Misfolding and subsequent aggregation of endogeneous proteins constitute essential steps in many human disorders, including Alzheimer and prion diseases. In most prion protein-folding studies, the posttranslational modifications, the lipid anchor in particular, were lacking. Here, we studied a fully posttranslationally modified cellular prion protein, carrying two N-glycosylations and the natural GPI anchor. We used time-resolved FTIR to study the prion protein secondary structure changes when binding to a raft-like lipid membrane via its GPI anchor. We observed that membrane anchoring above a threshold concentration induced refolding of the prion protein to intermolecular ␤-sheets. Such transition is not observed in solution and is membrane specific. Excessive membrane anchoring, analyzed with molecular sensitivity, is thought to be a crucial event in the development of prion diseases.FTIR ͉ membrane anchoring ͉ prion protein ͉ protein aggregation ͉ secondary structure

Mammalian myosin IXb (Myo9b) has been shown to exhibit unique motor properties in that it is a single-headed processive motor and the rate-limiting step in its chemical cycle is ATP hydrolysis. Furthermore, it has been reported to move toward the minus-and the plus-end of actin filaments. To analyze the contribution of the light chain-binding domain to the movement, processivity, and directionality of a single-headed processive myosin, we expressed constructs of Caenorhabditis elegans myosin IX (Myo9) containing either the head (Myo9-head) or the head and the light chain-binding domain (Myo9-head-4IQ). Both constructs supported actin filament gliding and moved toward the plus-end of actin filaments. We identified in the head of class IX myosins a calmodulin-binding site at the N terminus of loop 2 that is unique among the myosin superfamily members. Ca 2؉ /calmodulin negatively regulated ATPase and motility of the Myo9-head. The Myo9-head demonstrated characteristics of a processive motor in that it supported actin filament gliding and pivoting at low motor densities. Quantum dot-labeled Myo9-head moved along actin filaments with a considerable run length and frequently paused without dissociating even in the presence of obstacles. We conclude that class IX myosins are plus-end-directed motors and that even a single head exhibits characteristics of a processive motor.Myosins form a large superfamily of actin-based molecular motors that is composed of at least 35 classes (1). Class IX myosins arose in metazoa after the separation of the fungi (1). Invertebrates contain a single myosin class IX gene with the exception of the Drosophila species that have lost their class IX myosin. Bony fishes contain four myosin IX genes and other vertebrates, including mammalia two genes. The two class IX myosins in mammals, Myo9a 2 and Myo9b, exist in multiple splice variants (2). Myo9a has been shown to play a role in epithelial differentiation and morphology whereas Myo9b regulates the migration of macrophages and possibly other immune cells (3, 4). Class IX myosins share a similar structure with the myosins of the other classes, containing a head region, a calmodulin/light chain-binding domain, and a tail region.Additionally, class IX myosins carry some unique features, including a large N-terminal extension preceding the head domain and a long insertion within the head domain in loop 2. The tail region comprises a C1 zinc-binding domain and a RhoGAP domain. Because of this RhoGAP domain, class IX myosins are involved in signal transduction regulating the dynamics of the actin cytoskeleton (2, 5).Mammalian Myo9b, the only class IX myosin studied so far in vitro, exhibits unique mechano-chemical properties. It has been reported to take multiple successive steps along actin filaments without dissociating, indicating that it is a processive motor (6 -8). This is remarkable because Myo9b is a singleheaded myosin. Other myosins that move processively on actin filaments, such as myosin V, dimeric myosin VI, and myosin VII, are two-...

The mammalian class IX myosin Myo9b can move considerable distances along actin filaments before it dissociates. This is remarkable, because it is single headed and because the ratelimiting step in its ATPase cycle is ATP hydrolysis. Thus, it spends most of its cycling time in the ATP-bound state that has a weak affinity for F-actin in other myosins. It has been speculated that the very extended loop 2 in the Myo9b head domain comprises an additional actin-binding site that prevents it from dissociation in the weak binding states. Here we show that two regions in the loop 2 determine the F-actin concentrations needed to maximally activate the steady-state ATPase activity. Together these two regions regulate the amount capable of binding F-actin and the affinity of the nucleotide-free state. The isolated loop 2 behaved like an entropic spring and bound stoichiometrically and with high affinity to F-actin. Subfragment 1 from skeletal muscle myosin II bound to F-actin simultaneously with the isolated loop 2 of Myo9b and could not displace it. Furthermore, the present results imply also a regulatory role for the tail region. Taken together, the results demonstrate that the extended loop 2 in Myo9b binds F-actin and influences the binding of the conventional stereo-specific actin-binding site.Myosin 9b (Myo9b, myr 5) 2 has been reported to move processively along actin filaments, i.e. upon binding to an actin filament it takes multiple consecutive steps before it dissociates (1-3). This is remarkable, because Myo9b is a single-headed myosin. Other myosins that move processively are two-headed and coordinate movement between the two heads (4, 5). Myo9b is also unique in that ATP hydrolysis is the rate-limiting step in the ATPase cycle (6, 7). This means that Myo9b spends a considerable amount of its cycling time in the ATP-bound state that represents a typically weak actin affinity state. However, Myo9b in the ATP-bound state binds with a relatively high affinity to F-actin (6, 7). Nevertheless, kinetic data do not unequivocally support processive movement.It has been speculated that the exceptionally long insertion at the position of loop 2 in the myosin head tethers Myo9b to F-actin and prevents it from diffusing away. The loop 2 in myosins is a surface loop that has been implicated in the initial weak electrostatic interaction with F-actin. In the processive myosin Va this loop is a little longer and more positively charged than in the non-processive class II myosins. An increase in the net positive charge of loop 2 increased the affinity of myosin Va for F-actin in all nucleotide states, whereas a decrease in its net positive charge reduced the affinity (8). Similar findings were also obtained with naturally occurring splice variants of the non-processive myosin V from Drosophila melanogaster and by modifying the loop 2 in class II myosins (8 -12). The processive run length of myosin Va varied with the affinity for F-actin in the weak binding states (13,14). A higher net positive charge of loop 2 increased the pro...

Mammals contain two class IX myosins, Myo9a and Myo9b. They are actin-based motorized signalling molecules that negatively regulate RhoA signalling. Myo9a has been implicated in the regulation of epithelial cell morphology and differentiation, whereas Myo9b has been shown to play an important role in the regulation of macrophage shape and motility.

Although class IX myosins are single-headed, they demonstrate characteristics of processive movement along actin filaments. Double-headed myosins that move processively along actin filaments achieve this by successive binding of the two heads in a hand-over-hand mechanism. This mechanism, obviously, cannot operate in single-headed myosins. However, it has been proposed that a long class IX specific insertion in the myosin head domain at loop2 acts as an F-actin tether, allowing for single-headed processive movement. Here, we tested this proposal directly by analysing the movement of deletion constructs of the class IX myosin from Caenorhabditis elegans (Myo IX). Deletion of the large basic loop2 insertion led to a loss of processive behaviour, while deletion of the N-terminal head extension, a second unique domain of class IX myosins, did not influence the motility of Myo IX. The processive behaviour of Myo IX is also abolished with increasing salt concentrations. These observations directly demonstrate that the insertion located in loop2 acts as an electrostatic actin tether during movement of Myo IX along the actin track.