In May 2013, the SLAC National Accelerator Laboratory and Stanford ChEM-H solicited seed grant proposals from faculty and permanent-level staff scientists at SLAC and Stanford. Successful proposals will leverage the X-ray facilities (SSRL and LCLS) at SLAC for high-impact studies in the biological sciences that give deep insight into human physiology or disease. Three projects were selected for funding:
"Structural basis for high-affinity binding of a potent engineered angiogenesis inhibitor"
Jennifer Cochran, Bioengineering
Irimpan Mathews, SLAC
The formation of new vasculature, known as angiogenesis, plays a critical role in numerous diseases, including the wet form of age-related macular degeneration (AMD) and cancer. Disease pathogenesis is mediated by networks of intracellular signaling cascades that regulate endothelial cell proliferation, adhesion, and migration. Current therapeutics, which target angiogenesis by blocking ligand-receptor interactions that activate a single cell signaling pathway, have shown limited therapeutic efficacy. To more effectively target the complexity of pro-angiogenic cell signaling networks, we engineered a dual-specific antagonist capable of inhibiting two crossregulating pathways that are key drivers of angiogenesis. In addition to its significant therapeutic potential, this antagonist also exhibits unprecedented ultra-high affinity and specificity for both of its receptor targets, and thus is an important molecular tool for structure-function analysis. Consequently, we will use x-ray crystallography to elucidate the molecular basis for high affinity and dual-specificity of this engineered protein, and to understand structural determinants of native ligand-receptor interactions in this system. Insight into the molecular mechanism of action will complement the use of this engineered protein as a tool for investigating angiogenic cell signaling pathways and disease pathogenesis, and its translation to the clinic.
Kariolis M.S., Miao Y.R., Diep A., Nash S.E., Olcina M.M., Jiang D., Jones D.S., Kapur S., Mathews I.I., Koong A.C., Rankin E.B., Cochran J.R., Giaccia A.J. (2017) Inhibition of the GAS6/AXL pathway augments the efficacy of chemotherapies. Journal of Clinical Investigation. 127(1):183-198. doi: 10.1172/JCI85610. Read More
Kariolis, M.S., Miao, Y.R., Jones, D.S., Kapur, S., Mathews, I.I., Giaccia. A.J., Cochran, J.R. (2014) An Engineered Axl ‘decoy receptor’ effectively silences the Gas6-Axl signaling axis. Nature Chemical Biology, 10(11), 977-83. doi:10.1038/nchembio.1636 Read More
"Directing active site architecture to influence mechanics of O2 activation: an extradiol-intradiol switch"
Aina E. Cohen, Stanford Synchrotron Radiation Lightsource, SLAC
Edward I. Solomon, Chemistry
Soichi Wakatsuki, Structural Biology, SLAC
Activation of O2 from its abundant but relatively non-reactive state to that of a powerful oxidant is a fundamentally important biological process. Oxidases and oxygenases have evolved to activate molecular O2 using various mechanistic strategies, producing specialized reagents able to carry out diverse spectrum of chemical transformations in metabolism, mammalian physiology and biodegradation processes. Tight regulation and mechanistic precision of these reactions are imperative for maintaining the integrity of biological systems by minimizing the deleterious effects of reactive oxygen species (O2−, H2O2, HO2and HO) that are linked to a variety of cellular pathologies (e.g. aging). Homoprotocatechuate 2,3-dioxygenase (2,3-HPCD) from B. fuscum is an extradiol dioxygenase that utilizes molecular O2 to catalyze oxidative ring-opening of catecholic substrates, and represents the model system for diverse mononuclear Fe(II) metalloenzymes from the 2-His-1-carboxylate facial triad family. This proposal will investigate the mechanistic origins and structural basis for an unprecedented regiospecificity of the aromatic ring cleavage by extradiol dioxygenases, and in particular, factors involved in directing O2 activation. This collaborative project combines technical and scientific expertise in structural (X-ray and femto-second crystallography) and spectroscopic (single crystal UV-Vis-NIR, Raman, CD and MCD) methods for investigation of enzymatic reaction mechanisms, with specific emphasis on the correlation of molecular and electronic structures of transient reaction intermediates trapped in crystallo.This new partnership between Professor Edward Solomon (Stanford Department of Chemistry), SLAC staff scientist Dr. Aina Cohen (SSRL macromolecular crystallography) and Professor Soichi Wakatsuki (Stanford School of Medicine and SLAC Photon Science division) is a first step towards building a strong collaborative research program at SLAC that combines advanced X-ray/crystallographic techniques with spectroscopic analysis to understand the electronic and geometric structures of catalytic metal centers, particularly, biological catalysts utilizing molecular oxygen.
"Understanding Antibiotic Resistance: Time-Resolved XFEL Structural Studies of Wild-type and Mutant Ribosomes"
Michael Soltis, Structural Molecular Biology, SLAC
Joseph Puglisi, Structural Biology
During the two year funding period, we will use the unique characteristics of the XFEL to carry out serial femtosecond crystallography (SFX) experiments on the Coherent X-ray Imaging (CXI) endstation of LCLS to determine the high-resolution structures of ribosome. We will determine the structure of wild-type T. thermophilus 30S subunits from two different crystal forms and make direct comparisons with ribosome structures previously determined using synchrotron-generated X-rays. These experiments will be a proof-of-concept demonstration of ribosome structural studies using the XFEL beam. We will also determine the structures of antibiotic-resistant and antibiotic-dependent mutant ribosomes to determine how the mutations influence conformational dynamics to create drug resistance. We will also examine ribosomes with severe defects in the decoding process that require the presence of the antibiotic streptomycin in order to function. Our long term goal is to conduct time-resolved SFX kinetic crystallography studies of aminoglycoside antibiotic binding to the 30S ribosomal subunit and investigate the mechanism by which these drugs perturb the fidelity of the decoding process. Our collaborators at Brown University have identified several aberrant conformational changes resulting from drug binding. Ultimately, we will cross correlate structural states observed by SFX with dynamic states that are observed using single-molecule fluorescence methods.