 
Project #1: Biochemical and NMR Studies of
the HIV Envelope Proteins
The HIV envelope proteins gp41 and
gp120 play critical roles in HIV infection, the causative
agent of AIDS. Recently, it has become apparent that
current AIDS therapies fail in up to 50% of patients;
consequently, it is of interest to search for new viral
targets such as the envelope proteins. While at the
NIH, I characterized the structural and dynamic properties
of the SIV gp41 ectodomain, which is functionally analogous
to the HIV gp41 ectodomain (Caffrey et al., 1997; Caffrey
et al., 1998ab; Caffrey et al., 1999; Caffrey et al.,
2000). Presently, we are extending our biochemical and
NMR studies to HIV gp41 and gp120. The specific aims
of this project are: (i) determine the structure and
dynamic properties of the HIV gp41 ectodomain; (ii)
determine the mechanism of gp41 peptide inhibition of
HIV infection; (iii) develop a mutagenesis system to
relate HIV gp41 structure to function; (iv) determine
the structure and dynamic properties of the HIV gp41/gp120
complex. As a first step we have generated a high-resolution
model of the HIV gp41 ectodomain, which was based on
the SIV gp41 ectodomain structure (Caffrey, 2001). Recently,
we have determined the first structure of the gp120
C5 domain (Guilhaudis et al., 2002) and have started
to characterize the structural and thermodynamic properties
of the gp41 ectodomain by analytical ultracentrifugation
and calorimetry (Jacobs et al., 2004; Jacobs et al.,
submitted). In addition, we have recently analyzed the
effects of site-directed mutants of gp41 and gp120 on
viral entry (Jacobs et al., 2005; Sen et al., in preparation).
Project #2: Biochemical and NMR Studies of the
Coxsackie and Adenovirius Receptor
The extracellular domain of the Coxsackie
and Adenovirus Receptor protein (CAR-D1D2) has been
shown to be the receptor for the Coxsackie B virus (CBV),
which is a major cause of viral heart infections. Moreover,
CAR is the receptor for Adenovrius (Ad), which is an
important vector for gene therapy. In this project the
biochemical and structural properties of CAR-D1D2 will
be characterized with the long-term goal of developing
structure-based drug therapies that are directed against
chronic CBV infection. The specific aims of this project
are: (i) to prepare a recombinant forms of CAR-D1D2
that are amenable for biochemical and NMR studies; (ii)
determine the structural and dynamic properties of CAR-D1D2;
(iii) determine the binding site of CBV by NMR spectroscopy.
We have successfully determined the structure and dynamics
of the CAR-D1 (Jiang and Caffrey, 2002; Jiang et al.,
2004), the domain that binds to CBV and Ad. Recently,
we have characterized the structure of CAR-D2 by NMR
(Jiang and Caffrey, 2005; Jiang et al., in preparation).
Project #3: Biochemical and NMR Studies of Protein
Transduction Domains
Proteins containing protein transduction
domains (PTD) have been shown to rapidly traverse biological
membranes in a relatively nonspecific fashion without
the aid of protein receptors. Importantly, PTD offer
a tissue-independent vehicle to introduce biologically
active materials (e.g. drugs or therapeutic proteins)
across biological membranes. At present, little is known
about the PTD structure and mechanism, which is the
goal of the present project. The specific aims of this
project are: (i) construction of model PTD fusions for
biochemical and NMR studies; (ii) determine the structure
and dynamic properties of PTD in solution by NMR spectroscopy;
(iii) determine the structure and dynamic properties
of PTD in lipid environments by NMR spectroscopy. To
date a fusion protein containing the PTD from HIV tat
has been generated (PTD-tat) and the structural and
dynamic properties in solution have been characterized
by NMR spectroscopy (Hakansson and Caffrey, 2003). Moreover,
we have recently demonstrated that PTD-tat binds to
heparin (Hakansson et al., 2001; Hakansson and Caffrey,
2003), which may play a role in mediating PTD function
in vivo.
Project #4: Drug Discovery Studies of the Bacillus
anthracis Protective Antigen
Bacillus anthracis is the causative
agent of anthrax disease, which has recently generated
interest as an agent of bio-terrorism. The protective
antigen domain 4 of B. anthracis (PA-D4) plays a critical
role in the entry of the anthrax toxins and consequently
is an attractive target for the development of anti-toxins.
The specific aims of this project are: (i) determine
the structure and dynamic properties of PA-D4 by NMR
spectroscopy; (ii) use the complementary approaches
of phage display and in silico drug discovery to identify
peptides and small drug-like molecules that bind to
PA-D4; (iii) assay candidate peptides, peptoids, and
drug-like compounds for their ability to disrupt PA-D4
activity in vivo; (iv) characterize PA-D4/antagonist
complexes by biophysical techniques for future optimization
as lead drug candidates. In a first step we have developed
an expression system for the generation of PA-D4 in
amounts amenable for biophysical studies (Krishnanchettiar
et al., 2002).
Project #5: Biochemical and
Structural Studies of the SARS Coronavirus Envelope
Proteins.
The envelope proteins of SARS Coronavirus,
which are termed S1 and S2, play similar roles to the
HIV envelope proteins gp120 and gp41. Recently, we have
undertaken study of S1 and S2 domains by biochemical
and structural methods. In a first step we have determined
the solution structure of the S2 heptad repeat 2 domain,
which represents the first characterization of a transient
intermediate step (Hakansson et al., in preparation).
Selected References
Caffrey, M. (2001) Model of the HIV
gp41 ectodomain: insight into the intermolecular interactions
of gp41. Biochimia Biophysica Acta 1536, 116-122.
Caffrey, M., Braddock, D., Louis, J.,
Abu-Asab, M., Kingma, D., Liotta, L., Tsokos, M., Tresser,
N., Pannell, L., Watts, N., Steven, A., Simon, M., Stahl,
S., Wingfield, P. and Clore, G. (2000) Biophysical characterization
of gp41 aggregates suggests a model for the molecular
mechanism of HIV-associated neurological damage and
dementia. J. Biol. Chem. 275, 19877-19882.
Caffrey, M., Cai, M., Kaufman, J.,
Stahl, S., Wingfield, P., Gronenborn, A. and Clore,
G. (1997) Determination of the secondary structure and
global topology of the 44 kDa ectodomain of gp41 of
the simian immunodeficiency virus by multidimensional
nuclear magnetic resonance spectroscopy. J. Mol. Biol.
271, 819-826.
Caffrey, M., Cai, M., Kaufman, J.,
Stahl, S., Wingfield, P., Covell, D., Gronenborn, A.
and Clore, G. (1998a) Three-dimensional solution structure
of the 44 kDa ectodomain of SIV gp41. EMBO J. 17, 4572-4584.
Caffrey, M., Kaufman, J., Stahl, S.,
Wingfield, P., Gronenborn, A. and Clore, G., (1998b)
3D NMR experiments for measuring 15N relaxation data
in large proteins: application to the 44 kDa ectodomain
of SIV gp41. J. Magn. Reson., 135, 368-372.
Caffrey, M., Kaufman, J., Stahl, S.,
Wingfield, P., Gronenborn, A. and Clore, G. (1999) Monomer-trimer
equilibrium of the ectodomain of SIV gp41: insight into
the mechanism of peptide inhibition of HIV infection.
Prot. Sci. 8, 1904-1907.
Guilhaudis, L., Jacobs, A. and Caffrey,
M. (2002) Solution structure of the HIV gp120 C5 domain.
Eur. J. Biochem. 269, 4860-4867.
Hakansson, S. and Caffrey, M. (2003)
Structural and dynamic properties of the HIV-1 tat protein
transduction domain in the free and heparin bound states.
Biochemistry 42, 8999-9006.
Hakansson, S., Jacobs, A. and Caffrey,
M. (2001) Heparin binding by the HIV-1 tat protein transduction
domain. Prot. Sci. 10, 2138-2139.
Jacobs, A., Hartman, K., Laue, T. and
Caffrey, M. (2004) Sedimentation velocity studies of
the high molecular weight aggregates of HIV gp41. Protein
Sci. 13, 2811-2813.
Jacobs, A., Sen, J., Rong, L. and Caffrey,
M. (2005) Alanine scanning mutagenesis of the HIV gp41
loop, J. Biol. Chem., in press.
Jiang, S. and Caffrey, M. (2002) NMR
assignment of the Coxsackievirus and Adenovirus receptor
domain 1. J. Biomolec. NMR 24, 365-366.
Jiang, S. and Caffrey, M. (2005) NMR
assignment and secondary structure of the Coxsackievirus
and Adenovirus receptor domain 2, Prot. Pept. Lett.,
in press.
Jiang, S., Jacobs, A., Laue, T. and
Caffrey, M. (2004) Solution structure of the Coxsackievirus
and Adenovirus receptor domain 1. Biochemistry 43, 1847-1853.
Krishnanchettiar, S., Sen, J. and Caffrey,
M. (2002) Expression and purification of the Bacillus
anthracis protective antigen domain 4. Prot. Express.
Purif. 27, 325-330.
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