Ligand-induced conformational rearrangements regulate the switch between membrane-proximal and distal functions of Rho kinase 2

Abstract

Rho-associated protein kinase 2 (ROCK2) is a membrane-anchored, long, flexible, multidomain, multifunctional protein. Its functions can be divided into two categories: membrane-proximal and membrane-distal. A recent study concluded that membrane-distal functions require the fully extended conformation, and this conclusion was supported by electron microscopy. The present solution small-angle X-ray scattering (SAXS) study revealed that ROCK2 population is a dynamic mixture of folded and partially extended conformers. Binding of RhoA to the coiled-coil domain shifts the equilibrium towards the partially extended state. Enzyme activity measurements suggest that the binding of natural protein substrates to the kinase domain breaks up the interaction between the N-terminal kinase and C-terminal regulatory domains, but smaller substrate analogues do not. The present study reveals the dynamic behaviour of this long, dimeric molecule in solution, and our structural model provides a mechanistic explanation for a set of membrane-proximal functions while allowing for the existence of an extended conformation in the case of membrane-distal functions.

ROCK Inhibition as Potential Target for Treatment of Pulmonary Hypertension

“…This distribution suggests different functions, as described below [ 24 ]. Both isoforms of ROCK possess a similar tridimensional structure composed of three main regions: an N-terminal kinase domain, a Coiled-coil region containing the Rho-binding domain (RBD) and a C-terminal Pleckstrin homology domain (PHD), with an internal cysteine-rich zinc-finger domain [ 25 ]. The homology between ROCK1 and ROCK2 is equivalent to 65% along the entire protein but reaches over 90% in the kinase domain and they are almost identical in the ATP-binding site [ 23 , 24 , 25 ].…”

“…Both isoforms of ROCK possess a similar tridimensional structure composed of three main regions: an N-terminal kinase domain, a Coiled-coil region containing the Rho-binding domain (RBD) and a C-terminal Pleckstrin homology domain (PHD), with an internal cysteine-rich zinc-finger domain [ 25 ]. The homology between ROCK1 and ROCK2 is equivalent to 65% along the entire protein but reaches over 90% in the kinase domain and they are almost identical in the ATP-binding site [ 23 , 24 , 25 ]. The kinase domain of ROCK is generally thought to be active, as indicated after cleavage by caspase-3 (for ROCK1) or granzyme B (for ROCK2), despite the slight catalytic activity of the enzyme.…”

“…The kinase domain of ROCK is generally thought to be active, as indicated after cleavage by caspase-3 (for ROCK1) or granzyme B (for ROCK2), despite the slight catalytic activity of the enzyme. This effect results from an interaction between both the N- and C-terminus, resulting in autoinhibition [ 23 , 25 ]. The binding of phosphatidic lipids or arachidonic acid to the PHD or interaction of GTP-bound RhoA with the RBD, anchor the enzyme in the plasma membrane and increase the phosphorylation of intracellular ROCK substrates [ 23 , 24 , 25 ].…”

“…This effect results from an interaction between both the N- and C-terminus, resulting in autoinhibition [ 23 , 25 ]. The binding of phosphatidic lipids or arachidonic acid to the PHD or interaction of GTP-bound RhoA with the RBD, anchor the enzyme in the plasma membrane and increase the phosphorylation of intracellular ROCK substrates [ 23 , 24 , 25 ].…”

Beta-Secretase 1 Recruits Amyloid-Beta Precursor Protein to ROCK2 Kinase, Resulting in Erroneous Phosphorylation and Beta-Amyloid Plaque Formation

“…Although the structure of the whole molecule is only known at low resolutions [ 12 ], it is evident that for enzymatic activity, only the kinase domains are required. Recently, we constructed a functional model to explain the membrane-proximal and membrane-distal activities of ROCK2, where the weak complex between its terminal domains can be dissociated by natural substrates, allowing the kinase domain to access the substrate [ 13 ]. The concept of the present study is based on this model.…”