Uncomplexed Actin
Created by Mina Antonious
Uncomplexed actin from the organism Oryctolagus Coniculus is a globular protein that performs important cellular functions. The molecular weight of uncomplexed Actin is 41811.70 with an isoelectric point of 5.23. Actin performs numerous biological functions, including maintaining cellular shape(10), interacting with myosin to perform muscle contraction(6), assisting in cytokinesis during cell division(7), acting as a transport track for other proteins(11) and assisting with cell signaling(7). Cellular shape is maintained via actin microfilaments attaching the cytoplasm of cells to the surroundings. For cellular transport within organisms, Myosin uses the actin filaments as a track for transporting vesicles(11). In the cytoplasm of cells, actin and myosin assist in the process of cytoplasmic streaming which results in the flow of the cytoplasm inside cells(16). Cytoplasmic streaming allows for the transfer of nutrients to organelles within the cell and cellular motion.
Uncomplexed actin (PDB = 1j6z) is a crystallized protein in a monomeric form (1). The protein consists of four subdomains which contain alpha helices, beta sheets, and beta turns. Subdomains 1 and 3 contain five stranded beta pleated sheets, while subdomains 2 and 4 contain anti-parallel beta sheets(5).The subdomains meet at the center of the protein at an adenosine diphosphate ligand. The ligand is surrounded by a binding pocket which consists of the residues Lys-213, Glu-214, Thr-303, Met-305, Tyr-306, and Lys-336(5). The adenosine diphosphate ligand is a product of adenosine triphosphate hydrolysis which produces adenosine diphosphate and an inorganic phosphate(2). In addition to the adenosine diphosphate ligand, calcium ions and tetramethylrhoamine-5-maleimide are other associated ligands present along with water molecules. One calcium ion found near the center of the molecule is known as the primary calcium ion. The ion serves as a catalyst in reactions such as ATP hydrolysis13. The primary calcium ion is located within residues Asp 11, Gln 137, and Asp 154(5). The other calcium ions further from the center of the molecule can serve as secondary cation binding sites1. Tetramethylrhoamine-5-maleimide binds to actin in subdomain one near the carboxylic acid terminal, specifically at Cys-374 (2). In addition to the ligands, a 4-methylhistidine positioned at residue 73 is secreted in urine when muscle proteins break down and therefore is used by researchers to show the rate of breakdown of muscle protein (14).
Actin can either exist as monomeric G-actin or in a polymeric form known as F-Actin(4). During the polymerization process, ATP is hydrolyzed to ADP(2). The binding of tetramethylrhoamine blocks actin's ability to polymerize allowing for the crystallization of an uncomplexed actin monomer in the ADP state1. X-ray analysis of actin's three-dimensional structure can only be achieved if actin is crystallized therefore tetramethylrhoamine provides a useful function by allowing for the crystallization of actin(5). Uncomplexed actin's ability to polymerize can be blocked in other ways using proteins such as Deoxyribonuclease I, commonly referred to as DNase I. DNase I binds to actin at subdomain 2 residues. Residues His40-Gly48 are known as the DNase I binding loop(1).
At the center of the molecule where the four subdomains meet is the active site of the protein(1). Nucleotide differences at the active site lead to conformational changes in the protein at subdomain two (1). This is the site where ATP is converted to ATP during polymerization(1). The conformational differences between uncomplexed actin in the ATP state and the ADP state is as follows. There is a rotation of 10 degrees in subdomain four and a rotation of five degrees in subdomain two in the ADP actin state compared to the ATP state structure(1). Before the change, the DNase I binding loop exists either as a random shape or as a beta turn(1). The hydrolysis of ATP produces an inorganic phosphate, which stabilizes the F-actin filaments that are formed1 when polymerization of actin occurs. Polymerization of actin is an essential process for cells as the F-actin filaments formed performs many biological functions. The actin filaments assist in muscle contraction(6), cytokinesis(7), and cell to cell adhesion(8). The protein myosin uses the actin filaments as a track for transporting vesicles(11). Actin filaments assist in maintaining cellular shape by attaching the cytoplasm to its surroundings(10). Furthermore, polymerization allows actin to bind to certain regulatory proteins(2). Proteins such as coflin bind to actin in the adenosine diphosphate state(2).
Uncomplexed actin in the ADP state contains many alpha helices, beta sheets, turns, and random coils. Furthermore, some of the amino acids present exhibit hydrogen bonding(1). Hydrogen bonding occurs between Ser-14 and Asp-157, Ser-33 and Gly-13, and Arg-183 and Glu-721. There are differences in the hydrogen bonds between actin in the ATP state and the ADP state. For example, in the ATP state, the Ser-14 exhibits hydrogen bonding with the gamma phosphate of ATP1. In the ADP state, Ser-14 exhibits hydrogen bonding with an oxygen atom of the beta phosphate(1). This change in the hydrogen bonding of the protein leads to a change in the shape of the protein as residue Ser14 is shifted toward the central nucleotide(1). The shift disrupts hydrogen bonds at the ends of subdomain two (1).
Latrunculin-A, known as LAT-A is a drug that is known to bind to Actin (3). Latrunculin A binds to actin its monomeric form(3). The drug binds to actin adjacent to actin's nucleotide binding site where actin binds to adenosine triphosphate(3). In the Actin -Latrunculin-A complex, hydrogen bonds occur in the uncomplexed actin at Tyr 69, Thr 186, and Arg 210(9). LAT-A produces structural changes in the actin morphology(3). The drug is used for research to study the function and structure of Actin in various organisms(3).
DNase I is a protein that is structurally similar to actin . DNase I cleaves DNA at certain strands leading to DNA fragmentation(5). When bound to actin , DNase I prevents actin from polymerizing allowing actin to be crystallized (5). This allows for the analysis of the actin structure. DNase I binds to actin at subdomain 2 with interactions such as hydrogen bonds and hydrophobic interactions. Specifically the residues involved in these interactions in actin are Arg-39, Gln-41, Val-43, Val-45, Lys-61, Gly-63, Glu-207, and Thr-203. Residues Gly-42, Val-43, and Met-44 integrate into a beta sheet with residues in DNase I(5). Furthermore, Lys-61 forms a salt bridge with DNase I(5). Researchers use the ability of DNase I to prevent actin from polymerizing to study three-dimensional crystallized structures of actin(5).
Skeletal muscle myosin II is a protein with a similar tertiary structure to actin. Actin and myosin II interact in the skeletal muscles of cells to provide the essential process of muscle contraction for organisms via a cycle called the cross-bridge cycle(6). In muscle cells, with the use of ATP as an energy source, skeletal myosin II attaches to actin thin filaments and eventually leads to actin filaments sliding past one another, generating a power stroke for the muscles, and resulting in muscle contraction(6).
Gelsolin is a protein with a similar tertiary structure to actin. Gelsolin binds to Actin with the activation of calcium ions(10). Calcium rearranges gelsolin, revealing the actin binding site, and allowing gelsolin and actin to bind (7). Upon binding, gelsolin causes structural changes and rearrangements in the actin protein. These rearrangements assist in many biological functions such as receiving cellular signals, maintaining cellular shape, and conducting cellular movement (7).