Carolyn Cohen, Ph.D. - US grants

The purpose of this grant request is to obtain a high resolution Philips 420 electron microscope with cold stage, eucentric goniometer and low dose kit. Our research programs in structural biology involve computerized image analysis of electron micrographs, coordinated with X-ray crystallography and biochemical techniques. To make full use of these capabilities, it is essential that we upgrade our electron microscope facilities. Our present microscope, a Philips 301, is overloaded with users and its performance is deteriorating due to age-related wear. Technological advances have been made in the field of high resolution electron microscopy and in electron microscope design; in particular, the emergence of cryomicroscopy and the development of improved lenses and goniometer stages. The four major programs requiring a new electron microscope are: (1) Structural Studies of Biological Complexes and Structural Studies of Cellular Actin; (2) Assembly of Viruses, Membranes and Tissue; (3) Muscle Structure and the Contractile Mechanism; and (4) Physical-chemical Basis for the Contractile Mechanism. There are eleven individual research projects involving twenty investigators that depend heavily on the use of an electron microscope. In each of these research projects, the primary goal is to determine the three-dimensional structure of organized macromolecular systems and to understand their functions in terms of dynamic states of their structures. Specimens that have been dehydrated and contrasted with heavy metals suffer various distortions, and are not suitable for high resolution structure analysis. A low-temperature stage would allow samples to be examined in an unstained frozen-hydrated state; an eucentric goniometer stage permits collection of high resolution images from tilted specimens for three-dimensional structure determination. Low electron doses are required to minimize radiation damage. The Philips 420 microscope has the full range of capabilities required for progress in our research programs in structural biology.

This research program is focused on three inter-related questions concerning regulation of muscle contraction: the molecular basis for calcium-dependent control by myosin, the role of protein phosphorylation and filament architecture in regulating long-lasting contractions, and the structure and dynamics of the calcium-pumping protein assembly in the sarcoplasmic reticulum membrane. In each case, the goal is to relate the detailed structure and properties of individual protein molecules to the architecture and dynamics of the native protein assemblies in muscle. Drs. Vibert and Cohen have established that certain invertebrate muscles are especially suitable for these investigations, and that their structural and regulatory properties are of general significance for contraction in many muscle types, including those of vertebrates. In the next phase of this program, application of advanced methods of electron microscopy - especially cryo-electron microscopy of vitrified samples - should provide images of higher resolution and fidelity than have been produced in the past. Coupled with the use of proteins altered by site-directed mutagenesis, and of specific antibodies, these structural approaches should allow them to integrate findings at the molecular level into a picture of regulation in whole muscle. Knowledge of the structure and switching of the protein assemblies that regulate muscle contraction involves understanding inter-related control systems at a higher level of organization than any yet achieved.

DESCRIPTION (provided by applicant): The overall aim of the project is to determine atomic structures of proteins critical for muscle contraction and its regulation, including regions that can be selectively targeted by small molecule drugs. X-ray crystallography, supplemented by biochemical techniques and computer analyses, will be the primary methods used. Atomic resolution structures of the myosin head have revealed various states of the contractile cycle. A major aim is to obtain high-resolution structural information for the off-state of molluscan, vertebrate smooth muscle, and other related regulated myosins, with a focus on a nearly complete myosin and on heavy meromyosin(HMM), a large soluble regulated subfragment. Since regions of vertebrate smooth muscle myosin containing the regulatory light chain (RLC) have not yet been crystallized, specialized regulatory domains containing truncated yet phosphorylatable RLC's will also be studied, in order to compare the conformations of this domain in myosins regulated by different triggers. Our understanding of thin filament structure and its stabilization will be improved by the second aim of the project, to extend crystallographic analyses of tropomyosin to smooth muscle isoforms, to heterodimeric products of different tropomyosin genes, and to a portion of the TnT-TnI complex. Analyses of both myosin and tropomyosin proteins will also focus on specific regions implicated in various myopathies. A third aim is to focus X-ray crystallographic and computer analyses on selected alpha-helical coiled-coil segments from these and other proteins that contain specialized features such as alanine staggers, cavities, and trigger sequences. The goal of these studies is to provide a deeper understanding of how these factors influence the geometry, stability and flexibility of the molecules in relation to their function. Alpha-helical coiled coils have been shown to play a role in many oncogenic proteins. Here we have the opportunity to use knowledge gained from these studies of muscle protein alpha-helical coiled coils to help develop small molecule drugs aimed at targeting these structures. One example we focus on is a region of the human smooth-muscle myosin rod that is implicated in leukemia. PUBLIC HEALTH RELEVANCE: The overall aim of the project is to determine atomic structures of proteins critical for muscle contraction and its regulation. Mutations in these proteins are involved in diseases of the heart and other muscles, and have also been shown to cause some leukemias. A deeper understanding of the factors controlling the conformation and stability of these proteins - in particular the widespread alpha-helical coiled-coil motif - will be sought in order to help develop small molecule drugs that selectively target oncoproteins in diseased cells.

This research is intended to elucidate the mechanism by which force is developed by the interaction of myosin and actin molecules in muscle (and in a number of other motile systems) and will focus on the dynamics of the changes taking place in the crossbridges in striated muscle during contraction. It has long been established that muscle contraction is brought about by a sliding filament mechanism, in which partially overlapping arrays of actin and myosin filaments are acted on by a relative sliding force which leads to shortening of the muscle. It is generally believed that this force is generated by a cyclic interaction of myosin crossbridges with sites on the actin filaments. However, the structural details of the process have proved to be particularly difficult to establish, partly because of the transient and unsynchronized nature of many of the changes taking place, and partly because of the technical difficulty of obtaining submicroscopic structural information about any rapidly changing biological system. The development of rapid freezing techniques by Heuser and others has now made it possible in principle to obtain electron microscope images of these transient structural states and suitable variations of these techniques will be developed to enable correlations to be made with evidence derived from time-resolved x-ray diffraction studies. These approaches should provide important new information about the structural behavior of the crossbridges as they interact with actin and develop force. When combined with new information from biochemical and physiological studies, the results of the present work should materially advance our understanding of this very fundamental biological mechanism.

Scallop myosin is a regulated molecule that is switched on by the direct binding of Ca2+. The globular head portion of myosin (subfragment-1, S1) is the motor that contains the ATPase site and interacts with actin. The regulatory and the essential light chains, which form part of the head, act as inhibitory subunits. Scallop myosin is therefore uniquely suited for studies of the structural states that correspond to rest and activity, for characterizing the architecture of its unusual Ca2+-binding site, and for following light chain rearrangements and other conformational changes that result from Ca2+ binding. We have grown X-ray quality crystals of the 'native' regulatory domain of the scallop myosin head, consisting of a 10 kD fragment of the heavy chain and both light chains, and have obtained an interpretable electron density map of the structure. The atomic-level structure of the native regulatory domain will be determined by X-ray crystallography in the presence and absence of bound Ca2+ ions in order to describe the structure of the Ca2+-binding site, and to identify the sequences responsible for binding the light chains to the heavy chain. The regulatory domain has also been reconstituted from isolated chains and complete function is restored. We will attempt to crystallize and determine the structure of such domains reconstituted with mutant light chains and with light chains derived from both regulated and unregulated myosins. Such studies should clarify the mechanisms of light chain function. Small crystals of S1 have also been obtained, and we will attempt to improve these crystals in order to determine the structure at the atomic level. This result would allow us to describe the Ca2+- and ATP-induced structural changes of the myosin head. Preparation of a regulated S1 will also be attempted. The results of these studies will be applicable to the activation of all regulated myosins (smooth muscle and non-muscle myosins) and will help to clarify the structural changes involved in contraction.

In this application we describe research programs in structural biology at two different institutions, each of which requires the ability to analyze data obtained from our high resolution electron microscopes. In order to be able to fully utilize the capabilities of our EM Facilities and to overcome a major gap in the technological resources available to the life sciences communities at Brandeis University and at Boston University School of Medicine, we are proposing to purchase a high resolution precision microdensitometer scanning system. Considerable effort has been devoted to insure that the Rosenstiel Basic Medical Sciences Research Center (the Center) and BUSM, Department of Biophysics have the EM facilities necessary to pursue high-resolution structural studies. Over the past few years several projects have developed specimens extremely well suited for Cystallographic and single particle study using EM techniques. Optical diffraction of film recorded EM images and/or direct observation of film recorded electron diffraction patterns of a variety of these specimens have indicated that the information content of much of this data is great and that Programs for high-resolution structural determination are feasible. Central to working with any film recorded data is the ability to convert this information into digital form for computer-based processing and analysis. In neither institution is there currently a device able to digitize EM film recorded data in a form suitable for high-resolution structural studies. (The digitization equipment currently available is inadequate even for moderate resolution studies.) The few EM based high-resolution reconstructions performed on biological samples (bacteriorhodopsin, light harvesting complex and porin) in other laboratories were exclusively determined with data (images and diffraction patterns) digitized through precision microdensitometers. Both the Center's high resolution electron microscopes (Philips CM12 and Model 420) are routinely providing high resolution images for our structural biology research programs. However, in order to digitize these images for computer analysis it has been necessary for our investigators to travel to Albany, NY as there are no suitable scanners in New England. The instrument of choice for this application is currently being manufactured by Orbital Sciences Corporation (Pomona, CA). It is a flat-bed microdensitometer and is essentially identical to the Perkin-Elmer instrument which is no longer manufactured. It is widely regarded as the state-of-the-art instrument for image digitization and far exceeds the capabilities of our current scanner. The technique used in the Orbital microdensitometer system is almost completely free of optical aberrations and produces high fidelity data. The major advantage of this instrument is that an illuminated area of the specimen is optically projected onto a sample defining aperture as small as 3~m. Only that light which passes through the aperture is measured and digitized. Scanning is accomplished by laterally translating the specimen relative to the optical axis. The aperture maintains a fixed location on the optical axis, thereby minimizing distortions and the effects of optical aberrations. The Center's only available instrument is an Eikonix digitizer which operates by projecting a real image of the specimen to be digitized using a 35mm photographic lens. This real image is digitized via a linear photodiode array which is translated across the image plane. This approach, though relatively inexpensive, is subject to a multitude of optical and mechanical defects. The Eikonix scanner cannot support our high resolution structural studies reliably.

structural biology; proteins; biomedical resource; biological products;

In the past year, Doolittle and colleagues have determined the structures of the terminal human fragment D domain (M.W. 86 kDa) and the crosslinked D-D fragment obtained from fibrin (Spraggon et al., 1997). Using molecular replacement techniques (with X-ray data collected both at the Brookhaven and at CHESS), we have succeeded in positioning the fragment D domains in the unit cells of the flash-frozen (as well as 4 C) crystals of the intact bovine fibrinogen molecule. We find that the end-to-end contacts made between symmetry-related molecules in our crystals are the same as those found by Doolittle and colleagues in the human D-D dimer. Using electron density difference maps, we clearly see coiled-coil regions connecting the terminal D and central E regions of the molecule, and can now locate the bends in these coiled coils. These bends appear to be the primary points of flexibility of the molecule. Using a variety of techniques, including improved data processing programs, refinement, and density modification, we are now extending the resolution of the electron density map to 3.5[unreadable] (the present limit of the flash-frozen data sets). We are now tracing the chains in the Fragment E and connecting coiled coil regions, and are on the threshold of the first near-atomic resolution picture of virtually the whole fibrinogen molecule (Brown et al., in preparation). Using protein supplied to us by L. Medved, we have also crystallized the central Fragment E of bovine fibrinogen (which consists of a disulphide bridge-stabilized globular domain connecting two short coiled coils). Data to 2.8[unreadable] resolution have been collected at CHESS from a native and 7 heavy metal-soaked Fragment E crystals. In attempting to solve this crystal structure, we are also using models of Fragment E derived from our results on the whole molecule. This information is vital for understanding the assembly of fibrinogen into the blood clot and for establishing a comprehensive rational drug design approach for treatment of clotting disorders.

Scallop S1 In order to visualize the conformational changes occurring in the myosin head during ATP hydrolysis, we are determining the structures of the native scallop myosin head fragment (S1) (produced by proteolysis) complexed in the active site with various nucleotide analogs. The current view is that different positions of the lever arm will be found. Thus far, a 2.5[unreadable] data set collected at CHESS allowed us to visualize the ADP-bound form of the molecule.This structure represents the highest resolution obtained to date for a myosin S1 fragment. We have also obtained a 4.2[unreadable] data set and determined the structure of the nucleotide-free state of scallop myosin S1 which corresponds to the last step of the contractile cycle. For the first time, we have been able to describe an ~35 degree tilting movement of the lever arm by comparing two states of the same myosin isoform. Moreover, we have been able to establish at the atomic level the nature of the conformational changes occurring in the motor domain (Houdusse et al., in preparation). A 3.8[unreadable] native data set of scallop S1 complexed with a MgADP.Pi analog (MgADP.vanadate) has also been collected. Structure determination of these crystals should lead to the characterization of a third conformation for the myosin head, that of the pre-power stroke . Vertebrate Smooth Muscle Myosin Head Fragments In parallel with the studies on invertebrate myosin S1 prepared by proteolysis, we have obtained crystals of several subfragment-nucleotide complexes of expressed smooth muscle myosin, a low velocity but high force vertebrate muscle myosin. Using data collected at CHESS, the crystal structures of an expressed vertebrate smooth muscle myosin motor domain (MD) and a motor domain-essential light chain (ELC) complex (MDE) with a transition state analog (MgADP.AlF4-) in the active site were determined to 2.9[unreadable] and 3.5[unreadable] resolution, respectively. The MDE structure with an ATP analog (MgADP.BeFx) was determined to 3.6[unreadable] resolution. In all three structures, the converter domain in the C-terminal region is rotated /70 from that in skeletal subfragment 1 (S1), although the presence of the ELC affects the precise position of the converter. A comparison of the lever arm positions in MDE-AlF4- and in skeletal S1 shows that a potential displacement of /13 nm can be achieved during the power stroke. The MDE-BeFx and MDE-AlF4-structures are almost identical, consistent with the fact that they both bind weakly to actin. These results imply that MgATP binding, and not hydrolysis, primes the lever arm for the power stroke (Dominguez et al., submitted). Non-Muscle Myosins Some members of the myosin superfamily are regulated by direct binding of calcium on the calmodulin subunits in their lever arm. We have grown crystals of a lever arm fragment of myosin V containing two calmodulins in the absence of calcium. A native data set to 2.9[unreadable] resolution as well as Pt and Au derivative data sets to about 3.5[unreadable] resolution have been collected at CHESS. We expect to determine this structure by MIR methods in the near future. The results will allow us to see how calmodulin can bind to its target on myosin under these conditions. These results will provide the first structure of the lever arm of an unconventional myosin, and are the starting point for understanding regulation in this system.

In the past year, Doolittle and colleagues have determined the structures of the terminal human fragment D domain (M.W. 86 kDa) and the crosslinked D-D fragment obtained from fibrin (Spraggon et al., 1997). Using molecular replacement techniques (with X-ray data collected both at the Brookhaven and at CHESS), we have succeeded in positioning the fragment D domains in the unit cells of the flash-frozen (as well as 4 C) crystals of the intact bovine fibrinogen molecule. We find that the end-to-end contacts made between symmetry-related molecules in our crystals are the same as those found by Doolittle and colleagues in the human D-D dimer. Using electron density difference maps, we clearly see coiled-coil regions connecting the terminal D and central E regions of the molecule, and can now locate the bends in these coiled coils. These bends appear to be the primary points of flexibility of the molecule. Using a variety of techniques, including improved data processing programs, refinement, and density modification, we are now extending the resolution of the electron density map to 3.5[unreadable] (the present limit of the flash-frozen data sets). We are now tracing the chains in the Fragment E and connecting coiled coil regions, and are on the threshold of the first near-atomic resolution picture of virtually the whole fibrinogen molecule (Brown et al., in preparation). Using protein supplied to us by L. Medved, we have also crystallized the central Fragment E of bovine fibrinogen (which consists of a disulphide bridge-stabilized globular domain connecting two short coiled coils). Data to 2.8[unreadable] resolution have been collected at CHESS from a native and 7 heavy metal-soaked Fragment E crystals. In attempting to solve this crystal structure, we are also using models of Fragment E derived from our results on the whole molecule. This information is vital for understanding the assembly of fibrinogen into the blood clot and for establishing a comprehensive rational drug design approach for treatment of clotting disorders.

myosins; protein structure function; biomedical resource; structural biology; analytical method;

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We have collected crystal data from both the head and tail regions of scallop myosin. Following our recent study on the high-resolution crystal structure of the actin-detached, internally-uncoupled state of the scallop myosin head (S1) (Himmel, et al., in preparation;reported in our CHESS Progress Report of 2001), we have now obtained a 2.6A resolution structure of S1 in the pre-power stroke or primed conformation (Gourinath, et al., in preparation). Comparison of these two structures, which differ greatly in subdomain interactions, will provide key information on the flexibility of linkages in different states of the contractile cycle. Until now, there has not been a detailed structure reported for any part of the tail of myosin. Using data collected to 2.6A resolution at CHESS, we are determining the structure of a 50-residue-long segment of the scallop myosin tail, located adjacent to the myosin head. This region of the tail is critical for the regulatory properties of the myosin molecule (Li et al., in preparation). Based on data collected at CHESS during the past few years, we have also published reports on crystal structures of key fragments of tropomyosin and fibrinogen. Following our work on an N-terminal fragment of tropomyosin (Brown et al., 2001), we have now established the structure of a 31-residue C-terminal segment of striated-muscle tropomyosin, which shows an unusual splayed conformation. These results reveal a specific recognition site for troponin T and clarify the physical basis for the unique regulatory mechanism of striated muscles (Li et al., PNAS, in press). In our efforts to understand the molecular basis of blood clotting, we have also pursued crystal structures of fibrinogen. We previously reported the 4A structure of the 285 kDa backbone of bovine fibrinogen (Brown et al., 2000), and have now achieved a 1.4A resolution structure of the central domain of the molecule (Madrazo et al., 2001). This structure has a remarkable dimeric interface, which could not previously be visualized. Taken together, these results have improved our understanding of the assembly of the molecule into the fibrinogen clot.