P. John Hart Laboratory

Researchers in the Hart laboratory combine the use of single crystal X-ray diffraction with a wide range of molecular biological, biochemical, and biophysical methods to characterize macromolecules of fundamental biological and medicinal interest. Below is an enumeration of current research topics and an indication of future research directions.

 

1. Copper-Zinc Superoxide Dismutase (SOD1) Mechanistic Studies

    We have studied SOD1 for approximately ten years. Initial work focused on understanding the enzymatic mechanism of superoxide disproportionation using an approach where we cryogenically trapped reaction intermediates during various steps of the reaction cycle and determined their high resolution structures using the well established tools of single crystal X-ray diffraction. More recent work has sought to elucidate the mechanism bicarbonate-mediated peroxidation catalyzed by SOD1 in the presence of hydrogen peroxide, an alternative reactivity that has been postulated to play an important role in the etiology of motor neuron disease (ALS). Future efforts are aimed at understanding the interaction of nitric oxide (NO) and peroxynitrite (ONOO-) with the catalytic copper ion of SOD1, as these chemistries are also postulated to play a role in ALS.

 

2. Mutations in SOD1 and Familial ALS

    Since the link between mutations in SOD1 and familial ALS was first described approximately 10 years ago, laboratories worldwide have sought to understand how the ~100 distinct point mutations found in FALS families render the SOD1 protein toxic to motor neurons. Our group has made significant strides in this area recently through examination of pathogenic SOD1 structure, where we find that many of the mutants demonstrate conformational changes that give rise to non-native SOD1-SOD1 protein-protein interactions that in turn lead to higher order, amyloid-like filamentous assemblies. Because fibrillar aggregates of pathogenic SOD1 are observed in FALS patients and murine models of the disease, we suggest that our crystallographic observations may represent the molecular basis for pathogenic SOD1 aggregation in vivo.  The discovery of SOD1 misfolding and filamentous assembly has opened a completely new and exciting avenue of research for our group, as we now arecharacterizing rigorously the self-association properties of pathogenic SOD1 proteins using light scattering, surface plasmon resonance (BIAcore), analytical ultracentrifugation, congo red binding assays, etc.

 

3. The Copper Chaperone for SOD1

    Copper is required for the activation of dioxygen, which is essential for the survival of all living organisms. Paradoxically, the electron structure of copper which allows its direct interaction with oxygen also renders it quite toxic. Cells therefore tightly regulate the amount of copper allowed into the cytoplasm through the actions of the transmembrane high-affinity copper transporter, CTR1. In addition, cells are armed with a variety of proteins (such as the metallothioneins) that scavenge free copper ions. Proteins like SOD1 that use copper ion as a cofactor must somehow acquire it in the face of these scavenging molecules. Our knowledge of how this occurs has increased substantially in the last several years with the discovery of a class of molecules termed “copper chaperones”. The copper chaperones acquire copper from CTR1, protect it from the scavenging molecules (and the cellular environment from it), and deliver and insert it into specific target proteins thereby activating them. The copper chaperone for SOD has been identified in humans (hCCS) and yeast (yCCS or LYS7). The genes encoding both human and yeast CCS have been cloned. LYS7 knockout yeast display oxygen sensitivity, but produce copper-free SOD at normal polypeptide levels. Both yCCS and hCCS supplied in trans in these LYS7 knockout yeast rescue the oxygen sensitive phenotype and restore the biosynthesis of holoSOD1 in vivo. Thus, a logical extension of our SOD1 work is to understand molecular basis for copper transfer from CCS to SOD1. Our initial efforts have focused on yCCS, where we used a combination of X-ray crystallography and analytical ultracentrifugation to derive a model for the copper transfer process. Future efforts will focus on structural and biochemical studies of the human CCS protein. In particular, we would like to obtain structures of complexes between SOD1 and CCS. Such information will undoubtedly shed considerable light on the copper transfer process, and, given the possible role of copper in familial ALS (see above) the abrogation of SOD1/CCS interactions could represent a novel therapeutic avenue.

 

4. Mononuclear Blue Copper Proteins of the Phytocyanin Class

    Phytocyanins are a special class of redox-active, mononuclear blue copper proteins that are encoded by one of the largest gene families so far identified in plants. They are known to be highly and specifically expressed in a variety of different plant tissues, but their physiological roles have yet to be identified. My research group is engaged expressing and purifying recombinant phytocyanins for use in  3-D structural studies.  We compare and contrast these new phytocyanin structures with those of the more “traditional” blue copper proteins such as plastocyanin and azurin, as well as with the structures of two recently characterized phytocyanins, cucumber basic protein and cucumber stellacyanin. This combination of biological, spectroscopic, and structural approaches is synergistic, and will undoubtedly have significant impact on our understanding of the evolutionary and functional relationships of phytocyanins across a variety of plant species. A further benefit of these studies is realized in that the new information we obtain on phytocyanin copper binding sites will further our understanding of related sites human multidomain proteins such as ceruloplasmin and blood coagulation factor VIII.

 

5. Targets for Drug Design in Mycobacterium Tuberculosis

The Hart group is part of the Mycobacterium tuberculosis Structural Genomic Consortium (http://www.doe-mbi.ucla.edu/TB/). The goal of the Consortium is to provide the structural basis for the development of therapeutics for tuberculosis. Peptide methionine sulfoxide reductase (Msr) repairs oxidative damage to methionine residues arising from reactive oxygen species and reactive nitrogen intermediates. Msr activity is found in a wide variety of organisms, and it is implicated as one of the primary defenses against oxidative stress. In M. tuberculosis, MsrA and MsrB are enzymes that reduce the S- and R- forms of methionine sulfoxide to methionine, respectively. Disruption of the genes encoding MsrA and MsrB yields an organism that is incapable of infecting and/or surviving in host cells. Although in M. tuberculosis these enzymes have a similar activity, they demonstrate practically no similarity in their amino acid sequences. We recently determined and refined the structure of M. tuberculosis MsrA to 1.5 Å resolution. The structure reveals a methionine from one MsrA molecule in the crystal lattice bound to the active site of the neighboring molecule, and as such, serves as an excellent model for protein-bound methionine sulfoxide recognition and repair.

 

6. Targets for Drug Design from Francisella tularensis

    F. tularensis has been an organism of concern as a biological threat agent since the large state-funded biological weapons programs of the 1950s, when the United States first evaluated the organism as a biological weapon, and it was subsequently incorporated into weapons by the U.S.S.R. Now that the emphasis has shifted towards defending against biological terrorism, gene products in F. tularensis facilitating infection are of intense interest. We are undertaking the structural study of several gene products, which function in F. tularensis infectivity and survival in host cells. Although we have just recently begun this work, we have successfully purified these proteins from recombinant expression systems. The longer term goal is to develop a research program that provides structural information in important gene products such as these for use in synergy with the microbiological in vivo studies already underway in microbiological laboratories.