Kinesin-1 is a dimeric motor that transports cargo along microtubules, taking 8.2-nm steps in a hand-over-hand fashion. The ATP hydrolysis cycles of its two heads are maintained out of phase by a series of gating mechanisms, which lead to processive runs averaging ∼1 μm. A key structural element for inter-head coordination is the neck linker (NL), which connects the heads to the stalk. To examine the role of the NL in regulating stepping, we investigated NL mutants of various lengths using single-molecule optical trapping and bulk fluorescence approaches in the context of a general framework for gating. Our results show that, although inter-head tension enhances motor velocity, it is crucial neither for inter-head coordination nor for rapid rear-head release. Furthermore, cysteine-light mutants do not produce wild-type motility under load. We conclude that kinesin-1 is primarily front-head gated, and that NL length is tuned to enhance unidirectional processivity and velocity.
In cells, molecules are moved from one location to another by motor proteins. Kinesins are a large family of such motors that transport their cargos along long filaments known as microtubules. Most kinesin molecules are formed from two identical protein chains. Each chain has a motor region at one end (called the head) that can attach to microtubules. The other end of each protein chain wraps around its partner to form a common stalk region (called the tail) that links to the cargo being carried.
The two kinesin heads are connected to the tail via a ‘neck linker’ region, and they advance along the microtubule in strict alternation, similar to the way our legs move when walking. During each step, the front head remains tightly associated with the filament as the trailing head releases itself, advances beyond the front head, and reattaches to become the new leading head. The two heads need to coordinate their activities, so that at any given time, they're not at the same stage in the process. For example, if both heads remained bound to the microtubule at the same time, the motor would not be able to advance. If they both released, the motor would fall off the filament and diffuse away. However, the process by which the heads coordinate is not fully understood, and different models for how this process works have been proposed.
Now, Andreasson, Milic et al. have examined the role played by the neck linker in coordinating the motor's movement using a technique known as ‘optical trapping’. The experiments involved attaching microscopic beads to the motor proteins, which serve as markers that can be tracked. The beads can also be used to exert controlled forces on the kinesin molecules, to see how they respond to different loads.
Andreasson, Milic et al. extended the length of neck linker by inserting extra amino acids (which are the building blocks of proteins) into this region of the protein. It was found that kinesins can still walk even when each neck linker was extended by up to six additional amino acids. However, introducing even a single amino acid into the linker relaxed the normal tension that exists between the heads when these are both bound to the filament. This resulted in slowed speeds, shorter distances of travel, and less ability to sustain loads. The experimental results suggest that the length of the neck linker in naturally occurring kinesins may be optimized to support maximum movement. Based on their data, Andreasson, Milic et al. propose a general framework for understanding the communication that needs to take place between the heads in order to walk in a coordinated manner. Further work is required to understand if motor proteins other than kinesins can also be understood with this same framework.