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Protein Expression Laboratory
The NIAMS Protein Expression Laboratory supports intramural NIH scientists in studying the structure and function of Human Immunodeficiency Virus (HIV) proteins. Most structural biology techniques, especially those for studying the three-dimensional structures of proteins, require large quantities of highly purified, monodisperse, and correctly folded proteins. The Protein Expression Laboratory responds to this need by analyzing and providing HIV proteins to NIH and collaborating scientists. Recent research includes the following.
The human immunodeficiency virus (HIV) consists of a number of proteins with regulatory and structural roles. In the Protein Expression Laboratory, HIV proteins important for the virus life cycle, and proteins that have anti-HIV activity, are expressed in bacteria using recombinant DNA methods (Wingfield et al., 1997). The proteins are purified and studied to establish their chemical and physical properties. Well-characterized proteins are made available to NIH investigators who study the molecular structure of these proteins. This structural information may provide impetus for targeted drug design and development.
The surface proteins of the HIV, and related simian immunodeficiency virus (SIV), consist of two non-covalently associated glycoproteins: gp120 and gp41. The gp120 mediates viral entry into the host cell by binding to receptors located on the host cell surface. This binding changes the shape of the transmembrane gp41, which facilitates or induces fusion between the viral and host membranes. Using both nuclear magnetic resonance spectroscopy and X-ray crystallography (Caffrey et al., 1998; Yang et al., 1999), the three-dimensional structure of the gp41 molecule was determined by scientists at the Protein Expression Laboratory and their collaborators. Based on structural and biochemical information, scientists at the Laboratory have suggested a mechanism for membrane fusion (Caffrey et al., 1999). Structural studies also provide an explanation for the in vivo formation of high molecular weight aggregates that may be responsible for HIV-associated neurological damage and dementia (Caffrey et al., 2000).
The HIV-1 protease consists of two identical subunits and is an important target for anti-HIV HIV-1 drugs. The virus gradually evolves to evade these drugs, lessening their efficacy. Based on the study of protease residues susceptible to the oxidative process, namely the sulfur-containing residues methionine and cysteine, it has been proposed that drugs which target the region between the subunits (interfacial) may provide an alternate site of intervention (Davis et al., 2000).
Related to the HIV-1 virus is the Hepatitis B Virus (HBV), the major worldwide cause of cancer. Liver cancer is one of the most common infectious diseases of humans. Although a vaccine has been developed, it is not universally available and, since the virus is vertically transmitted from mothers to infants, chronic HBV is often acquired in childhood. The HBV nucleocapsid plays an important structural and metabolic role in the life cycle of the virus. Studies of the molecular structure of the HBV nucleocapsid (Conway et al., 1997) indicate that drugs targeting the assembly of the capsid protein could prevent formation and the assembly of the virus (Zlotnick et al., 1999). Ongoing structural studies of the unassembled protein subunits are being carried out.
Scientific research at the Protein Expression Laboratory includes examination of microtubules that comprise one of the major cytoskeletal systems of the cells. These microtubules play an essential role in cytoplasmic organization and cell division. Investigators have shown that Rev, a key HIV-1 regulatory protein (Wingfield et al., 1991), binds strongly to microtubules. This results in the formation of large ring structures, the structural determination of which is being pursued (Watts, et al., 2000). The in vivo interaction between Rev and microtubules provides further insight into the role of Rev during HIV infection.
Current research also includes investigation into the catalytic mechanism of MAP3O, a plant protein obtained from melons using protein engineering methods that appears to have anti-HIV and anti-tumor actions. MAP 30 was originally isolated from the mature seeds of the bitter melon Momordica charantia, a medicinal plant found in China and Southeast Asia. Genomic DNA was isolated from the leaves of freshly grown bitter melon and was used to clone the MAP30 protein. A colleague in the project, Dr. Sylvia Lee-Huang, School of Medicine, New York University, performed the initial purification and cloning of this interesting protein. The structure of the protein in solution was solved recently by collaborators of the Laboratory (Wang et al., 2000).
(Note: References are listed under Selected Publications.)
Fang X, Wang J, O'Carroll IP, Mitchell M, Zuo X, Wang Y, Yu P, Liu Y, Rausch JW, Dyba MA, Kjems J, Schwieters CD, Seifert S, Winans RE, Watts NR, Stahl SJ, Wingfield PT, Byrd RA, Le Grice SF, Rein A, Wang YX. An unusual topological structure of the HIV-1 Rev response element. Cell. 2013 Oct 24;155(3):594-605. doi: 10.1016/j.cell.2013.10.008.
Bereszczak JZ, Rose RJ, van Duijn E, Watts NR, Wingfield PT, Steven AC, Heck AJ. Epitope-distal Effects Accompany the Binding of Two Distinct Antibodies to Hepatitis B Virus Capsids. J Am Chem Soc. 2013 May 1;135(17):6504-12. doi: 10.1021/ja402023x.
Watts NR, Conway JF, Cheng N, Stahl SJ, Steven AC, Wingfield PT. Role of the Propeptide in Controlling Conformation and Assembly State of Hepatitis B Virus e-Antigen. J Mol Biol. 2011 Apr 2. [Epub ahead of print]
Uetrecht C, Watts NR, Stahl SJ, Wingfield PT, Steven AC, Heck AJ. Subunit exchange rates in Hepatitis B virus capsids are geometry- and temperature-dependent. Phys Chem Chem Phys. 2010 Nov 7;12(41):13368-71.
Roos WH, Gibbons MM, Arkhipov A, Uetrecht C, Watts NR, Wingfield PT, Steven AC, Heck AJ, Schulten K, Klug WS, Wuite GJ. Squeezing protein shells: how continuum elastic models, molecular dynamics simulations, and experiments coalesce at the nanoscale. Biophys J. 2010 Aug 9;99(4):1175-81.
Watts NR, Vethanayagam JG, Ferns RB, Tedder RS, Harris A, Stahl SJ, Steven AC, Wingfield PT. Molecular basis for the high degree of antigenic cross-reactivity between hepatitis B virus capsids (HBcAg) and dimeric capsid-related protein (HBeAg): insights into the enigmatic nature of the e-antigen. J Mol Biol. 2010 May 14;398(4):530-41.
Stahl SJ, Watts NR, Rader C, DiMattia MA, Mage RG, Palmer I, Kaufman JD, Grimes JM, Stuart DI, Steven AC, Wingfield PT. Generation and characterization of a chimeric rabbit/human Fab for co-crystallization of HIV-1 Rev. J Mol Biol. 2010 Apr 2;397(3):697-708.
DiMattia MA, Watts NR, Stahl SJ, Rader C, Wingfield PT, Stuart DI, Steven AC, Grimes JM. Implications of the HIV-1 Rev dimer structure at 3.2 A resolution for multimeric binding to the Rev response element. Proc Natl Acad Sci U S A. 2010 Mar 30;107(13):5810-4.
Jedidi I, Zhang F, Qiu H, Stahl SJ, Palmer I, Kaufman JD, Nadaud PS, Mukherjee S, Wingfield PT, Jaroniec CP, Hinnebusch AG. Activator Gcn4 employs multiple segments of Med15/Gal11, including the KIX domain, to recruit mediator to target genes in vivo. J Biol Chem. 2010 Jan 22;285(4):2438-55.
Dolinska MB, Sergeev YV, Chan MP, Palmer I, Wingfield PT. N-terminal extension of beta B1-crystallin: identification of a critical region that modulates protein interaction with beta A3-crystallin. Biochemistry. 2009 Oct 13;48(40):9684-95.See extended list of publications
Updated February 2, 2010