theoretical modeling of the force spectra have enabled us to develop a new theory to describe the non-equilibrium dynamic response of protein coiled-coils to external mechanical factors. The quantitative agreement between the theory and simulations of the systems studied allows for a new approach to rationally design coiled-coils with desired mechanical properties into novel biomaterials for specific technological applications. For example, experimentally manipulating protein systems to incorporate coiled coil segments with specific properties, such as the recombinant fibers containing vimentin fragments 26 and hagfish slime protein 27 coiled coil fragments, could take advantage of a rational design approach utilizing the theory of coiled coils’ functional dependencies and scaling laws we presented here to create better biomaterials, both faster and cheaper.

EXPERIMENTAL PROCEDURES Structural models of polypeptide coiled-coils: We used the atomic structural models of myosin (PDB entry: 2FXO 28 ), vimentin (1GK4 29 ), fibrin (3GHG 30 ), bacterial chemotaxis receptor (1QU7 24 ), and phenylalanine zippers (2GUV and 2GUS 25 ). All the models of polypeptide coiled-coils are displayed in Figure 1 (see SI for more details). Force spectroscopy in silico: We employed the all-atom Molecular Dynamics (MD) sim-

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CHARMM19 unified hydrogen force-field 31 (see SI). 9 In silico models of proteins were constructed using the CHARMM program. 32 We used a lower damping coefficient γ=0.15 ps−1 vs. γ=50 ps−1 for ambient water at 300K for more efficient sampling of the conformational space. 33 To mimic the experimental force-ramp conditions and to enable rotational motions of coiled-coils around the pulling axis, we implemented the pulling plane with harmonically attached tagged residues at one end of the molecular system and the resting plane with constrained residues at the other (Figure 2a). This approach ensures even tension distribution in polypeptide chains and suppresses chain sliding past each other. The pulling plane was connected to a virtual cantilever moving with a velocity vf =104 − 106 µm/s, thereby ramping up the applied force f =rf t with a loading rate rf =ks vf =10−3 −10−1 N/s (ks =100 pN/nm is the cantilever spring constant). Explicit solvent modeling: We also performed MD simulations in explicit solvent to validate the results of SASA implicit solvent based modeling (see SI for more detail).

RESULTS The α-to-β transition: We mechanically tested myosin, vimentin, fibrin, bacterial chemotaxis receptor and phenylalanine zippers (Figure 1) by employing Molecular Dynamics simulations of protein forced unfolding accelerated on Graphics Processing Units (GPUs). We implemented dynamic force ramp conditions of time-dependent pulling force as described in the Experimental Procedures. All the coiled-coils undergo the force-driven structural transition from the α-helices to the β-sheets regardless of the number of helices and parallel or anti-parallel architecture. In Figure 2, we display the force-strain (f ε) curves and structure snapshots for bacterial receptor and myosin. The f ε-curves are reminiscent of experimental force extension profiles that can be found elsewhere. 17,18 The plateau force for myosin f ∗ ∼100 pN (Figure 2d) is higher than the experimental value of ∼40 pN 18 due to 104 −105 fold faster pulling speeds used in silico. Indeed, experimental pulling speeds are typically

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low strain (f ∼ε; ε

Dynamic Transition from Î±-Helices to Î²-Sheets in Polypeptide Coiled

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