Friday, 15 June 2012

A Structural State of the Myosin V motor without nucleotide by Coureux

  • Myosin superfamily of molecular motors use ATP hydrolysis and actin activated product to produce directed movement and force
  • Conform of myosin without bound bt:
          --- nt-binding site has adopted new conform of nt-binding elements 
          --- reduce affinity for nt
          --- major cleft in mol has closed
          --- lever arm has assumed position consistent with actomyosin rigor complex
          --- change caused by movements of subdomains
  • Myosin V is a myosin motor
  • has str and kinetic features that let it act as 2-headed processive motor protein
  • has long tandem repeat of 6 calmodulin/light chain binding sites
  • form a long lever arm
  • let myosin V take multiple 36 nm steps along an actin filament without detachment (processive movement)
  • lets mol function as vesicle transporter
  • or processive movement, rates of key kinetic steps on Myosin V are diff from myosin II
  • each head spends most of ATP cycle strongly bound to actin
  • Myosin coupling ATP hydrolysis and actin binding to movement is lever arm hypothesis
  • Nt binding, hydrolysis and product release are coupled to small movements in myosin motor core
  • movements are amplified and transmitted by a region called the converter
          --- to a lever arm consisting of a light chain binding helix and assoc light chains
          --- lever arm further amplifies converter's motions into large directed movements
  • Absence of actin: ATP hydrolysis
          --- product release is slow
          --- trap lever arm in primed or pre-power-stroke position
  • Actin binding
          ---- product released
          --- lever arm moves
          --- force generation
         --- strong binding between actin and myosin (see Supplementary Fig. 1).


  • Cystals with vanadate or AlF4 and MgADP reveal "Transition" state
  • mimics pre-powerstroke state of cyosin compativle with ATP hydrolysis and before actin binding
  • near rigor state has been seen with Mg ADP,  AbATP, ATP analogies or no nt in active site
  • proposed to reveal position of myosin lever arm at end of power stroke on release of MgADP
  • rigor state
  • Myosin V binds rapidly to actinin concentration dependent manner
  • not temperature dependent
  • doe not saturate over actin concentrations examined
  • kinetics of myosin V binding appear to be diffusion limnited
  • in absence of nt and actin may be in state nearly equivalent to rigor state formed on actin addition
  • Lever arm is similar top that of near rigot state
Fig 1 Positions of subdomains and connectors in the three myosin states and closure of the 50-kDa cleft.  a, A comparison of the myosin V motor domain to the Dictyostelium myosin II in the near-rigor and transition states shows the different positions of the subdomains, nucleotide-binding elements and connectors in each state. The structures have been superimposed on the N-terminal subdomains. Relative to this subdomain, the rotation necessary to move from the myosin V state to the near-rigor state is indicated for each subdomain of myosin V; similarly, the rotation necessary to move from the near-rigor to the transition state is indicated on the subdomains of the transition-state structure. Contours of the solvent-accessible cavities for the near-rigor (1,735 plusminus 173 Å3) and transition states (795 plusminus 140 Å3) are shown with a red contour, whereas the green contour (73 plusminus 27 Å3) depicted in all structures represents an internal cavity of myosin V. Pi, inorganic phosphate. b, Shown are specific hydrogen bonds involving the strut that result in cleft closure near the actin interface. Of note is the interaction of D570 with K405 and K246, which are residues conserved in all myosins. However, a number of residues involved in cleft closure in our structure (such as T571, K569, N398) are not absolutely conserved in the myosin superfamily. Variability in cleft interactions could alter the kinetics of cleft closure, and thus the rate of the weak-to-strong binding to actin.


  • Fig 1a shows movements of subdomains of myosin motor allow dif actin interface, closeure of a major clect (50kDa cleft) in mol and weakening of nt binding
  • Subdominas of motor proposed to move as units connected by flexible ocnnectors of joints
          --- are N terinal upper 50kDa and lower 50kDa subdomains 
          --- and converter (to wh lever arm is attached)


  • shows new conforms of previously defined connectors
          --- switch II, relay and SH1 helix
  •  shows importance of 4th connector (strut)
  • links lower and upper 50 kDa subdomains near actin interface
  • Connectors mediate precise interactions
  • diff for each myosin state
  • allow stabilisation of unique subdomain positions in each state
  • In new state, significant movement of upper 50kDa subdomain relative to N terminal subdomain
  • critical for rearranging nt-binding pocket and closure of internal cleft between 2 50 kDa subdomains
  • It is predicted that 50kDa cleft in myosin II must close to create rigor interaction
  • In new str, actin interface is diff from near rigor or transition states
  • due to relative rotation and translation of upper and lower 50kDa subdomains
  • cause cleft closure near actin- and nt-binding sites (Fig 1a)
  • closure near actin-binding sites requires conform change in a connector between upper and lower 50kDa subdomains (the strut) that lets it interact spec with both subdomains (fig 1b)
  • Changes in strut allow many direct interactions between upper and lower 50kDa subdomain
          --- vdW and spec side-chain interactions
  • mutagenesis shows changing strut length prevent strong binding to actin
  • In ou str N398 forms H bonds with residues of lower 50kDa subdomain and strut (Fig 1b)
  • formation of actin-myosin rigor complex requires conform change in myosin II
  • causes net exclusion of water mols from motor
  • cleft closure causes exclusion of a lot of water from mol
  • obliterates aqueous tunnels seen in near rigor str (fig 1a)
  • in our myosin V str P loop and switch I moved  6.5 A˚ apart
  • destroys ability to coordinate Mg
  •  destroys possibility of switch I interactions with nt.

FIGURE 2. Nucleotide-binding site and distortion of the bold beta-sheet at the interface of the N-terminal and upper 50-kDa subdomains.a, Shown is an overlay of the beta-sheet (N-terminal subdomain superimposed) between myosin V in blue and near rigor (Dictyostelium myosin II) in grey. Note that strands 5–7, which belong to the upper 50-kDa subdomain are distorted to allow the upper 50-kDa rotation that removes switch I from the nucleotide-binding site. b, The positions of the nucleotide-binding elements are shown for three myosin states. The yellow asterisk in the myosin V structure marks the position of the Mg2+ in the other structures. In the transition state, switch II contributes to coordination of the gamma-phosphate of the nucleotide, but it bends in myosin V in the opposite direction and forms direct interactions (broken green lines) with the fourth beta-strand and the P loop of the N-terminal subdomain.

  • New conform of switch II is stabilised by new interactions (fig 2b)
  • sitch 1 conform is not changed
  • follows movement of upper 50kDa subdomain relative to P loop and N terminal subdomain (fig 2a)
  • Position of P loop provide steric hindrance to nt entry (fig 2a)
  • as position in str is stabilised by weak interactions, in sol P loop can explore other conform and not significantly hinder ATP binding
  • 7-stranded b-sheet that couples N terminal and upper-50kDa distorts
  • essential ot let movement of upper 50kDa subdomain (inc switch I)
  • key interactions that maintain sheet not altered
  • switch I and II follow rotation of upper 50kDa subdomain
  • maintain upper 50kDa N terminal subdomain interactions on opposite side of sheet
  • distortion greatest for strang 5
  • strand 5 connects to switch II
  • also connects to a linker from longest helix (HO) of upper 50kDa subdomain that begins at actin interface (HCM loop)
  • thru helix, linker provide coupling between b-sheet and actin interface
  • linker distors in our new state compared to weak beingind states (transition or near-rigor states)
  • Switch II positions subdomains critical for cleft closure and lever arm position in new state
  • switch II promotes diff set of interactions between subdomains in TS str
  • cause partial cleft closure of TS compared to near-rigor state
  • traps phosphate after ATP hydrolysis
  • cause repositioning of lever arm in its pre-power-stroke conform
  • Interactions that close cleft in TS are not all maintained in new state
  • part of region closed in TS opens to form a small internal cavity in nt-free str (fig1a)
  • if interface between lower 50kDa subdomain and actin is same in all myosin states, new str form more extensive interface with actin
  • upper 50kDa subdomain moved close to actin surface
  • HSM loop contributes hydrophobic interactions to create strong actin binding
  • HSM loops and another loop (loop4) in position to make actin interactions (fig 3)

Fig3 Actin-myosin interface. a, Myosin V as viewed from the actin side of the interface reveals the positioning of putative actin-binding elements. b, The same view of myosin V is overlaid on the lower 50-kDa subdomains ofDictyostelium myosin II structures. Note the conformational change in the strut and the obvious rotation of the upper 50-kDa subdomain towards the actin filament (represented by an arrow) in the myosin V structure. c, The myosin V and transition state actin-binding elements are docked on an actin filament, maintaining the identical positioning of the lower 50-kDa subdomain in both cases. In myosin V, the HCM loop has been repositioned in such a way that it can directly contribute to actin binding.

  •  proposed that myosin V str is in rigor-like state
  • if rigorlike str rearranges to conform seen in near rigor state on ATP intro, explains how ATP binding actomyosin rigor complex weakens myosin affinity for actin
  • Seq of events:
          --- Phosphates of ATP bind to P loop
          --- inward movement of switch I coordinates g- and b-phosphates and Mg2+ ion
          --- switch I movement mediate by rearrangement of b-sheet
          --- switch II moves
          --- cleft reopens
          --- decrease affinity of myosin for actin
          --- myosin dissoc
          --- formation of near-rigor state
          --- isomerisation and dissoc occurs with minimal movement of converter and lever ark
          --- ATP binding does not reverse lever arm movement
          --- reversal or repriming occurs on isomerisation from near rigor state to TS
          --- while myosin is detached from actin
          --- immediately precedes ATP hydrolysis
          --- after phosphate release, myosin V populates a strong actin-binding site
          --- MgATP bound strongly to nt-binding site
          --- this is predominate steady-state intmt of myosin V's actin activated ATPAse cycle
  • myosin V populates a rigorlike conform in absence of both actin and nt
  • this str of myosin is closed cleft state

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