N-Terminus of HIV-1 Tat Protein Bound to the Active Site of Dipeptidyl Peptidase IV

Cassie Calabrese '07 and Sarah Watkins '07

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Contents:

I. Introduction

Human immunodeficiency virus (HIV) is of the retrovirus family and infects cells of the immune system, destroying them and the body’s ability to stave off other infections. HIV uses the host cell’s replication enzymes to copy itself, but it can also encode its own proteins. One of these proteins, trans activating factor (Tat) is a regulatory protein that is necessary for viral replication and functions by binding to an RNA element called that Tranactivating responsive sequence (TAR).
Apart from its important role in viral replication, Tat also exists extracellularly and participates in the immunosuppression of non-HIV-infected T-Cells in AIDS patients (Wrenger, S., et al 1997). Tat accomplishes this by binding to CD26, a T cell activation marker that is important in the regulation of lymphocyte growth, and inhibits its dipeptidyl peptidase IV (DP IV) activity. DP IV is essential for activating and proliferating lypmphoctyes. It is known that the N-teriminal amino acid sequence of HIV-1 Tat plays an important role in the inhibition of DP IV.

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II. General Structure of the N-terminus of Tat Protein and DP IV

Tat is an HIV-1 gene that encodes a 14-kilodalton nuclear protein which acts in trans to stimulated viral gene expression. It is a 101 amino acid protein that is synthesized from two exons. Exon one codes for amino acids 1-72 and exon two codes for amino acids 73-101. Within these exons there are four domains all of which, except the N-Terminal domain, are highly conserved in various strains of HIV-1 (Kuppuswamy, M., et al., 1989). The four domains are found in the first 58 amino acids of Tat and this truncated Tat protein is known to be fully functional (Seigel et al., 1886).

The N-terminus of Tat (want a closer look? ) appears to affect transactivation. This first domain contains 13 amino acids with both hydrophilic and hydrophobic characteristics (Ruben, et al., 1989).(zoom out ) What one views in this image is the first 9 residues of two Tat molecules bound to DP IV which are Met-Asp-Pro-Val-Asp-Pro-Asn-Ileu-Glu.

DP IV is an asymmetric serine protease that is made of two homodimers: monomer 1: , and monomer 2: . Each monomer consists of a C-terminal domain: called the alpha/beta hydrolase fold (residues 39-55 and 497-766) and the 8-blade beta propeller of the N-terminus (residues 61-495) . The two monomers are related by a twofold dyad axis. The active site is found in the center of each monomer, where two negatively charged glutamic acids recognize and bind the N-terminus of Tat. .

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III. The Importance of Tat's Interactions with CD26

Much more research is needed to fully explain Tat’s interactions with DP IV, which is also known as the T-Cell activation antigen, CD26. However, evidence suggests that the defects in the immune response of HIV-1 infected individuals are mediated by these Tat-DP IV interactions. DP IV catalyzes the cleavage of dipeptides from the N-terminus of oligopeptides and polypeptides with optimal efficiency if they contain a Pro or Ala in the second to last position. The N-terminus of DP IV substrates are known to adopt extended polyproline-like helical structures stabilized by having a proline as every third residue in order to bind to DP IV (Wrenger, S., et al 1997).

The N-terminus of Tat is not a recognized DP IV substrate because it contains neither Pro nor Ala in this analogous position. Research has shown, however, that Tat protein is a competitive inhibitor of DP IV and binds with high affinity that may be explained by a number of attractive hydrophobic interactions. Tat’s N-terminus is not very flexible because of two prolines at positions 3 and 6 , rendering it a rigid conformation which most likely contributes to the nature of its inhibitor interactions. These prolines are held to DP IV by Tyr 632 and Trp630. There is a hydrophobic interaction of the side chain of Met1 , with Trp 638. The negatively chargedAsp5 makes important hydrophillic interactions and Asp2 interacts with Trp628.

IV. Implications

The mechanism of DP IV's function in the proliferation and activation of lymphocytes has yet to be resolved. What is known is that Tat binds to the T-Cell activation marker, CD26, thereby inhibiting its DP IV activity which plays a key role in the growth regulation of lymphocytes. As a result of this, T-Cell proliferation, B and natural killer cell growth, immunoglobulin secretion and cytokine production can be inhibited. T-Cells are mature lymphocytes that can belong to one of two subsets. The type of T-Cell discussed here is distinguished by the presence of a surface glycoprotein called CD4. CD4+ T-Cells are essential for both the cell-mediated and antibody-mediated branches of the immune system. Unfortunately, AIDS presents a clear illustration of the importance of T Cells. An uninfected human averages 1,000 CD4+ T-Cells per microliter of blood, but in AIDS patients, HIV binds to these CD4 molecules, invading them and therefore causing a decline in their number. When CCD4+ T-Cells levels decline below 400 per µl, the immune system can no longer mount an effective response to infections, eventually leading to death from opportunistic infection. With this knowledge that the N-terminal sequence of Tat acts as a competitive inhibitor and acts directly with CD26, future studies could focus on substances that interfere with the interactions of Tat and the active site of CD26 as a target for AIDS therapy.


V. References

Gutheil W.G., et al. Human Immunodeficiency Virus 1 Tat binds to Dipeptidyl Aminopeptidase IV (CD26): A possible mechanism for Tat's immunosuppressive activity. Proceedings of the National Academy of Science 1994; 91:6594-6598

Jeang K. HIV-1 Tat: Structure and Function. Molecular Virology Section, LMM, NIAID, National Institutes of Health 1996.

Lorey, S., et al. Different Modes of Dipeptidyl Peptidase IV (CD26) Inhibition by Oligopeptides Derived from the N-terminus of HIV-1 Tat Indicate at Least Two Inhibitor Binding Sites. European Journal of Biochemistry 2003; 270:2147-2156.

Pugliese A., et al. A Review of HIV-1 Tat Protein Biological Effects. Cell Biology and Function 2005; 23: 223-227.

Ruben S., et al. Structural and Functional Characterization of Human Immunodeficiency Virus Tat Protein. Journal of Virology 1989; 63:1-8.

Wrenger S., et al. The N-terminal Structure of HIV-1 Tat is Required for Suppression of CD26-dependent T Cell Growth. Journal of Biological Chemistry 1997; 272(48):30283-30288. 28:4568-4574.


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