James H. Hurley, Ph.D. : NIDDK

James H. Hurley, Ph.D.

NIDDK, National Institutes of Health
Building 50, Room 4517
50 South Dr.
Bethesda, MD 20892-0580
Tel: 301-402-4703
Fax: 301-480-0639
Email: hurley@helix.nih.gov

Education / Previous Training and Experience:
B.A., San Francisco State University, 1984
M.S., San Francisco State University, 1986
Ph.D., University of California, San Francisco, 1990

Research Statement:

Structural Membrane Biology

Eukaryotic cells are divided into compartments by internal membranes. The main goal of our work is to provide mechanistic insights into fundamental questions in eukaryotic membrane biology at the highest possible level of structural detail and quantitative rigor.

The lysosome is the main locus for the breakdown of membrane proteins, lipids, and endocytosed material in cells. Soluble lysosomal hydrolases are sorted from the TGN to lysosomes via transmembrane sorting receptors. These receptors are rescued from destruction in the lysosome and recycled to the TGN via the retromer complex. The structures of the human retromer subunit VPS26 and the retromer subcomplex VPS29:VPS35 C-terminal fragment were determined in the lab. The overall structure of the full VPS26:VPS29:VPS35 cargo-recognition complex was elaborated by electron microscopy and bioinformatics-based modeling. This resulted in a model for retromer as a novel class of vesicular coat adapted for the tubular endosomal network, operating on completely different principles than the previously-characterized clathrin and COPII coats of spherical vesicles. Together with NIH collaborators Juan Bonifacino and Alasdair Steven, we are extending this analysis to the full retromer complex and its interactions with partners using a fully integrated approach that ranges from crystallography, cryoelectron microscopy, protein-protein and protein-lipid interactions, to in vitro reconstitution and cell imaging.

Ubiquitination is the best characterized marker for protein sorting to lysosomes, and can also serve as an endocytic signal. The Rab5 GTP exchange factor Rabex-5 is an early endocytic protein that recognizes the ubiquitin signal. The structures of three classes of ubiquitin binding domain, the GAT domain of GGA3, and the A20 ZNF and MIU domains of Rabex-5, were characterized by the lab in complex with monoubiquitin. The A20 NZF work was one of the first demonstrations of ubiquitin recognition outside the canonical Ile-44 patch on ubiquitin. Ubiquitination is the major signal directing membrane proteins to multivesicular bodies (MVBs), which subsequently fuse with the lysosome. The lab is currently studying the mechanisms by which monoubiquitination and Lys-63 linked polyubiquitination direct cargo into the ESCRT pathway.

The ESCRT pathway directs the sorting of ubiquitinated cargo into MVBs. There are three major soluble heteroligomeric protein complexes in the ESCRT pathway: ESCRT-0, ESCRT-I, and ESCRT-II. The laboratory has dissected all three of these complexes, determined the core architecture of each of them. Taking this information together with the contributions of others in domain studies, this has advanced understanding of how ESCRTs assemble on membranes and cluster cargo for packaging into MVBs. Analysis of the structure and mechanism of the ESCRT system is one of the major directions in the lab. As for the retromer complex, we take a fully integrated approach. We have an especially strong focus on 1) reconstitution of the system in vitro and 2) integrated structural and cell biological analysis of the role of ESCRTs in MVB biogenesis (via genetics and cell biology within the lab), HIV-1 budding (with Eric Freed), and cytokinesis (with Craig Blackstone and Jennifer Lippincott-Schwartz).

The lab has a strong interest in the role of membrane traffic in the HIV-1 life cycle. The ESCRT pathway is hijacked by HIV-1 and many other viruses to bud from host cells. The structure of the Bro1 domain of the ESCRT-related protein Alix was determined in our lab and its binding site for ESCRT-III elucidated for the yeast proteins, paving the way for seminal insights into the role of the Bro1 domain in HIV-1 budding. We mapped the binding site for the HIV-1 YPXL-class late domain to the V-domain of the ESCRT-associated protein ALIX, and determined its structure. The analysis of interactions between the ESCRTs and other human membrane trafficking complexes on the one hand, and HIV-1 on the other, are a major focus in the lab.

Of all membrane components, cholesterol has received special attention, due to its importance in human health. Cholesterol is moved around the cell both by vesicular and non-vesicular mechanisms. The non-vesicular routes used carrier proteins that include the START domain and Oxysterol binding protein-Related Protein (ORP) families. The crystal structures of the archetypal START domain protein MLN64 and the ORP family member Osh4 were determined at high resolution in the lab. The latter was solved in complex with cholesterol, ergosterol, and various oxysterols. A conformational change involved in sterol transfer was characterized, the structure of the open conformation was also determined, and the dynamics of the conformational change analyzed. Working with NIDDK collaborator Will Prinz, a model for the biological function of Osh4 in subcellular sterol transport was proposed. These studies provide for the first time a structural and functional framework for understanding the important, but until-now poorly understood, ORP class of sterol transport and sensing proteins. The lab has a strong ongoing interest in cholesterol-protein interactions.

Membranes contain the precursors of key lipid and soluble messenger molecules. The prototypical membrane-based messenger system is the hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) to the lipid messenger diacylglycerol and the soluble messenger inositol (3,4,5)-trisphosphate (Ins(3,4,5)P3). Diacylglycerol activates C1 domain-containing proteins such as protein kinase C (PKC) and the chimaerin family of Rac GTPase-activating proteins. One of the first accomplishments of this lab was to show how activated C1 domain proteins such as PKC bound to membranes. Since then we have sought to understand the allosteric activation of PKC and other C1-domain containing enzymes. We solved the structure of the full-length diacylglycerol-activated RacGAP, b2-chimaerin. The structural and functional data led us to a detailed model for the allosteric activation of b2-chimaerin when its C1 domain engages the membrane and disengages from a series of autoinhibitory interactions that block the Rac binding site. This work is still the only complete structural-level account of the diacylglcyerol-dependent activation of a signaling protein. Structural analysis of full-length PKC is the lab’s longest standing goal.

Selected Publications:

Canagarajah, B., Hummer, G., Prinz, W. and Hurley, J. H. Dynamics of Cholesterol Exchange in the OSBP-Related Protein Family. J. Mol. Biol. 378 , 737-748 (2008).

Hurley, J. H. ESCRT Complexes and the Biogenesis of Multivesicular Bodies. Curr. Opin. Cell Biol. 20, 4-11 (2008).

Hurley, J. H. and Yang, D. MIT Domainia. Dev. Cell. 14, 6-8 (2008).

Bonifacino, J. S. and Hurley, J. H. Retromer. Curr. Opin. Cell Biol. 20, 427-436 (2008).

Im, Y. J. and Hurley, J. H. Integrated Structural Model and Membrane Targeting Mechanism of the Human ESCRT-II Complex Dev. Cell. 14, 902-913 (2008).

Yang, D., Rismanchi, N., Renvoise, B., Lippincott-Schwartz, J., Blackstone, C. and Hurley. J. H. Structural Basis for Midbody Targeting of Spastin by the ESCRT-III Protein CHMP1B. Nat. Struct. Mol. Biol 15, 1278-1286 (2008).

Lee, H., Elia, N., Ghirlando, R., Lippincott-Schwartz, J. and Hurley. J. H. Midbody Targeting of the ESCRT Machinery by a Non-Canonical Coiled-Coil in CEP55. Science
322, 576-580 (2008).

Lindwasser OW, Smith WJ, Chaudhuri R, Yang P, Hurley JH, Bonifacino JS A Diacidic Motif in HIV-1 Nef is a Novel Determinant of Binding to AP2. J Virol, 2007. [Full Text/Abstract]

Munshi UM, Kim J, Nagashima K, Hurley JH, Freed EO An Alix fragment potently inhibits HIV-1 budding: characterization of binding to retroviral YPXL late domains. J Biol Chem(282): 3847-55, 2007. [Full Text/Abstract]

Fujii K, Hurley JH, Freed EO Beyond Tsg101: the role of Alix in ''ESCRTing'' HIV-1. Nat Rev Microbiol(5): 912-6, 2007. [Full Text/Abstract]

Chaudhuri R, Lindwasser OW, Smith WJ, Hurley JH, Bonifacino JS Downregulation of CD4 by human immunodeficiency virus type 1 Nef is dependent on clathrin and involves direct interaction of Nef with the AP2 clathrin adaptor. J Virol(81): 3877-90, 2007. [Full Text/Abstract]

Hierro A, Rojas AL, Rojas R, Murthy N, Effantin G, Kajava AV, Steven AC, Bonifacino JS, Hurley JH Functional architecture of the retromer cargo-recognition complex. Nature(449): 1063-7, 2007. [Full Text/Abstract]

Kostelansky MS, Schluter C, Tam YY, Lee S, Ghirlando R, Beach B, Conibear E, Hurley JH Molecular architecture and functional model of the complete yeast ESCRT-I heterotetramer. Cell(129): 485-98, 2007. [Full Text/Abstract]

Lee S, Joshi A, Nagashima K, Freed EO, Hurley JH Structural basis for viral late-domain binding to Alix. Nat Struct Mol Biol(14): 194-9, 2007. [Full Text/Abstract]

Prag G, Watson H, Kim YC, Beach BM, Ghirlando R, Hummer G, Bonifacino JS, Hurley JH The Vps27/Hse1 complex is a GAT domain-based scaffold for ubiquitin-dependent sorting. Dev Cell(12): 973-86, 2007. [Full Text/Abstract]

Leonard TA, Hurley JH Two kinase family dramas. Cell(129): 1037-8, 2007. [Full Text/Abstract]

Hurley JH Membrane binding domains. Biochim Biophys Acta (1761): 805-11, 2006. [Full Text/Abstract]

Raychaudhuri S, Im YJ, Hurley JH, Prinz WA Nonvesicular sterol movement from plasma membrane to ER requires oxysterol-binding protein-related proteins and phosphoinositides. J Cell Biol (173): 107-19, 2006. [Full Text/Abstract]

Wang H, Yang C, Leskow FC, Sun J, Canagarajah B, Hurley JH, Kazanietz MG Phospholipase Cgamma/diacylglycerol-dependent activation of beta2-chimaerin restricts EGF-induced Rac signaling. EMBO J (25): 2062-74, 2006. [Full Text/Abstract]

Kostelansky MS, Sun J, Lee S, Kim J, Ghirlando R, Hierro A, Emr SD, Hurley JH Structural and functional organization of the ESCRT-I trafficking complex. Cell (125): 113-26, 2006. [Full Text/Abstract]

Lee S, Tsai YC, Mattera R, Smith WJ, Kostelansky MS, Weissman AM, Bonifacino JS, Hurley JH Structural basis for ubiquitin recognition and autoubiquitination by Rabex-5. Nat Struct Mol Biol (13): 264-71, 2006. [Full Text/Abstract]

Hurley JH, Emr SD The ESCRT complexes: structure and mechanism of a membrane-trafficking network. Annu Rev Biophys Biomol Struct (35): 277-98, 2006. [Full Text/Abstract]

Shi H, Rojas R, Bonifacino JS, Hurley JH The retromer subunit Vps26 has an arrestin fold and binds Vps35 through its C-terminal domain. Nat Struct Mol Biol (13): 540-8, 2006. [Full Text/Abstract]

Hurley JH, Lee S, Prag G Ubiquitin-binding domains. Biochem J (399): 361-72, 2006. [Full Text/Abstract]

Hierro A, Kim J, Hurley JH Polycistronic expression and purification of the ESCRT-II endosomal trafficking complex. Methods Enzymol (403): 322-32, 2005. [Full Text/Abstract]

Miller GJ, Wilson MP, Majerus PW, Hurley JH Specificity determinants in inositol polyphosphate synthesis: crystal structure of inositol 1,3,4-trisphosphate 5/6-kinase. Mol Cell (18): 201-12, 2005. [Full Text/Abstract]

Kim J, Sitaraman S, Hierro A, Beach BM, Odorizzi G, Hurley JH Structural basis for endosomal targeting by the bro1 domain. Dev Cell (8): 937-47, 2005. [Full Text/Abstract]

Im YJ, Raychaudhuri S, Prinz WA, Hurley JH Structural mechanism for sterol sensing and transport by OSBP-related proteins. Nature (437): 154-8, 2005. [Full Text/Abstract]

Prag G, Lee S, Mattera R, Arighi CN, Beach BM, Bonifacino JS, Hurley JH Structural mechanism for ubiquitinated-cargo recognition by the Golgi-localized, gamma-ear-containing, ADP-ribosylation-factor-binding proteins. Proc Natl Acad Sci U S A (102): 2334-9, 2005. [Full Text/Abstract]

Chi Y Zhou B Wang WQ Chung SK Kwon YU Ahn YH Chang YT Tsujishita Y Hurley JH Zhang ZY Comparative mechanistic and substrate specificity study of inositol polyphosphate 5-phosphatases SPsynaptojanin and SHIP2. J Biol Chem , 2004. [Full Text/Abstract]

Miller GJ, Hurley JH Crystal structure of the catalytic core of inositol 1,4,5-trisphosphate 3-kinase. Mol Cell (15): 703-11, 2004. [Full Text/Abstract]

Canagarajah B, Leskow FC, Ho JY, Mischak H, Saidi LF, Kazanietz MG, Hurley JH Structural mechanism for lipid activation of the Rac-specific GAP, beta2-chimaerin. Cell (119): 407-18, 2004. [Full Text/Abstract]

Hickenbottom SJ Kimmel AR Londos C Hurley JH Structure of a lipid droplet protein; the PAT family member TIP47. Structure (Camb) (12): 1199-207, 2004. [Full Text/Abstract]

Hierro A, Sun J, Rusnak AS, Kim J, Prag G, Emr SD, Hurley JH Structure of the ESCRT-II endosomal trafficking complex. Nature (431): 221-5, 2004. [Full Text/Abstract]

Fritz TA, Hurley JH, Trinh LB, Shiloach J, Tabak LA The beginnings of mucin biosynthesis: the crystal structure of UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferase-T1. Proc Natl Acad Sci U S A (101): 15307-12, 2004. [Full Text/Abstract]

Corbin JD Blount MA Weeks JL 2nd Beasley A Kuhn KP Ho YS Saidi LF Hurley JH Kotera J Francis SH 3H]sildenafil binding to phosphodiesterase-5 is specific, kinetically heterogeneous, and stimulated by cGMP. Mol Pharmacol (63): 1364-72, 2003. [Full Text/Abstract]

Shih SC Prag G Francis SA Sutanto MA Hurley JH Hicke L A ubiquitin-binding motif required for intramolecular monoubiquitylation, the CUE domain. EMBO J (22): 1273-81, 2003. [Full Text/Abstract]

Zhang G Hurley JH Crystallization of the protein kinase Cdelta C1B domain. Methods Mol Biol (233): 299-304, 2003. [Full Text/Abstract]

Hurley JH GAF domains: cyclic nucleotides come full circle. Sci STKE (2003): PE1, 2003. [Full Text/Abstract]

Hurley JH Leucine in the sky with diamonds. Structure (Camb) (11): 1192-3, 2003. [Full Text/Abstract]

Trievel RC Flynn EM Houtz RL Hurley JH Mechanism of multiple lysine methylation by the SET domain enzyme Rubisco LSMT. Nat Struct Biol (10): 545-52, 2003. [Full Text/Abstract]

Prag G Misra S Jones EA Ghirlando R Davies BA Horazdovsky BF Hurley JH Mechanism of ubiquitin recognition by the CUE domain of Vps9p. Cell (113): 609-20, 2003. [Full Text/Abstract]

Hurley JH Membrane proteins: adapting to life at the interface. Chem Biol (10): 2-3, 2003. [Full Text/Abstract]

Miller GJ Mattera R Bonifacino JS Hurley JH Recognition of accessory protein motifs by the gamma-adaptin ear domain of GGA3. Nat Struct Biol (10): 599-606, 2003. [Full Text/Abstract]

Hurley JH Structural analysis of protein kinase C: an introduction. Methods Mol Biol (233): 289-90, 2003. [Full Text/Abstract]

Suer S Misra S Saidi LF Hurley JH Structure of the GAT domain of human GGA1: a syntaxin amino-terminal domain fold in an endosomal trafficking adaptor. Proc Natl Acad Sci U S A (100): 4451-6, 2003. [Full Text/Abstract]

Hurley JH Wendland B Endocytosis: driving membranes around the bend. Cell (111): 143-6, 2002. [Full Text/Abstract]

Aasland R Abrams C Ampe C Ball LJ Bedford MT Cesareni G Gimona M Hurley JH Jarchau T Lehto VP Lemmon MA Linding R Mayer BJ Nagai M Sudol M Walter U Winder SJ Normalization of nomenclature for peptide motifs as ligands of modular protein domains. FEBS Lett (513): 141-4, 2002. [Full Text/Abstract]

Kato Y Misra S Puertollano R Hurley JH Bonifacino JS Phosphoregulation of sorting signal-VHS domain interactions by a direct electrostatic mechanism. Nat Struct Biol (9): 532-6, 2002. [Full Text/Abstract]

Misra S Puertollano R Kato Y Bonifacino JS Hurley JH Structural basis for acidic-cluster-dileucine sorting-signal recognition by VHS domains. Nature (415): 933-7, 2002. [Full Text/Abstract]

Hurley JH Anderson DE Beach B Canagarajah B Ho YS Jones E Miller G Misra S Pearson M Saidi L Suer S Trievel R Tsujishita Y Structural genomics and signaling domains. Trends Biochem Sci (27): 48-53, 2002. [Full Text/Abstract]

Trievel RC Beach BM Dirk LM Houtz RL Hurley JH Structure and catalytic mechanism of a SET domain protein methyltransferase. Cell (111): 91-103, 2002. [Full Text/Abstract]

Misra S Miller GJ Hurley JH Recognizing phosphatidylinositol 3-phosphate. Cell (107): 559-62, 2001. [Full Text/Abstract]

Tsujishita Y Guo S Stolz LE York JD Hurley JH Specificity determinants in phosphoinositide dephosphorylation: crystal structure of an archetypal inositol polyphosphate 5-phosphatase. Cell (105): 379-89, 2001. [Full Text/Abstract]

Hurley JH Meyer T Subcellular targeting by membrane lipids. Curr Opin Cell Biol (13): 146-52, 2001. [Full Text/Abstract]

Hurley JH Tsujishita Y Pearson MA Floundering about at cell membranes: a structural view of phospholipid signaling. Curr Opin Struct Biol (10): 737-43, 2000. [Full Text/Abstract]

Hurley JH Misra S Signaling and subcellular targeting by membrane-binding domains. Annu Rev Biophys Biomol Struct (29): 49-79, 2000. [Full Text/Abstract]

Page last updated: January 06, 2009

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