Kinesin, dynein and myosin constitute the three superfamilies of molecular motors that generate force along cytoskeletal filaments by converting the energy from ATP hydrolysis into mechanical work. The kinesin superfamily contains more than 100 different motor proteins that move along microtubule tracks and power intracellular motile processes such as organelle transport and cell division. The long term goal of this laboratory is to elucidate the conformational changes that kinesin-superfamily members undergo as they hydrolyze ATP and translocate along microtubules. Kinesin-superfamily members contain a highly conserved catalytic domain that possesses the ATP hydrolytic and microtubule binding activities. Kinesin, the archetypal member of the superfamily, has two identical motor domains. It is a processive motor that undergoes many ATP hydrolysis cycles without dissociating from the microtubule. Despite much progress, there is limited information available on the conformational changes kinesin undergoes during ATP hydrolytic cycles and on the mechanism responsible for kinesin's processivity. This proposal addresses these two fundamental issues. The structure and configuration of the two motor domains will be characterized at different points in the ATPase cycle using cryo- electron microscopy and fluorescence polarization microscopy on kinesin molecules fluorescently labeled on the motor domain. Currently, the favored hypothesis to explain kinesin processivity is a hand over hand mechanism. We wilt investigate this hypothesis by testing structural predictions of the model such as the presence of alternating conformations for both motor domains. We will also look for direct structural evidence of the key conformational changes that are though to cause kinesin translocation. We seek to answer the following specific questions 1) Does ATP binding to kinesin moves forward one of the two motor domains? 2) Does ADP binding to kinesin cause an order disorder transition that could move the motor forward? 3) Do the two motors of kinesin alternate conformations as they walk along a microtubule? The results of these studies will contribute to elucidate at a molecular level the structural basis by which kinesins transduce ATP hydrolysis into mechanical work. Given the central role of kinesins in many cellular processes and the importance of microtubules as a target for anti-cancer therapy, this work will provide new insights into normal cellular function and may lead to the identification of new chemotherapeutic targets.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR048620-02
Application #
6623337
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Wang, Fei
Project Start
2002-07-01
Project End
2007-06-30
Budget Start
2003-07-01
Budget End
2004-06-30
Support Year
2
Fiscal Year
2003
Total Cost
$323,980
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Physiology
Type
Schools of Medicine
DUNS #
071036636
City
Bronx
State
NY
Country
United States
Zip Code
10461
Asenjo, Ana B; Sosa, Hernando (2009) A mobile kinesin-head intermediate during the ATP-waiting state. Proc Natl Acad Sci U S A 106:5657-62
Tan, Dongyan; Rice, William J; Sosa, Hernando (2008) Structure of the kinesin13-microtubule ring complex. Structure 16:1732-9
Tan, Dongyan; Asenjo, Ana B; Mennella, Vito et al. (2006) Kinesin-13s form rings around microtubules. J Cell Biol 175:25-31
Asenjo, Ana B; Weinberg, Yonatan; Sosa, Hernando (2006) Nucleotide binding and hydrolysis induces a disorder-order transition in the kinesin neck-linker region. Nat Struct Mol Biol 13:648-54
Peterman, Erwin J G; Sosa, Hernando; Moerner, W E (2004) Single-molecule fluorescence spectroscopy and microscopy of biomolecular motors. Annu Rev Phys Chem 55:79-96