Introduction
Intensive research has been conducted in the area of HIV pathogenesis during the past few decades. That research lead to the discovery of the Nef protein coded for by the RNA of HIV-1. Nef, which is short for negative factor, originally got its name because researchers thought that it played a role in the inhibition of HIV pathogenesis. However, recent research has shown that Nef, rather that inhibition HIV proliferation , plays a key role in promoting the success of HIV in its host (Goldsmith, et al., 1995). Those patients with HIV -Nef strains lived much longer healthier lives, while those with +Nef strains developed a faster onset of AIDS. In addition, Rhesus monkeys injected with -Nef strains of SIV acquired long term immunity to subsequent injections of +Nef strains of SIV (Lee, et. al., 1996).
Many functions of Nef are unknown, however research has shown that Nef promotes CD4 down-regulation in HIV infected cells (Saksela, et al., 1995). This action decreases the amount of CD4 receptor sites on the cell surface, thereby decreasing the amount of viruses that attack a single cell and preventing cell super- infection. The viruses within the cell can then ensure that the cell does not die prematurely and maximizes the cells machinery in the production of progeny viruses.
An even more interesting aspect of Nef has been the recent discovery of Nef's role in certain forms of Leukemia. It has been shown that Nef binds to the SH3 domain of Src family proteins (Gmeiner, et al., 1996). One such tyrosine kinase is hematopoietic cellular kinase, a non-receptor signal transduction protein partly responsible for the control of cell proliferation. Nef binds tightly to the Hck SH3 domain (tightest known SH3 interaction with a Kd of 91 uM) preventing inactivation of Hck and promoting oncogenesis (Lee, et al., 1995). This modeling project will cover the interaction between Nef and the SH3 domain of another Src protein, Fyn. The crystal structure of of Hck SH3 has not been obtained yet, therefore Hck SH3 could not be used in this protect. However, Fyn is closely related to Hck with differences lying outside of the SH3 domain, therefore studying the Fyn SH3-Nef interaction (shown above) will provide valuable insight into the Hck SH3-Nef interaction. In order to view various portrayals of the Nef /SH3 complex protein, click on the following buttons.
To view a color coded complex, right click on the molecule, select chain then C or D. Then go back and double click on the molecule again and select a color for the chain selected. Do the same for the next chain.
The Ramachandran plot is presented below showing the various secondary structures found within Nef and SH3. Click on the button below to view the secondary structures.
Nef Structure (Click below to view the structure of Nef)
Beginning at the N terminus, Nef contains a PP-II helix, which is then followed by two anti-parallel alpha helices. The two alpha helices , A and B, are connected by a peptide linkage, consisting of 10 residues. Click below to observe these alpha helices.
Note that the PP II helix does not show up in cartoon form well, rather it is depicted by the small, wire, helical tail prior to the first main A helix. In addition, the two helices are packed up against 4 anti-paralles beta strands. Click below to observe these beta sheets.
The C terminus consist of two helices, packing on the other side of the beta strands. It is the PP II helix along with the hydrophobic crevice created between the A and B helix that are responsible for the interaction with other molecules. Click below to view this hydrophobic crevice.
SH3 Structure (Click below for the structure of SH3)
The tertiary structure of the SH3 is a beta-barrel with an RT loop extending from the beta barrel structure. Click below to view the beta barrel and observe the beta barrel by rotating the molecule.
In regards to binding, the important SH3 structure is the RT loop extending from the beta barrel. Click below to observe the RT loop.
The RT loop has hydrophobic residues which are key to the interaction with Nef. Click below to observe the hydrophobic areas of SH3.
The center portion of the RT loop contains a isoleucine residue, which will play an important role in the interaction with the Nef structure. To view the isoleucine residues, right click on the molecule, select, residue, isoleucine. Then go back, right click again and select color.
Nef-SH3 Interaction (Click below to view the Nef- SH3 complex)
This interaction is much easier to observe in cartoon mode. Click below for this option.
The Nef-SH3 interactions are a result of the A and B alpha helices of the Nef and the RT loop of the SH3. Also, the PP-II of Nef interacts with the N-terminal side of the RT loop and the carbonyl portion of the beta barrel on the SH3 protein. Hydrogen bonding and electrostatic interactions do occur between the Nef and SH3 proteins, however, hydrophobic interactions are critical to Nef-SH3 docking. The total interactive surface area between Nef and SH3 is 1200 square angstroms. The majority of this surface area contributes to hydrophobic interactions between the SH3 and Nef residues. A large portion of the hydrophobic interactions occur between the Nef A and B alpha helices and the SH3 RT loop. Click below to observe the hydrophobic interactions.
The isoleusine residue extends from the RT loop and fits in a hydrophobic pocket created by the two A and B alpha helices of Nef, resulting in additional hydrophobic interactions. Click below to observe this isoleucine residue.
To view the isoleucine residues, right click on the molecule, select, residue, isoleucine. Then go back, right click again and select color.
The area at the Nef-SH3 interface that is not related to the hydrophobic regions on the A and B alpha helices and the RT loop contains polar residues are attributed to electrostatic interactions between the two complexes. The hydrophobic and electrostatic interactions between the Nef-SH3 complex results in a Kd = 90 uM.