Dr. Michal Caffrey
- PhD, University of Arizona, Tucson, Arizona
- Postdoctoral: Institut de Biologie Structurale, Grenoble France; National Institutes of Health, Bethesda, Maryland
The general interests of our laboratory concern biochemical and NMR studies of protein-protein interactions. We are especially interested in the mechanisms of viral entry and the mechanisms of toxin entry. Current viral systems under study include HIV, SARS Coronavirus, Ebola and Influenza. Current toxins under study include HIV tat and Bacillus anthracis anthrax toxin. Our general approach consists of 5 stages: (i) generation of protein samples suitable for biochemical and NMR studies; (ii) determination of the protein structural properties by NMR spectroscopy; (iii) characterization of the protein intermolecular interactions and dynamic properties by NMR spectroscopy; (iv) correlation of the protein structural and dynamic properties to protein activity in vivo by biochemical studies; (v) NMR-based drug discovery studies. We feel that our general approach, which includes classic biochemical and state of the art NMR studies, will lead to detailed structure-function information on our current projects, as well as other future projects of large and physiologically relevant protein systems. In what follows, I will discuss 4 systems that are currently being studied in our laboratory.
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).