Capping of F-actin by vinculin in its closed conformation and open II conformation.

<p>(A) The surface of Vt (orange) likely to cap F-actin is normally occluded by vinculin head domains (black). D1 of the vinculin head is shown in green and the flexible loop region is shown in yellow. Subunit <i>n</i> of actin is shown in blue, and subunit <i>n-2</i> of actin is shown in red. Simulation with full-length vinculin in its closed conformation oriented towards S1 and S3 and with the same orientation as used for binding of the occluded surface of Vt to S1 and S3 resulted in formation of a small interface between Vt and the barbed-end of actin (inset). (B) The occlusion of Vt from F-actin can be removed after a potential conformational change. After 15 ns of simulation with this full-length vinculin structure in an open II conformation an interface has formed between Vt and F-actin (inset). Several interactions stabilize this interface: E932 with K291, R935 and K889 with D288, N943 with D286, E883 with R147, R925 with E167, K956 with T351, K952 with Q354, and R945 with Q349.</p

Capping of the actin filament at S3.

<p>(A) Vt (orange) is simulated with its exposed surface initially oriented towards the S3 subdomain of the barbed-end of F-actin. After 15 ns a small interacting interface is formed (inset). There are three interactions at this interface: R1049 with D288, D1051 with R290, and E839 with R290. (B) Vt (orange) is simulated with its occluded surface initially oriented towards S3 of the <i>n</i> subunit. After 15 ns of simulation two interactions are formed between the occluded surface of Vt and S3 of the barbed-end (inset): R1060 and K911 interact with D288, and D907 interacts with K291. Subunit <i>n</i> is shown in blue, and subunit <i>n-2</i> is shown in red.</p

Capping of the actin filament at S1.

<p>(A) Vt (orange) is simulated interacting with the barbed-end of actin. The Exposed surface of Vt is initially oriented towards actin, and Vt is arranged to approach S1 of the barbed-end. After 15 ns of simulation two binding surfaces are produced: (i) electrostatic linkage between charged Vt residues and S1 (top inset), and (ii) hydrophobic insertion of 3 Vt residues into a hydrophobic patch between S1 and S3. (B) Vt is simulated interacting with the barbed-end of actin with its occluded surface initially oriented towards actin. After 15 ns of simulation a binding surface is produced between Vt and S1 of the barbed-end (inset). Several interactions are formed at this interface: R903 interacts with D2 and D3 of S1, K911 with D4, R1060 with E99 and E100, K881 with S350, E879 with Q354, R874 with E361, and E869 with R372. Subunit <i>n</i> is shown in blue, and subunit <i>n-2</i> is shown in red.</p

Structure of an actin filament.

<p>The actin filament is a polar double stranded polymer. Each subunit has a pointed (−) end and a barbed (+) end <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002995#pcbi.1002995-Oda1" target="_blank">[28]</a>. Actin filaments are formed mainly by addition of new actin subunits to the barbed (+) end. (A) The actin filament used in simulation for interacting with vinculin is three subunits in length: subunit <i>n</i> (blue), subunit <i>n-1</i> (white), and subunit <i>n-2</i> (red). The pointed-ends of all the subunits are aligned. An additional subunit <i>n-1</i> would add to the barbed-end of the filament. (B) Each actin subunit has four subdomains: S1 (green), S2 (cyan), S3 (pink), and S4 (tan). (C) View of the actin filament shown in (A) rotated 90 degrees. Actin subunit <i>n-1</i> lies behind <i>n</i> and <i>n-2</i> as viewed from (A). (D) View of the actin filament shown in (B) rotated 90 degrees. Each subunit of the actin filament is rotated relative to its neighboring subunits. This view shows the <i>n</i> subunit is rotated relative to the <i>n-2</i> subunit. Vinculin can bind actin at both along the filament and at the barbed end (arrows).</p

The vinculin binding regions on the surface of F-actin.

<p>The surface of F-actin is shown with all residues shown to interact vinculin colored by charge (basic residues in blue, acidic residues in red, and polar residues in green). The <i>n</i> subunit is shown in cyan, the <i>n-1</i> subunit in white, and the <i>n-2</i> subunit in pink. Three regions on the surface of F-actin are found to be involved in interaction with vinculin: (A) residues in S1 of subunit <i>n</i> are involved in both binding of vinculin along the filament and in capping of the filament by vinculin, (B) residues at S1 and S3 of the barbed-end are involved in capping of the filament by vinculin, and (C) residues at S3 of <i>n-2</i> and S4 of <i>n</i> are involved in binding to D1 of vinculin in its open conformation while binding F-actin along the filament.</p

The open II conformation allows for dynamic linking to F-actin.

<p>The interaction of talin VBS with D1 of vinculin is regulated by the conformation of vinculin. (I) In its closed conformation vinculin can only link to talin VBS. (II) Transition of vinculin form a closed conformation to an open I conformation would result form interaction of Vt with F-actin combined with partial linking of D1 to talin. (III) Formation of the open I conformation allows for complete talin VBS insertion in to D1. (IV) Following formation of open I vinculin, further forces from F-actin can cause formation of the open II conformation. With the open II conformation vinculin can form a dynamic link to F-actin.</p

Interaction of Vt with actin along the filament.

<p>The interaction between Vt (orange) and the F-actin is simulated using molecular dynamics. Vt forms two sets of interactions with F-actin along the filament: (1) an interaction with S3 of <i>n-2</i> (top inset), (2) and interaction with S1 of <i>n</i> (bottom inset). Four interactions are formed between Vt and <i>n-2</i>: T1004 with K328, T1000 with R147, K996 with S145, and R963 with S350. Five interactions are formed between Vt and <i>n</i>: K996 with G48, E986 with R28, K996 with D51, T973 with R95, and R978 with E93. Actin subunit <i>n</i> is shown in blue, and subunit <i>n-2</i> is shown in red.</p

Interaction between vinculin in its closed and open conformation with F-actin along the filament.

<p>(A) The interaction between full-length vinculin in its closed conformation and F-actin is simulated with molecular dynamics. Initially vinculin is with the same orientation relative to F-actin that was used for simulation of Vt. After 15 ns of simulation no stabilizing interactions are formed between vinculin in the closed conformation and F-actin. The closed vinculin makes contact with P333 and R147 of F-actin, as shown in the inset. The contacts result in steric clashes and are followed by movement of vinculin in its closed conformation away from F-actin. (B) A structure of vinculin in its open conformation has previously been suggested <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002995#pcbi.1002995-Golji3" target="_blank">[42]</a>. Using this structure, the interaction between vinculin in its open conformation and F-actin is simulated. The vinculin molecule is initially oriented towards actin with the same orientation that was used for simulation of Vt. After 15 ns of simulation the open conformation of vinculin has formed three sets of binding interactions along the actin filament: (1) an interaction if Vt (orange) with S2 of subunit <i>n</i> (top left inset), (2) and interaction of Vt with S1 of subunit <i>n</i> (top right inset), and (3) and interaction of D1 (green) mainly with S3 of subunit <i>n-2</i> (bottom right inset).</p

The Interaction of Vinculin with Actin

<div><p>Vinculin can interact with F-actin both in recruitment of actin filaments to the growing focal adhesions and also in capping of actin filaments to regulate actin dynamics. Using molecular dynamics, both interactions are simulated using different vinculin conformations. Vinculin is simulated either with only its vinculin tail domain (Vt), with all residues in its closed conformation, with all residues in an open I conformation, and with all residues in an open II conformation. The open I conformation results from movement of domain 1 away from Vt; the open II conformation results from complete dissociation of Vt from the vinculin head domains. Simulation of vinculin binding along the actin filament showed that Vt alone can bind along the actin filaments, that vinculin in its closed conformation cannot bind along the actin filaments, and that vinculin in its open I conformation can bind along the actin filaments. The simulations confirm that movement of domain 1 away from Vt in formation of vinculin 1 is sufficient for allowing Vt to bind along the actin filament. Simulation of Vt capping actin filaments probe six possible bound structures and suggest that vinculin would cap actin filaments by interacting with both S1 and S3 of the barbed-end, using the surface of Vt normally occluded by D4 and nearby vinculin head domain residues. Simulation of D4 separation from Vt after D1 separation formed the open II conformation. Binding of open II vinculin to the barbed-end suggests this conformation allows for vinculin capping. Three binding sites on F-actin are suggested as regions that could link to vinculin. Vinculin is suggested to function as a variable switch at the focal adhesions. The conformation of vinculin and the precise F-actin binding conformation is dependent on the level of mechanical load on the focal adhesion.</p></div

Formation of a vinculin open II conformation.

<p>In its closed conformation (A) vinculin is not likely to bind actin filament. Separation of D1 from Vt constitutes formation of the open I conformation (B) and molecular dynamics simulations suggest the open I conformation can bind along the actin filament. Another critical link between Vt and the vinculin head domains exists at the interface between D4 and Vt. Separation of Vt from D4 results in formation of an open 2 conformation (C). (D) When the open II conformation is equilibrated after being formed D1 moves towards Vt and the loop region reduces its strain. However, Vt remains detached from the vinculin head and D1 remains removed from Vt. In this open II conformation vinculin could interact with F-actin both along the filament and at its barbed-end. Using the umbrella sampling method <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002995#pcbi.1002995-Torrie1" target="_blank">[79]</a> the potential of mean force for two reaction coordinates were calculated: (E) the distance between D1 and Vt, and (F) the distance between D4 and Vt. Progress along reaction coordinate (E) leads to formation of the open I conformation. Progress along reaction coordinate (F) leads to formation of the open II conformation. Formation of the open I conformation has a free energy barrier of 90 KT and the open I conformation is not likely to stay open without persistent energy input. The formation of the open II conformation following formation of the open I conformation has a smaller free energy barrier of less than 20 KT. The plateau at the end of reaction coordinate (F) suggests the open II conformation could remain open without external force.</p