Charles V. Sindelar - US grants

musculoskeletal system; proteins; biomedical resource;

The kinesin motor protein family generates force and movement along microtubules by an as-yet unknown chemical mechanism. Key roles are served in kinesin's force-generation cycle by the power-giving ATP substrate and the tubulin against which motion is generated. We are conducting experiments to characterize the interactions between kinesin, nucleotide, and tubulin. The effect of chemical transitions in the bound nucleotide on the atomic structure of kinesin is being examined by X-ray crystallography studies of complexes between kinesin and chemical variants of ATP. No form of tubulin is known to be compatible with X-ray crystallography studies, but structural data indicate that a short C-terminal segment of b-tubulin contributes most of tubulin's so-called 'major' interaction with kinesin. Variants of this C-terminal peptide will be constructed and assayed for kinesin-binding activity, ability to catalyze kinesin ATPase, or ability to competitively inhibit the microtubule-stimulated ATPase reaction. Peptides with kinesin interactions that are functionally similar to those of tubulin will be co-crystallized with kinesin, in each of the high-tubulin-affinity nucleotide forms of kinesin, and these complexes characterized by X-ray crystallography. These experiments provide the first direct structural information describing the kinesin-microtubule binding interaction, and could provide a structural explanation for the kinesin-bound nucleotide's ability to modulate this interaction. Computer graphics resources are essential to this project because our ultimate goal is to generate a three-dimensional atomic-resolution model of the motor mechanism of kinesin. Our structural determination and refinement efforts require significant amounts of interactive graphics computing.

DESCRIPTION (provided by applicant): 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.

Abstract The Southeastern Center for Microscopy of MacroMolecular Machines (SECM4) is a consortium of 15 Universities/Medical Centers with a total of 19 investigators throughout the Eastern United States studying a wide range of important biomedical projects as variable as high resolution virus structure, membrane protein structure, macromolecular complexes of various types, some isolated in active form from cells, bacterial ultrastructure, muscle filaments, spliceosomes, ribosomes complexes all of which will benefit from ready access to a high resolution electron microscope such as a Titan Krios equipped with a direct electron detector (DED). Human health implications extend from virus and bacterial pathogens to the understanding of diseases resulting form genetic mutations. The basic biology of cancer and heart disease is being studied in several member laboratories. The Titan Krios at Florida State University has been in operation since 2009 and recently has had its image recording device upgraded from CCD camera to a Direct Electron LLC, DE-20 direct electron detector positioned ahead of an existing imaging filter which removes inelastically scattered electrons thereby improving the image quality. Although we propose a robust plan to enable members to come to Florida State University, we propose creating a facility based on the synchrotron template currently in use at multiple sites X-ray crystallography beam lines around the country whereby users ship specimens to us and watch the data being collected as it comes off the microscope from the familiar confines of their own laboratories. We will provide sufficient preprocessing that consortium members can evaluate the prospects for obtaining a final high resolution structure from damage and motion corrected ?movie? images of their samples. The result will be a model for high throughput structure determination utilizing high-end instrumentation that can reveal the inner workings of complex macromolecules and subcellular structures.

PROJECT SUMMARY: The spirochetes are a group of bacteria that are responsible for several deadly ailments including the notorious illnesses, Lyme disease and syphilis. These organisms have developed a special mode of propulsion, whereby their flagella wrap around the cell body to power a screw-like `drilling' motion made by the entire organism. This trait is especially useful for infecting people and other animal hosts, for two reasons: (1) it keeps the flagellum close to the cell body, where it can be covered up by the bacterial outer membrane; this helps spirochetes evade the host immune system, which usually recognizes flagella with ease as a `foreign invader'; and (2) the `drilling' action is highly effective at penetrating dense materials, such as host tissue, which speeds up and otherwise facilitates infection. Despite the clear relevance for disease, many aspects of spirochete motility remain mysterious, including the precise composition and three-dimensional structure of the molecular machinery that drives it. We formed a multidisciplinary, multinational team focused on understanding the detailed structure and function of a spirochete flagellum, working with a pathogenic strain called Leptospira that causes a deadly water-borne illness called leptospirosis. We are using an integrative approach that combines advanced cryo-electron microscopy and cryo-electron tomography, X-ray crystallography, molecular microbiology and genetics techniques, to address major open questions, including: (1) why do spirochete flagella contain many distinct types of protein components when their counterparts from other bacteria need only one; (2) what is the molecular basis of a distinctive, highly coiled flagellar morphology in Leptospira ; and (3) how do these features facilitate motility? By combining complementary top-down and bottom-up strategies, our approach should shed light on spirochetal biology and pathogenesis, and unveil novel molecular targets to develop drugs and vaccines with improved efficacy. 8

Project Summary Kinesin and myosin are so-called `motor proteins' that can use two `feet' to walk along microtubule and actin filaments (respectively) that make up the cytoskeleton. These motors support many vital functions with the cell, including pulling DNA structures apart during cell division and resupplying nerve junctions (the synapse) with neurotransmitters (such as seratonin). The precise mechanisms by which these elaborate molecular machines, which are composed of tens of thousands of exquisitely arranged atoms, are able to `walk' are complex and incompletely understood. To see how they work, it is necessary to visualize these complex structures in three dimensions at sufficient levels of detail to resolve individual atoms? and to follow molecular rearrangements that happen while the motors step forward. This goal, however, has long remained out of reach due to the extreme technical challenges involved. We have addressed this problem by developing new methods to analyze images of frozen motor-filament assemblies collected by latest-generation electron microscopes. This approach, known as cryo-electron microscopy, allows us to directly visualize the three dimensional shape of individual molecular motor proteins attached to their partner filaments. During the previous funding period, we solved 3D structures of truncated single `feet' (one motor domain) of kinesin and myosin motors attached to their partner filaments, showing in atomic detail how these structures changed when molecules of ATP fuel were bound and consumed. We also captured a 3D structure of an intact pair of kinesin molecules (dimer) caught in mid- step on a microtubule. This allowed us to visualize, for the first time, a way in which the two `feet' of kinesin can pull on each other in a way to stay coordinated while walking. In our ongoing research we are improving our methods to capture more intermediates in the stepping process of kinesin, in order to gain a complete more understanding of how it walks. We are improving our analysis methods to better resolve precise chemical details within these structures. Finally, we are extending our approach to understand how a pair of myosin molecules can walk along the actin filament. Results of our studies are expected to aid the development of a new generation of pharmaceutical agents for treating cancer and a wide variety of other diseases.