Kinesin molecular motors move along microtubules by taking alternating steps with a pair of catalytic head domains, where each step is powered by hydrolysis of a single molecule of ATP. This activity plays a key role in numerous cellular functions such as mitosis and neuronal vesicle transport. It is therefore of considerable interest to dissect the molecular details that underlie kinesin's motility functions, not only as a basis fo understanding how this motor's activity may be modulated in vivo by a large variety of regulating factors, but also to aid the development of pharmaceuticals that target these motors for cancer therapy and other therapeutic purposes. Despite intensive study, however, the conformational changes that underlie kinesin's motility cycle remain strongly debated. A particularly elusive question is how dimeric kinesin sustains continuous stepwise movement, because existing methods have not captured the structure of actively stepping kinesin dimers . We have recently made two breakthroughs in our studies of the kinesin motor. First, by using a combination of state of the art cryo-electron microscopy instrumentation together with our own novel image-processing methods, we have solved a new 3D reconstruction of the kinesin-microtubule complex at ~5-6? resolution, substantially improving on previous efforts. This map reveals an unanticipated rearrangement of kinesin's active site following microtubule-stimulated ADP release, suggesting a novel mechanism for this key step in the kinesin cycle and also informing the motor's power stroke. Second, we have devised a novel algorithm for producing high-resolution 3D reconstructions from cryo-EM images of imperfectly decorated, heterogeneous assemblies of kinesin with microtubules. This method has allowed us to solve the first 3D reconstruction of a kinesin dimer as it steps along a microtubule. We will combine our new cryo-EM methods with a host of other state of the art structural and functional techniques, including AFM and saturation-transfer EPR, to establish the detailed basis of kinesin motor function. By comparing structure and functional properties of dimeric kinesin in the presence or absence of mutations that cause loss of motor coordination, we will define the structural basis of inter-molecular tension control and other critical properties of kinesin that are enabled by dimerization. We will also apply cryo-EM to structure/function studies of site-directed mutants in the kinesin catalytic domain in order to test hypotheses for how kinesin's activity is regulated by microtubule binding, and how the motor regulates its affinity for the microtubule during its cycle. The methods developed during the course of this research will transform our ability to study many other large and previously intractable filament-binding proteins, including other molecular motor families as well as microtubule severing enzymes.

Public Health Relevance

The kinesin motor proteins are involved in nearly every step in mitosis and cytokinesis, a trait that has recently led to their targeting by a new generation of anti-cancer pharmaceutical compounds. By revealing how kinesins use ATP to move along filaments inside of cells, our studies will clarify the means by which inhibitors interfere with kinesin function, and how certain mutations can lead to failure of motor motility. Insights gained from our studies will facilitate our ability to design drug inhibitors of these kinesins in order t treat cancer, as well as understanding illnesses related to defects in kinesin function.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
1R01GM110530-01
Application #
8674409
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Flicker, Paula F
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Yale University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
New Haven
State
CT
Country
United States
Zip Code
06510