In spite of the prominence of the coiled coil as an important motif in the myosin superfamily, structural and functional details of these coiled coils are severely lacking, due to the general absence of crystal structures. Though most of the myosin coiled coils have typical heptad repeats, the strength of the interactions between the two α-helices forming the various segments of the coiled coil are likely to vary. Very recently, the participation of the coiled coil motif in thick filament assembly of yeast myosin II has been suggested to involve two pathways. These investigators have employed deletion mutants and in vivo assays to demonstrate the role of the coiled coil, and an evolutionarily conserved structural kink within, in forming cleavage furrow ingression at the division during cytokinesis.46
In the literature, demarcation of coiled coil regions into weak and strong regions, protein-protein interaction sites and the recognition of structural kinks are just emerging.
There are many prediction programs that are available to predict the rough positions of coiled coils given the amino acid sequence of a protein.47
There are also structural analysis programs that enable identification of the coiled coil boundaries following the ridges-grooves arrangement of amino acid side chains.48
However, most of the predictive programs assume a uniform and ideal strength to the predicted regions, albeit being sensitive to sequence breaks like stutters and stammers. The sequence signatures at the coiled coils have evolved to such an extent that one can hardly find any conserved motifs across the subtypes. Yet many conserved (at the amino acid level) motifs can be found in the predicted coiled coil tail (please see a multiple sequence alignment of myosin VI in Supplementary Fig. 1
We have calculated the strength of interactions between the coiled coil α-helices within dimeric myosins through the pseudo-energy function inscribed in COILCHECK.49
For this we applied an algorithm () that computationally ana lyzes the interaction strength of myosin at the coiled coil motif by three-dimensional modeling using MODELLER50
followed by retrospective calculations of interaction energies at the coiled coil interface using COILCHECK. Unlike general protein-protein interfaces51,52
that are largely dominated by hydrophobic interactions, the coiled coil interfaces are special, elaborate and retain alternating hydrophobic interactions and salt bridges built around a simple system of a pair of helices. Van der Waals and electrostatic interactions are measured all along the coiled coils across the dimer (see for Methods). Further, interactions such as hydrogen bonds and salt bridges also contribute to the stability of these domains. This method has been applied to 26 protein structural entries that contain coiled coil. The binding strength values correlate well with the structure and stability of coiled coils. Indeed, COILCHECK results are sensitive to demarcate DNA-binding proteins that structurally deviate from ideal coiled coils (). The guanine nucleotide exchange factor (GEF) domain of sec2p protein is a 22-nm long coiled coil and has a weakly interacting N-terminal region and a strongly intertwined middle region as evident from the crystal structure.53
Calculation of the interaction energies at different regions of the GEF domain using COILCHECK are consistent with the experimental results ().
Figure 5 Coiled coils interaction strength analysis protocol. Molecular models of myosin coiled coils were made using tropomyosin as template. The models were generated using MODELLER program.50 The long models were split into penta-heptad sized fragments and (more ...)
Figure 6 COILCHECK based inter-protomer interactions. (A) Left: Pictorial representation of parallel coiled coil crystal structures depicting the interaction energy. Each block corresponds to a 35 residue window. Green blocks are regions of low inter-protomer (more ...)
When COILCHECK was applied to the tropomyosin structure (PDB code: 1C1G), actin-interacting zones were observed to acquire relatively poor energies for coiled coil formation (). Furthermore, in order to probe the sensitivity of COILCHECK in detecting local structural perturbation due to the introduction of mis-sense mutations, we performed a series of virtual mutations (). Interestingly, COILCHECK energy differences between the wild type and ‘single-site mutants’ were not very high when permitted amino acid exchanges, such as Leu to Val, were performed at either ‘a’ or ‘d’ positions. On the other hand, if drastic amino acid changes, such as Leu to Asp or Val to Lys, were introduced in the coiled coil, COILCHECK energy differences were quite high (), suggesting that the method is sensitive to predict the effect of small sequence changes on the stability at the interface.
Using this approach, we found that the coiled coil regions of myosins V are generally interrupted by poorly interacting dimeric zones (). Myosins VI have only a couple of short predicted strong interactions in its α-helical tail (). These myosin VI results are consistent with experimental findings that the putative coiled coil region ( ie region predicted as coiled coil by COILS or PAIRCOIL algorithms) of myosin VI, are actually not coiled coil but stable single α-helices.54
Coiled coils of myosins II are predicted to have many segments of weak interactions that may have roles in oligomerization or protein-protein interactions ().
Figure 7 Inter-protomer interaction strength. Non-uniform inter-protomer interaction strength in predicted coiled coil regions are color coded and represented as boxes. Each box corresponds to 35 amino acids. Strongly interacting regions are shown in green and (more ...)