email:
trybus@physiology.med.uvm.edu

Structure/Function Studies of Conventional 
and Unconventional Myosin Motor and Actin

A major aim of my research program is to understand how molecular motors produce force and motion, and how the motor’s activity is regulated. We are also focusing on the structure and function of actin.

Myosin V:

Unconventional myosin V is a processive motor that moves cargo along actin for long distances without dissociating. Single molecule and biochemical techniques are being used to understand features of the molecule that enhance its processivity, including its interaction with the microtubule-based motor kinesin. The two class V myosins in budding yeast, which transport mRNA and membranous organelles, are being studied with complementary in vitro and in vivo approaches.

• Processive run lengths of kinesin are enhanced by myosin V bound to the same cargo. Kinesin returns the favor when myosin V transports cargo on actin. The non-working motor acts as an electrostatic tether to prevent cargo dissociation. Ali, M.Y., Lu, H., Bookwalter, C.S., Warshaw, D.M., and Trybus, K.M. (2008). Myosin V and Kinesin act as tethers to enhance each others' processivity. PNAS 105, 4691-4696.

• Myosin V surprisingly undergoes a one-dimensional diffusion on microtubules! This potentially allows it to hook up with cargo being transported by microtubule-based motors. Myosin V is also adept at maneuvering through actin intersections, a feature necessary for transport through the dense actin meshwork. Ali, M.Y., Krementsova, E.B., Kennedy, G.G., Mahaffy, R., Pollard, T.D., Trybus, K.M., and Warshaw, D.M. (2007). Myosin Va maneuvers through actin intersections and diffuses along microtubules. PNAS 104, 4332-4336.
• We showed that Myo4p, a class V myosin in budding yeast, is single-headed by virtue of its tight association with the adapter protein She3p. She3p binds to the rod portion of Myo4p, and potentially forms a hetero-coiled coil with it. Having the adapter protein become a subunit of the motor makes biological sense given that it is required for binding to all of Myo4p’s cargo, namely mRNA and cortical ER. Hodges, A.R., Krementsova, E.B., and Trybus, K.M. (2008). She3p binds to the rod of yeast myosin V and prevents it from dimerizing, forming a single-headed motor complex. J Biol Chem 283, 6906-6914.

• Myosin V, when not bound to cargo, forms an inhibited folded conformation in which the motor domain binds to the globular tail. This allows the motor to not wastefully use ATP unless it is actively transporting cargo. Cargo-binding unfolds and activates the molecule. Liu, J., Taylor, D.W., Krementsova, E.B., Trybus, K.M., and Taylor, K.A. (2006). Three-dimensional structure of the myosin V inhibited state by cryoelectron tomography. Nature 442, 208-211.

 

 

Actin:

• We have expressed functional vertebrate actin in the baculovirus/insect cell expression system. Using a mutational strategy, we produced a non-polymerizable monomeric actin, by virtue of two point mutations in subdomain 4 (called “AP-actin”). We have used this actin to examine nucleotide-dependent conformational changes, and to obtain an actin-cytochalasin D structure, which also revealed domain motions of actin consistent with those expected in the filament. Rould, M.A., Wan, Q., Joel, P.B., Lowey, S., and Trybus, K.M. (2006). Crystal structures of expressed non-polymerizable monomeric actin in the ADP and ATP states. J Biol Chem 281, 31909-31919, and Nair, U.B., Joel, P.B., Wan, Q., Lowey, S., Rould, M.A., and Trybus, K.M. (2008). Crystal structures of monomeric actin bound to cytochalasin D. J Mol Biol 384, 848-864.
• The non-polymerizable actin is being used for co-crystallization with other actin-binding partners. Expressed polymerizable actins are also being used to obtain defined small oligomers of actin for structural studies.
  

Smooth Muscle Myosin:

• Smooth muscle myosin is regulated by light chain phosphorylation The structure of the inhibited, dephosphorylated double-headed subfragment of smooth muscle myosin was obtained from 2-D crystals formed on lipid monolayers. An unexpected, asymmetric structure was observed that allowed us to propose a model by which the ATPase activity of both heads could be inhibited, albeit by different mechanisms. This structure has recently been shown to also occur in striated muscle myosins. (Wendt, T., Taylor, D., Trybus, K.M., and Taylor, K.A. (2001) 3-D image recons truction of dephosphorylated smooth muscle heavy meromyosin reveals asymmetry in the interaction between myosin heads and the placement of subfragment 2. Proc. Natl. Acad. Sci. USA, 98, 4361-4366).
• The properties of smooth muscle myosin with a single head phosphorylated were investigated. This species is prevalent during smooth muscle activation, and may also have relevance for the latch state. We showed that under unloaded conditions, myosin with one head phosphorylated activates both heads of the whole molecule. Rovner, A.S., Fagnant, P.M., and Trybus, K.M. (2006). Phosphorylation of a single head of smooth muscle myosin activates the whole molecule. Biochemistry 45, 5280-5289.
• We are pursuing the mechanism by which regulatory light chain phosphorylation activates the molecule via mutational approaches, coupled with enzymatic and motility assays. Ongoing crystallographic studies of the alpha-helical coiled-coil rod of myosin are being used to elucidate why this domain is critical for regulation.