Our earlier cryo-electron microscopic studies have provided detailed insights into the three-dimensional (3D) organization of icosahedral PDH complexes (Milne et al. EMBO J., 2002;Milne et al., J. Biol. Chem., 2006). We established that E1 or E3 components form an outer shell with an annular gap of 75-90 Angstrom between the outer surface of the E2 core and the regions at each 3-fold vertex where the inner linker joins the peripheral subunit binding domain to which E1 and E3 bind. The creation of this annular space or gap provides an improved means of substrate channeling, both by restricting the volume that the lipoyl domains of the E2 core must sweep through in cycling between the E1, E2 and E3 active sites and by lessening potentially deleterious interactions of a lipoyl domain with E1 or E3 as it shuttles back and forth between the E2 core and the outer layer of E1 and E3 enzymes. The methods developed in the course of this work have served as a platform for studies on the structure of HIV and SIV envelope glycoproteins and have resulted in a number of new structures that we have reported over the last year. In particular, the structures of both soluble and membrane-bound forms of trimeric Env have led to new and unexpected insights into mechanisms of HIV entry into target cells. Our contribution to these studies has resulted in significant methodological advances for automation of image processing and structure refinement, and these have led to a 10-fold increase in the speed of determining structures of these membrane proteins displayed on intact viruses using cryo-electron tomography. These studies have produced the first structures of trimeric Env immunogens and will be invaluable for rational vaccine design.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010412-13
Application #
8552688
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
13
Fiscal Year
2012
Total Cost
$282,176
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Banerjee, Soojay; Bartesaghi, Alberto; Merk, Alan et al. (2016) 2.3 Å resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition. Science 351:871-5
Merk, Alan; Bartesaghi, Alberto; Banerjee, Soojay et al. (2016) Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery. Cell 165:1698-1707
Bartesaghi, Alberto; Merk, Alan; Banerjee, Soojay et al. (2015) 2.2 Å resolution cryo-EM structure of ?-galactosidase in complex with a cell-permeant inhibitor. Science 348:1147-51
Merk, Alan; Subramaniam, Sriram (2013) HIV-1 envelope glycoprotein structure. Curr Opin Struct Biol 23:268-76
Harris, Audray K; Bartesaghi, Alberto; Milne, Jacqueline L S et al. (2013) HIV-1 envelope glycoprotein trimers display open quaternary conformation when bound to the gp41 membrane-proximal external-region-directed broadly neutralizing antibody Z13e1. J Virol 87:7191-6
Bartesaghi, Alberto; Merk, Alan; Borgnia, Mario J et al. (2013) Prefusion structure of trimeric HIV-1 envelope glycoprotein determined by cryo-electron microscopy. Nat Struct Mol Biol 20:1352-7
Tran, Erin E H; Borgnia, Mario J; Kuybeda, Oleg et al. (2012) Structural mechanism of trimeric HIV-1 envelope glycoprotein activation. PLoS Pathog 8:e1002797
White, Tommi A; Bartesaghi, Alberto; Borgnia, Mario J et al. (2011) Three-dimensional structures of soluble CD4-bound states of trimeric simian immunodeficiency virus envelope glycoproteins determined by using cryo-electron tomography. J Virol 85:12114-23
Khursigara, Cezar M; Lan, Ganhui; Neumann, Silke et al. (2011) Lateral density of receptor arrays in the membrane plane influences sensitivity of the E. coli chemotaxis response. EMBO J 30:1719-29
Butan, Carmen; Hartnell, Lisa M; Fenton, Andrew K et al. (2011) Spiral architecture of the nucleoid in Bdellovibrio bacteriovorus. J Bacteriol 193:1341-50

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