James Bayrer

The central role of LRH-1 in gastroenterological cancer pathogenesis makes it an attractive target for novel drug discovery. Research aims include structure determination of LRH-1 crystallized with inhibitor(s) to isolate and characterize the ligand-binding pocket(s) and regulatory mechanisms. LRH-1 inhibitors have been identified in the sponsor’s laboratory, and work is underway to obtain a LRH-1 LBD:inhibitor crystal complex. The sponsor’s laboratory has extensive experience crystallizing LRH-1 LDB alone and in complex with co-regulatory proteins, making this Aim immediately possible. Information gained from these experiments will enhance our understanding of ligand binding and the impact on protein structure as well as provide targets for combinatorial chemistry and lead compound optimization.

John Bruning (joint project with Robert Fletterick and Matt Jacobson)

Gata4 and Tbx5 are two of the three cardiac transcription factors that have been shown to reprogram cardiac fibroblasts into functional cardiomyocytes. A detailed molecular understanding of the underlying biology of these transcription factors could be crucial to developing novel biotherapies to treat cardiovascular disease. Previous experiments have demonstrated that Tbx5 and Gata4 not only interact with each other but also bind to the same DNA response element. My project will be to obtain a crystal structure of the Gata4/Tbx5/DNA complex.

Peter Hwang

is studying nuclear receptors and their associations with coactivator proteins. His project is to define critical interactions between the coactivators and domains of the nuclear receptors using surface plasmon resonance. Peter also manages the Biacore T100, part of the SPR Core Facility at Mission Bay

Kris Kuchenbecker

studies androgen receptor (AR) interactions using surface plasmon resonance.The androgen receptor (AR) is a ligand activated transcription factor that is essential for transduction of endocrine signaling by the natural hormones testosterone and dihydrotestosterone.  AR manifests this activity by coordinating the assembly of multiple components required for gene transcription.  Comparing AR with other regulated enzymes identifies hormone binding as the allosteric event that unlocks the catalytic activity of the protein-protein interaction surfaces.  

Binding of hormone to the C-terminal ligand binding domain (LBD) is a local process that affects the behavior of the full-length protein by controlling the mode of self-association and the interaction partners.  The LBD interacts with many different proteins in the cell.  Further, different ligands, both natural and synthetic, lead to distinct transcriptional programs.  Because of technical difficulties associated with handling AR, these interactions remain poorly characterized and, as a consequence, our mechanistic understanding of differential gene regulation is limited.  

Direct observation of the LBD's interactions with hormones and proteins has allowed measurement of the kinetics and thermodynamics that govern AR function.

Sabine Mocklinghoff

is studying transcriptional multi-component assemblies by focusing on the essential transcription factors Steroidogenic factor 1 (SF-1) and Liver Receptor Homolog 1 (LRH-1) and their co-regulators, which are critical to embryonic stem (ES) cells and to induced pluripotent stem (iPS) cells. The possibility to achieve stable transcriptional protein assemblies in vitro in the presence of appropriate DNA molecules or by the incorporation of posttranslational modifications (PTMs) will be evaluated with these transcription factors. The stability of full-length SF-1 and LRH-1 in the presence of optimized DNA fragments and co-regulating proteins is expected to improve and thus to gain access to structural studies. The introduction of PTMs can further increase protein stability and might help to identify novel protein binding partners. The remarkable advantage of the chosen semi-synthetic strategy to incorporate PTMs into proteins lies in the generation of homogeneously modified proteins that will allow crystallographic studies. The investigation of the resulting stabilized protein assemblies will provide necessary information for understanding the SF-1 and LRH-1 structure-function relationship and how one could design and use specific modulators to tune aberrant activities which underlie endocrine metabolic diseases and cancer.

 

Elena Sablin

studies two enigmatic orphan nuclear receptors, LRH-1 and SF-1, and their physiological regulatory partner, orphan nuclear receptor Dax-1. Dax-1 is indispensable in embryogenesis and vital for maintenance of pluripotent state of ES cells. Because of its critical role in human development and physiology, Dax-1 is the central target of current studies. We recently expanded the study of regulatory mechanisms by Dax-1 to Nanog, a key transcriptional regulator and marker of the pluripotent state of embryonic stem (ES) cells.

In collaboration with Dr. Holly Ingraham (UCSF), we have determined the crystal structures of the ligand binding domains (LBDs) of LRH-1 and SF-1 to 2.4 Å and 1.2 Å resolution, respectively. These structural studies suggested a model for constitutive transcriptional activity of these receptors and revealed phosphatidyl inositols as potential functional ligands for LRH-1 and SF-1. We have also determined the first structure of Dax-1 in a trimeric complex with its physiological  target, LRH-1. This unusual trimeric assembly might explain how Dax-1 regulates multiple partners in reproductive tissues and embryonic stem cells.

Sablin EP, Blind RD, Krylova IN, Ingraham JG, Cai F, Williams JD, Fletterick RJ, and HA Ingraham (2009) Structure of SF-1 bound by different phospholipids: evidence for regulatory ligands. Mol Endocrinol. 23, 25-34.

Sablin EP, Woods A, Krylova IN, Hwang P, Ingraham HA, and R.J. Fletterick (2008) The structure of corepressor Dax-1 bound to its target nuclear receptor LRH-1. Proc Natl Acad Sci USA 105, 18390-5.  

Krylova IN., Sablin EP, Moore J, Xu RX, Waitt GM, McKay JA, Juzumiene D, Bynum JM, Madause K, Montana V, Lebedeva L, Suzawa M, Williams JD, Williams SP, Guy RK, Thornton JW, Fletterick RJ, Willson TM and HA Ingraham (2005) Structural analyses reveal phosphatidyl inositols as ligands for the NR5 orphan receptors SF-1 and LRH-1. Cell 120, 343-355.

Sablin EP, Krylova IN, Fletterick RJ, and HA Ingraham (2003) Structural basis for ligand-independent activation of the orphan nuclear receptor LRH-1.  Mol Cell 11, 1575-85.

Fumiaki Yumoto

obtained his PhD from the Univ. of Tokyo and joined theFletterick lab in 2007. He studies the molecular mechanisms of the synergy between the nuclear receptor, LRH-1, and beta-catenin using X-ray crystallography. This structure-function study is in progress in collaboration with the labs of Dr. Paul Webb of The Methodist Hospital Research Institute (TMHRI) in Houston. Fumiaki is interested in stem cell biology and because LRH-1 is an up-regulator of Oct4 expression in embryonic stem cells (ES cells), it contributes to maintaining the pluripotent state. Fumiaki has expanded his research to identify LRH-1-interacting molecules from mouse ES cells by mass spectrometry-based proteomics through collaboration with Nevan Krogan’s lab at UCSF. Fumiaki was awarded a postdoctoral research fellowship from the Sandler Program for Breakthrough Biomedical Research (UCSF) for this project. He is also working on a collaborative study on cell reprogramming with the labs of Dr. Bruce Conklin and Dr. Shinya Yamanaka at the Gladstone Institute for Cardiovascular Disease.

 

Maia Vinogradova

works on the regulation of the kinesin motor. Her two projects on kinesin regulation
are: 1) crystallization of the kinesin-like calmodulin binding protein (KCBP) in the complex with its regulator, either calmodulin or a novel single EF-hand, plant specific protein KIC, and 2)  crystallization of the complex of the kinesin CENP-E and its cargo binding protein – protein kinase BUBR1 which is involved in mitotic checkpoint. She also works on defining ligands in binding to androgen receptor by X-ray crystallography.

Maia is also studying the skeletal muscle ternary complex in both relaxed and calcium-activated forms. The goal of this project is to find a drug capable of increasing the muscle strength and Ca2+-sensitivity of muscle contractions.

Maia previously determined the structure of troponin with and without calcium.

Vinogradova MV, Malanina GG, Reddy AS, Fletterick RJ. (2009)  Structure of the complex of a mitotic kinesin with its calcium binding regulator.  Proc Natl Acad Sci U S A. May 19; 106(20):8175-9.

Vinogradova M, Malanina G, Reddy V, Reddy A and RJ Fletterick. (2008)  Structural dynamics of the microtubule binding and regulatory elements in the kinesin-like calmodulin binding protein. J Structural Biology 2008 J Struct Biol. 2008 Jul;163(1):76-83.

Vinogradova MV, Stone DB, Malanina GG, Mendelson RA, Fletterick RJ. (2007) Ca ion and the troponin switch. Adv Exp Med Biol.592:47-57. Review.