Myosin

Myosin-11 (PBD 3J04) is a protein that exists in Gallus gallus, or more commonly known as chicken. Myosin-11 is a smooth muscle heavy meromyosin (smHMM) that is comprised of 909 residues, with a molecular weight of 275.8 kDa and isoelectric point of approximately 5.42, as obtained from ExPASy (6). SmHMM plays a crucial role in ATP hydrolysis and muscle contractions in organisms. It naturally exists in two structurally different forms in biological systems; phosphorylated and dephosphorylated. These two forms of myosin-11 affect the activity of ATPase, which in turn has significant effects on the contraction of different muscles. These two forms of smHMM are exemplary of the fundamentals of the biochemistry in proteins, that is; how the primitive functions and properties of proteins are affected by the structures each protein possesses. SmHMM, in the phosphorylated form, has the ability to interact in a head to head manner with molecules intermolecularly. Conversely, the dephosphorylated form interacts in a head to head manner with molecules intramolecularly (9).

Smooth muscles myosin (smM) is a class II myosin with two heavy chains (HC), two light chains, which include the essential light chain (ELC) and regulatory light chain (RLC) (2). The heavy chain has an N-terminus that forms the globular motor domain, site for ATP hydrolysis and actin binding (2). Contraction of muscle is dictated by the interaction of myosin cross-bridge with actin. The myosin cross-bridge acts as a communication functional unit and includes the actin-binding site, the site for hydrolysis, and the lever arms (5). All of these units contribute to the movement of chicken muscle. The cross-bridge initially binds with actin, and undergoes a swinging motion in which the myosin rows the actin filament along one direction. The binding of actin to myosin strongly influences the binding of ATP to the hydrolysis site on the myosin cross-bridge. Once ATP binds to the ATP binding site, it is used to drive the movement of myosin along the actin filament (2). The lever arm amplifies these small changes that occur at the active site into larger signals that transport the myosin head along the actin filament. Following this step, ATP dissociates from the myosin-actin complex, and it is hydrolyzed by myosin (5). This cycle is recycled infinite amounts of times to allow movement in organisms. As such, molecules that are structurally compatible to perform these functions in smooth muscle myosin are vital in biological systems.

The light chain domain of Smooth muscle myosin (smM) consists of an alpha helical segment to which light chains can bind. Another structurally important segment of smM is S-2. It contains the two C-terminal halves of the heavy chain, which forms a long alpha helix coiled coil. This structurally enables smM to dimerize, allowing it to have the option to associate with other proteins and form a quaternary structure (2). Often times, proteins form quaternary structures to increase the entropy of the system and decrease the surface area to volume ratio of the protein. Decreasing this ratio allows proteins to experience fewer problems on the surface, and increases the structural stability of proteins as a result of the burying of hydrophobic core. Myosin-11 comprises of six subunits. The secondary structure of smHMM consists of 17 α-helix, 22 antiparlllel β-sheet, 3 parallel β-sheet, 24 β-turns, and 34 random coil(2). All of these structures are mainly stabilized by hydrogen bonding. Having stable secondary structure allows the smHMM to form a tertiary structure in 3D space, which can further be stabilized by dimerization through the formation of a quaternary structure.

The final major component of smM is the regulatory light chain (RLC). ATPase activity that is activated by actin is regulated by the phosphorylation of Ser-19 on the RLC. Phosphorylated and Dephosphorylated myosins are both activated by the regulatory light chain (2). Dephosphorylated smHMM assumes a closed conformation, which prevents ATPase activity in actin. 2D crystallography reveals that the dephosphorylated form of smHMM has an asymmetric structure and can only participate in head to head interactions with different motor domains. The myosin heads in the dephosphorylated state are referred to as “free” and “blocked” due to their abilities to bind with the actin filament without steric hindrance from the associated head (2). The free myosin head in the dephosphorylated smHMM is only able to bind to the actin-binding interface of the blocked myosin head, which exists on the same molecule. Consequently, dephosphorylated smHMM tends to have a 3 to 4 times lower affinity for actin in the presence of MgATP (7). Ultimately, both myosin heads are inhibited in the dephosphorylated state because the binding of the blocked head prevents the motion of the converter domain, which is required for fast phosphate release in the hydrolysis of ATP. This property makes unphosphorylated smHmm soluble and structurally incompatible with the filament formation in the myosin-actin complex. However, when the smHMM is phosphorylated, the actin filament binds to the “free” head of the myosin rather than the “blocked” head. As a result, smHMM is able to exhibit intermolecular interactions. The intermolecular interaction the phosphorylated smHMM tends to exhibit is a product of crystal packing forces present within the native structure of myosin (10). This increases ATPase activity in the phosphorylated smHMM by about 1000 times relative to the unphosphorylated (2). Consequently, phosphorylated smHMM is able to catalyze the proper interactions with actin, which results in muscle contraction. It is also observed that the phosphorylation of 20 kDa light chains results in a 60 fold increase in the activity of MgATPase activity. Since the unphosphorylated smHMM does not exhibit these characteristics, it hydrolyzes ATP very slowly in the presence of high concentrations of actin filament (8).

The heads of the phosphorylated smHMM can be referred to as “free-like” and “blocked-like” to relate these structures to the dephosphorylated form of smHMM (2). Phosphorylated and unphosphorylated smHMM interact with lipid monolayers through the actin-binding surface of the free/free-like myosin head. In this interaction, the blocked-like head motor domain and S-2, are held away from the monolayer. This is because the interaction between the monolayer and the free heads is solely based on the polarization of charge. The monolayer has a positive net charge, which allows only the free heads of the smHMM to interact with its surface. Since only the free-heads interact, most of the protein’s mass is held away from the layer, allowing only a small amount of contact on the surface of the monolayer. The smHMM interacts with the monolayer at residues 395-400 of the “free-like” head, and residues 76-79 of the “blocked-like” head on the RLC (2). There tends to arise a difference in the configuration of the phosphorylated and unphosphorylated “free” heads in the ways in which they align themselves with respect to the monolayer. The unphosphorylated free head aligns more vertically than the phosphorylated free head. Although these are notable differences, it is also important to note the similarities of both free heads interacting with the monolayer while the blocked head and S-2 are held away in both forms of smHMM (2).

Myosin 5A (PBD: 2DFS), a closely related cousin of Myosin 11, is also found in chicken. While Myosin-11 is Heter-6-mer protein, Myosin 5A is a Heter-4-mer (2). Myosin 5A is comprised of 1080 residues with a molecular weight of 453.5 kDa (4), and has an isoelectric point of approximately 8.3 (6). Its structure consists of two heads that have an amino-terminal motor domain that can bind up to six calmodulins (4). Like Myosin-11, Myosin 5A also comprises of a unit that can dimerize to form more stable structures. A unit that is unique to Myosin 5A is the carboxyl-terminal globular cargobinding domain. It serves the purpose of transporting organelles, mRNA, and mediating membrane trafficking in biological systems. Myosin 5A has the ability to exhibit an alternate conformation, Calmodulin (PDB 2K0E), under conditions that allow it to perform a more specialized cellular function (3). It also exhibits a similar secondary structure to Myosin 11. It has various beta sheets, alpha helices, and random coils, all of which are stabilized by hydrogen bonds. Since Myosin 5A lacks basic residues, ionic interactions are not possible. Myosin 5A is comprised of thirteen subunits that primarily act as elastic linkers between the motor and cargo. Functionally important residues include Pro-117 - Pro 118, all of which abut the entrance of the ATP binding pocket, loop 1 and beta bulge during different interactions it has with varying substrates (4).

The two databases that were used to obtain results for the investigation of these proteins are PSI-BLAST and Dali Server. The purpose of PSI-BLAST was to search and find proteins that had a similar primary structure to Myosin-11. The server did this by searching for sequence similarities and homologies between Myosin-11 and other proteins. It also identified any sequential differences by placing “gaps” in the protein's primary sequence. The purpose of the Dali Server was to compare the assigned protein’s tertiary structure to other proteins. The Z score generated by the Dali was helpful in understanding how similar proteins were based on their corresponding intramolecular distances. A Z score of 30.6 was generated for myosin-11 and myosin 5A which indicated that there was a sufficient enough difference and similarity in intramolecular distance between these two proteins for a valid comparison. A Z value of less than 72 was considered significant, as that indicated sufficient similiarities in overall tertiary structure for comparison. An E value of 3 x 10^-34 was generated by the PSI-BLAST which indicated similarities in primary structure between these proteins which led to further analysis of structure and function. In order for the E value to be significant, it had to be less than 0.005, as that indicated sufficient enough differences and similarities in primary strucuture for a valid comparison. These two servers helped in choosing a proper comparison protein (Myosin 5A) to the original assigned protein (Myosin-11) based on structural and functional similarities and differences.