Center for Proteomics and Bioinformatics:
Department of Pharmacology:
|1998||B.S. Cell and Molecular Biology, Tulane University|
|2004||Ph.D., Molecular Biology, University of Texas at Austin|
|2005-2011||Postdoctoral Trainee, Department of Pharmacology, Case Western Reserve University|
|2012-||Tenure-track Assistant Professor, School of Medicine, Case Western Reserve University, Cleveland|
|2010||K99/R00 Pathway to independence award by the National Eye Institute "Structural Studies in Rhodopsin and G Protein Activation"|
|2012||Mt. Sinai Foundation Scholar|
The Lodowski laboratory employs a combination of x-ray crystallography and electron microscopy techniques to explore the mechanisms of GPCR and G protein activation.
1. Structural studies of rhodopsin activation
While several structures of activated (agonist-bound) rhodopsin and other GPCRs have been determined, the mechanism by which the agonist-initiated signal propagates through the receptor to initiate nucleotide exchange and consequent activation of Gα subunits is not fully understood. Because all non-rhodopsin GPCRs determined to date have required significant protein engineering to enable their crystallization and structure determination, bovine rhodopsin provides a model system to understand the activation process.
Information flow during rhodopsin activation. Upon absorption of a photon of light, the 11-cis-retinylidene chromophore is isomerized to its all-trans-state, driving all subsequent activation steps. Deprotonation of the Schiff base linkage follows photoisomerization, and through small-scale changes within the transmembrane region, the activation signal is propagated to the D(E)RY (Glu134, Arg135, and Tyr136) region, resulting in disruption of the "ionic lock" and uptake of a proton from the cytoplasm (most likely onto Glu181, which protrudes toward the chromophore from the one of the β-strands of the plug domain), leading to fully activated meta II rhodopsin. Meta ll catalyzes nucleotide exchange upon the G protein α-subunit of transducin heterotrimers, propagating the activation signal inside the cell. Three regions important in activation and other GPCR functions are highlighted within the transmembrane region: the D(E)RY motif, the NPxxYx(5,6)F motif. and the chromophore binding site. The three insets detail the interactions present within these conserved motifs. For ease of nterpretation, helices are depicted in the following colors: H-I, red; H-II, orange; H-III, yellow; H-IV, lime green; H-V, dark green; H-VI, teal; H-VII, blue; and H-8, purple.
From: Salon JA, Lodowski DT, Palczewski K. "The significance of G protein-coupled receptor crystallography for drug discovery." Pharmacol Rev. 2011 Dec;63(4):901-37.
2. Combining biochemistry, modeling, X-ray crystallography and electron microscopy to understand G protein activation.
While structures for many of the components of the G protein activation complex have been determined, this is by design a dynamic complex, so static X-ray and EM methods for structure determination provide only a snapshot of the dynamic continuum over which this complex exists during the G protein activation process. Stabilization of states along this continuum has allowed structural information to be determined for the Rhodopsin:transducin heteropentamer by electron microscopy as well as a recent crystal structure of the β2-adrenergic receptor solved by the Kobilka group.
3D map of the Rho*•Gt complex at 21.6 Å resolution. (A) Class averages of the Rho*•Gt complex obtained by manual selection of particles prepared by the sandwich negative staining method. This set was used to calculate an initial model. Asterisks mark class averages, which document the bipartite morphology of the complex. Box dimensions, 250 Å 250 Å. (B) Top row: The 3D map calculated from projections of crosslinked Rho*•Gt complexes has a resolution of 21.6 Å according to the FSC function, 50% criterion. The views were generated by Chimera (Pettersen et al., 2004), rotating the Rho*•Gt complex around its long axis in 45° increments. Bottom row: Independent 3D map having a resolution of 28.4 Å calculated from projections of native Rho*•Gt complexes. Scale bar, 100 Å. (C) Semi-empirical model of the Rho*•Gt complex fitted into a 3D envelope established by single particle analysis. Because a single photon can activate the complex, the Rho molecule, which directly interacts with the Gt C-terminus is depicted in yellow to denote that it is the activated subunit. The second Rho is denoted in red. Gtα, Gtβ, Gtγ are colored dark blue, green and magenta, respectively.
From: Jastrzebska B, Ringler P, Lodowski DT, Moiseenkova-Bell V, Golczak M, Müller SA, Palczewski K, Engel A. Rhodopsin-transducin heteropentamer: three-dimensional structure and biochemical characterization. J Struct Biol. 2011 Dec;176(3):387-94. Epub 2011 Sep 6.
3. Understanding the macromolecular machines that underlie cellular signaling processes.
Traditional biochemistry and structural study has centered on the study of individual proteins and protein complexes in isolation. With the advent of systems biology, we can now begin to extend these isolated structural studies to encompass the larger scale cellular machinery responsible for the entire signaling process, providing a structural and relational framework upon which to place the wealth of biochemical data for these diverse components.