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<Structure load='1atn' size='500' frame='true' align='right' caption='Actin structure complexed with DNAI' scene='Insert optional scene name here' /> | |||
== Introduction == | == Introduction == | ||
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=Structure= | =Structure= | ||
[[Image:Actin Image.gif]] | [[Image:Actin Image.gif]] | ||
Figure 2: Ribbon diagram of actin with ADP and the divalent cation highlighted. | |||
Actin exists in both its monomeric (globular, G-actin) and polymeric (filamentous or F-actin) form. The actin monomer is approximately pear shaped. Actin is composed of 376 residues that are folded into two large domains, each comprised of two subdomains. Its <scene name='Sandbox_Reserved_471/Secondary_structure/3'>secondary structure</scene> is 41% helical (22 helices; 153 residues) and 20% beta sheet (20 strands; 77 residues). The actin filament is a double-stranded, right-handed helix with a half-pitch of 37 nm and a one-start left-handed genetic helix with a rise of 2.75 nm per monomer. The width of the filament is within the range of 7–10 nm. <ref>PMID: 20672362</ref> The large domains are organized to form a hinged molecule with a deep cleft. Subdomains 1 and 3 are structurally related, whereas subdomains 2 and 4 can be viewed as large insertions into subdomains 1 and 3, respectively. There is moderately little contact between the two major domains of actin; the polypeptide chain passes twice between these domains at the loop centered at residue Lys336 and at the linker helix Gln137-Ser145, which functions as the hinge axis between the domains. As a result, two clefts are formed between the domains. Within the upper cleft are the cofactors, an adenine nucleotide and a divalent metal ion, usually magnesium. It is thought that the cofactors interact with the domains on either side, increasing their connectivity. The lower cleft between domains 1 and 3 is lined by residues Tyr143, Ala144, Gly146, Thr148, Gly168, Ile341, Ile345, Leu346, Leu349, Thr351, and Met355, which are predominantly <scene name='Sandbox_Reserved_471/Hydrophobic_polar/1'>hydrophobic residues</scene>. The <scene name='Sandbox_Reserved_471/Hydrophobic_polar/1'>hydrophilic residues</scene> can also be seen, however they are not as involved in the binding site. This cleft constitutes the major binding site for most ABPs, and is thus called the targetbinding or hydrophobic cleft.Most of the interaction that occur in the subunit are electrostatic in character but there are also hydrophobic interactions.<ref>PMID: 2395459</ref> | Actin exists in both its monomeric (globular, G-actin) and polymeric (filamentous or F-actin) form. The actin monomer is approximately pear shaped. Actin is composed of 376 residues that are folded into two large domains, each comprised of two subdomains. Its <scene name='Sandbox_Reserved_471/Secondary_structure/3'>secondary structure</scene> is 41% helical (22 helices; 153 residues) and 20% beta sheet (20 strands; 77 residues). The actin filament is a double-stranded, right-handed helix with a half-pitch of 37 nm and a one-start left-handed genetic helix with a rise of 2.75 nm per monomer. The width of the filament is within the range of 7–10 nm. <ref>PMID: 20672362</ref> The large domains are organized to form a hinged molecule with a deep cleft. Subdomains 1 and 3 are structurally related, whereas subdomains 2 and 4 can be viewed as large insertions into subdomains 1 and 3, respectively. There is moderately little contact between the two major domains of actin; the polypeptide chain passes twice between these domains at the loop centered at residue Lys336 and at the linker helix Gln137-Ser145, which functions as the hinge axis between the domains. As a result, two clefts are formed between the domains. Within the upper cleft are the cofactors, an adenine nucleotide and a divalent metal ion, usually magnesium. It is thought that the cofactors interact with the domains on either side, increasing their connectivity. The lower cleft between domains 1 and 3 is lined by residues Tyr143, Ala144, Gly146, Thr148, Gly168, Ile341, Ile345, Leu346, Leu349, Thr351, and Met355, which are predominantly <scene name='Sandbox_Reserved_471/Hydrophobic_polar/1'>hydrophobic residues</scene>. The <scene name='Sandbox_Reserved_471/Hydrophobic_polar/1'>hydrophilic residues</scene> can also be seen, however they are not as involved in the binding site. This cleft constitutes the major binding site for most ABPs, and is thus called the targetbinding or hydrophobic cleft.Most of the interaction that occur in the subunit are electrostatic in character but there are also hydrophobic interactions.<ref>PMID: 2395459</ref> | ||
<Structure load='1j6z' size='200' frame='true' align='right' caption='Crystal structure of G-actin' scene='Insert optional scene name here' /> | |||
<Structure load='1rfq' size='200' frame='true' align='right' caption='Structure of F-actin' scene='Insert optional scene name here' /> | |||
Under physiologic conditions, G-actin is transformed to F-actin by ATP. ATP is one of the most common <scene name='Sandbox_Reserved_471/Ligand/1'>ligands</scene> binding in the cleft of actin. After G-actin is bound to an ATP molecule it can then bind to another ATP bound monomer to form an unstable dimer. It can then add a third ATP bound monomer to create a stable trimer that serves as a basis for building fibrous actin (F-actin). Once the F-actin is formed the ATP can be hydrolyzed, keeping the bound ADP and releasing an inorganic phosphate. The polymer can undergo a process known as treadmilling. This is where the polymer continuously grows at its positive end but disassembles at its negative end. This process requires a permanent source of energy such as ATP <ref>Boron, W., & Boulpaep, E. (2008). Medical physiology. (2 ed., pp. 14-20). Saunders Elsevier. Retrieved from http://books.google.com/books/about/Medical_Physiology_E_Book.html?id=HlMJRw08ihgC</ref> | Under physiologic conditions, G-actin is transformed to F-actin by ATP. ATP is one of the most common <scene name='Sandbox_Reserved_471/Ligand/1'>ligands</scene> binding in the cleft of actin. After G-actin is bound to an ATP molecule it can then bind to another ATP bound monomer to form an unstable dimer. It can then add a third ATP bound monomer to create a stable trimer that serves as a basis for building fibrous actin (F-actin). Once the F-actin is formed the ATP can be hydrolyzed, keeping the bound ADP and releasing an inorganic phosphate. The polymer can undergo a process known as treadmilling. This is where the polymer continuously grows at its positive end but disassembles at its negative end. This process requires a permanent source of energy such as ATP <ref>Boron, W., & Boulpaep, E. (2008). Medical physiology. (2 ed., pp. 14-20). Saunders Elsevier. Retrieved from http://books.google.com/books/about/Medical_Physiology_E_Book.html?id=HlMJRw08ihgC</ref> | ||
[[Image:Actin + atp.jpg]] | [[Image:Actin + atp.jpg]] | ||
Figure 5: Structure of actin bound with ATP. | |||
Unlike G-actin, F-actin does not form crystalline arrays that can be analyzed by X-ray crystallography. Instead it can be found by using fiber diffraction, electron-microscopy data and mathematical models. The structure can then be refined and remodeled as necessary. <ref>PMID: 8970724 </ref> The first high-resolution structural model of the actin filament was at a resolution of 8 A ˚ and was proposed by Holmes et al., 1990. Recently, an improved high-resolution F-actin model was created by Oda et al., 2009 with a resolution of 3.3 A ˚ in the radial and 5.6 A ˚ in the equatorial directions. | Unlike G-actin, F-actin does not form crystalline arrays that can be analyzed by X-ray crystallography. Instead it can be found by using fiber diffraction, electron-microscopy data and mathematical models. The structure can then be refined and remodeled as necessary. <ref>PMID: 8970724 </ref> The first high-resolution structural model of the actin filament was at a resolution of 8 A ˚ and was proposed by Holmes et al., 1990. Recently, an improved high-resolution F-actin model was created by Oda et al., 2009 with a resolution of 3.3 A ˚ in the radial and 5.6 A ˚ in the equatorial directions. | ||
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=Mechanism of Action= | =Mechanism of Action= | ||
Polymerization mechanism | ==Polymerization mechanism== | ||
At increased ionic strength, G-actin molecules polymerize into filaments, known as F-actin. F-actin is the main component of the thin filaments in sarcomeres of muscle cells. Muscle sarcomeres use ATP hydrolysis to produce conrtractions by sliding the thin actin filament | At increased ionic strength, G-actin molecules polymerize into filaments, known as F-actin. F-actin is the main component of the thin filaments in sarcomeres of muscle cells. Muscle sarcomeres use ATP hydrolysis to produce conrtractions by sliding the thin actin filament | ||
past thick myosin filaments. The entire process is initiated by the slow formation of actin dimmers and trimers. These serve as a base for filament elongation. During elongation more actin monomers associate to than dissociate from either of the two ends, which results in the net growth of both filament ends. The steady-state phase is characterised by a dynamic equilibrium where the length of the actin filaments remains constant. This means while monomers are associating, other monomers are dissociating from the ends. In this dynamic equilibrium a stationery population of free actin monomers is established and is called the critical concentration. The polarity of the ends of the actin filaments play a key role in elongation. The ends of the filament are referred to as "barbed" and "pointed" according to the polarity of the arrowhead-like structure generated on binding with myosin subfragment 1.<ref>PMID: 1388079</ref> The barbed or plus end binds actin monomers faster than the pointed or minus end. Although its ATPase activity is not crucial for actin polymerisation, actin self-assembly is associated with the ATPase cycle, which powers this treadmilling process. <ref>PMID: 20672362</ref> After the actin molecule is changed into a filament, the bound ATP is hydrolysed to ADP and inorganic phosphate is realeased.<ref>PMID: 1388079</ref> The formation of the F-actin is what stimulates the actin ATPase. The release of the inorganic phosphate happens more slowly than the formation of the filament so that the growing filament has a cap of ATPactin at its barbed end, while monomers containing ADP and inorganic phosphate accumulate in the rest of the filament. | past thick myosin filaments. The entire process is initiated by the slow formation of actin dimmers and trimers. These serve as a base for filament elongation. During elongation more actin monomers associate to than dissociate from either of the two ends, which results in the net growth of both filament ends. The steady-state phase is characterised by a dynamic equilibrium where the length of the actin filaments remains constant. This means while monomers are associating, other monomers are dissociating from the ends. In this dynamic equilibrium a stationery population of free actin monomers is established and is called the critical concentration. The polarity of the ends of the actin filaments play a key role in elongation. The ends of the filament are referred to as "barbed" and "pointed" according to the polarity of the arrowhead-like structure generated on binding with myosin subfragment 1.<ref>PMID: 1388079</ref> The barbed or plus end binds actin monomers faster than the pointed or minus end. Although its ATPase activity is not crucial for actin polymerisation, actin self-assembly is associated with the ATPase cycle, which powers this treadmilling process. <ref>PMID: 20672362</ref> After the actin molecule is changed into a filament, the bound ATP is hydrolysed to ADP and inorganic phosphate is realeased.<ref>PMID: 1388079</ref> The formation of the F-actin is what stimulates the actin ATPase. The release of the inorganic phosphate happens more slowly than the formation of the filament so that the growing filament has a cap of ATPactin at its barbed end, while monomers containing ADP and inorganic phosphate accumulate in the rest of the filament. | ||
[[Image:Filament_formation.png]] [[Image:Treadmilling.gif]] | [[Image:Filament_formation.png]] Figure 6: Filament formation | ||
[[Image:Treadmilling.gif]] | |||
Figure 7: Image demonstrating the addition of monomers to barbed ends and removal from pointed ends. | |||
The mature filament contains only ADP-actin. The tendency of actin to polymerize depends upon the affinity of actin monomers for filament ends. There is an actin monomer concentration below which actin will not polymerize known as the Critical Concentration (CC). At monomer concentrations above the CC, the actin will polymerize until the free monomer concentration is equal to the CC. It is thought that the strong binding to the ATP is due to the amide proton of a serine 14 amino acid forming hydrogen bonds with the phosphate of ATP, thus stabilizing the nucleotide protein complex.<ref>PMID: 8970724 </ref> Pure actin can be switched between these states in the test-tube by altering the salt concentration. Actin in low salt conditions is in the G-state, while adding salts causes the actin to polymerise.<ref>Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). Molecular biology of the cell. (3 ed.). New York: Garland Science.</ref> Because actin polymerization is reversible, filaments can depolymerize by the dissociation of actin subunits, allowing actin filaments to be broken down when necessary. Therefore an apparent equilibrium exists between actin monomers and filaments, which is dependent on the concentration of free monomers. At this critical concentration, monomers and filaments are in apparent equilibrium. | The mature filament contains only ADP-actin. The tendency of actin to polymerize depends upon the affinity of actin monomers for filament ends. There is an actin monomer concentration below which actin will not polymerize known as the Critical Concentration (CC). At monomer concentrations above the CC, the actin will polymerize until the free monomer concentration is equal to the CC. It is thought that the strong binding to the ATP is due to the amide proton of a serine 14 amino acid forming hydrogen bonds with the phosphate of ATP, thus stabilizing the nucleotide protein complex.<ref>PMID: 8970724 </ref> Pure actin can be switched between these states in the test-tube by altering the salt concentration. Actin in low salt conditions is in the G-state, while adding salts causes the actin to polymerise.<ref>Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). Molecular biology of the cell. (3 ed.). New York: Garland Science.</ref> Because actin polymerization is reversible, filaments can depolymerize by the dissociation of actin subunits, allowing actin filaments to be broken down when necessary. Therefore an apparent equilibrium exists between actin monomers and filaments, which is dependent on the concentration of free monomers. At this critical concentration, monomers and filaments are in apparent equilibrium. | ||
Power Stroke Mechanism | ==Power Stroke Mechanism== | ||
Myosin II molecules bound to actin filaments generate force in skeletal muschles through a mechanism known as the power stroke. The mechanism is comprised of a cycle of ATP-binding, hydrolysis and phosphate release. At the beginning of the first step, the myosin head lacks an ATP and is attached to an actin filament in a conformation known as the “rigor confirmation.” In step two, the ATP binds to the myosin head which induces a conformational shift in the actin-binding site that reduces its binding to actin and cause myosin to release the actin. In the third step the binding of ATP leads to the myosin head being positioned further along the filament. The ATP is then hydrolysed and the inorganic phosphate and ADP remain bound to myosin. In the fourth step, the myosin head makes contact with the actin filament leading to another conformational change that promotes the release of inorganic phosphate. In step five, the binding between myosin and actin is reinforced and triggers the power stroke. Forces are generated on the actin filament as the myosin head returns to its original position. In the final step, the ADP is released and the myosin head remains bound to the filament at a new position from where it began therefore starting the cycle again.<ref>PMID: 11810692</ref> | Myosin II molecules bound to actin filaments generate force in skeletal muschles through a mechanism known as the power stroke. The mechanism is comprised of a cycle of ATP-binding, hydrolysis and phosphate release. At the beginning of the first step, the myosin head lacks an ATP and is attached to an actin filament in a conformation known as the “rigor confirmation.” In step two, the ATP binds to the myosin head which induces a conformational shift in the actin-binding site that reduces its binding to actin and cause myosin to release the actin. In the third step the binding of ATP leads to the myosin head being positioned further along the filament. The ATP is then hydrolysed and the inorganic phosphate and ADP remain bound to myosin. In the fourth step, the myosin head makes contact with the actin filament leading to another conformational change that promotes the release of inorganic phosphate. In step five, the binding between myosin and actin is reinforced and triggers the power stroke. Forces are generated on the actin filament as the myosin head returns to its original position. In the final step, the ADP is released and the myosin head remains bound to the filament at a new position from where it began therefore starting the cycle again.<ref>PMID: 11810692</ref> | ||
[[Image:Myosin powerstroke.jpg]] | [[Image:Myosin powerstroke.jpg]] | ||
Figure 7: Image of the power stroke mechanism | |||
=Function= | =Function= | ||
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Many of the functions of the cytoskeleton involve cytoskeleton and plasma membrane interactions. Proteins that exist between the two help to control cell shape, define the membrane domains and regulate cell/cell interactions and adhesion. There are at least 15 major protein species involved in the membrane-cytoskeleton of the human red blood cell. A mutation in any of these proteins can lead to cell fragility and cell death. Homologs of the erythrocyte membrane-skeleton can be found in many other cell types, which suggests the significance of these proteins. One of particular importance is the actin associated protein dystrophin. Dystrophin is an important part of the sarcolemmal membrane where it links the membrane to the sides of the actin filament bundles. Mutations in this protein leads to muscular dystrophies such as Duchenne’s disease or Becker muscular dystrophy. A homolog of dystrophin called utropin is thought to play a similar role in non-muscle cells. <ref>Boron, W., & Boulpaep, E. (2008). Medical physiology. (2 ed., pp. 14-20). Saunders Elsevier. Retrieved from http://books.google.com/books/about/Medical_Physiology_E_Book.html?id=HlMJRw08ihgC</ref> | Many of the functions of the cytoskeleton involve cytoskeleton and plasma membrane interactions. Proteins that exist between the two help to control cell shape, define the membrane domains and regulate cell/cell interactions and adhesion. There are at least 15 major protein species involved in the membrane-cytoskeleton of the human red blood cell. A mutation in any of these proteins can lead to cell fragility and cell death. Homologs of the erythrocyte membrane-skeleton can be found in many other cell types, which suggests the significance of these proteins. One of particular importance is the actin associated protein dystrophin. Dystrophin is an important part of the sarcolemmal membrane where it links the membrane to the sides of the actin filament bundles. Mutations in this protein leads to muscular dystrophies such as Duchenne’s disease or Becker muscular dystrophy. A homolog of dystrophin called utropin is thought to play a similar role in non-muscle cells. <ref>Boron, W., & Boulpaep, E. (2008). Medical physiology. (2 ed., pp. 14-20). Saunders Elsevier. Retrieved from http://books.google.com/books/about/Medical_Physiology_E_Book.html?id=HlMJRw08ihgC</ref> | ||
The genome of many mammals, including humans contains six actin genes, ACTA1, ACTA2, ACTB, ACTC, ACTG1 and ACTG2. Four of these are differentially expressed in cardiac (ACTC), smooth (ACTA2) enteric (ACTG2) and skeletal muscles (ACTA1); two are described as cytoplasmic actin genes (ACTB and ACTG1) and are expressed in all cells. <ref>Remedios, C., & Chhabra, D. (2008). Actin-binding proteins and diseases. (8 ed., pp. 16-18). New York: Springer.</ref> Actin mutations were not really known until the last 6-10 years. Mutations that have been found to cause human diseases have been found in the ACTC cardiac muscle gene which can cause dilated or hypertrophic cardiomyopathies including congenital fiber-type disproportion (CFTDP). Recently, mutations in ACTG1 have been associated with autosomal dominant deafness. Majority of actin disease mutations have been found to be dominant mutations. This has been the case for actin mutations is the Drosophila flight muscle-specific actin gene ACT88F. Scientists believe the common dominance of actin mutations is because the major functions of actin occur when it is in its F-form, associated with other proteins. The dominant actin mutations that lead to disease are mostly missense mutations. Actin mutations have been found in a variety of species such as yeast, nematodes and flies whose genomes are more accessible to mutagenesis. It is thought that there are three reasons why actin mutations are likely to be at very low frequencies in human populations: 1. actin is a ubiquitously expressed protein, 2. it is a highly conserved protein with many binding partners, so mutations in a large fraction of actin residues appear to cause a severe phenotype in humans and other organisms and 3. most actin mutations are dominant and given the usually severe effects, are not passed on to offspring. Thus familial actin mutants are likely to be relatively mild dominant alleles or recessive. Severe dominant alleles are likely to be de novo mutations. In the case of cytoplasmic actin, five ACT1G disease-causing mutant alleles are known so far and no disease-causing ACTB alleles have yet been described. | |||
=References= | =References= | ||
