Proceedings of Bioc 462a Honors

Abstract

For years it has been known that HIV binds to human immune cell CD4 receptors via a protein on the HIV glycoprotein envelope, gp120 (short for glycoprotein 120). Medical researchers at the US National Institute for Allergies and Infectious Diseases have recently been able to get antibody b12 to bind to gp120 and have shown structurally how this was accomplished. Initially it was believed by many that gp120 had to undergo a conformational change in the presence of the immune cells before the binding site was accessible, rendering any antibody binding extremely difficult at best. However, new research indicates that the initial binding site for CD4 cells is present and available before the conformational change, with the conformational change serving only to lock in the cell once the connection is made. With this evidence, scientists targeted the CD4 binding loop (site of CD4 attachment on protein gp120) as a site for antibody response. The CD4 loop is one of two parallel loops on the surface of the HIV virus; two loop arrangements such as this is a secondary structure frequently recognized by antibodies. Several key residues on b12 bind to gp120 in ways similar to CD4. By binding to gp120 at key sites of attachment used by CD4, antibody b12 can inhibit the binding of the HIV virus to human immune cells. This research indicates that there may be an effective method for developing an HIV/AIDS vaccine.

Introduction

As the AIDS epidemic rages on, scientists continue to search for answers to this most plaguing problem. Over the years, critical steps have been made on the journey toward finding a cure, and progress is still being made: research is probing into the physical structure of the HIV virus, the mechanisms of infection are under constant scrutiny, and the body’s response to the foreign invader is a subject that still has much to be discovered. The ultimate goal in AIDS research would be to find a cure and rid the world of this horrible disease. While ongoing research is definitely making progress on that journey, scientists may be close to achieving an important intermediate – prevention.
The HIV virus attacks the human immune system. There are two components involved in joining of host and viral cells – glycoprotein 120 and CD4 receptors. CD4 receptors are glycoproteins expressed on the surface of a variety of human immune cells, such as lymphocytes (most notably T lymphocytes, or T cells), monocytes, and macrophages (Bour et al, 1995). The other component of binding is gp120, part of a glycoprotein spike that project from the surface of the virus. Most of HIV’s surface proteins are constantly evolving, which is part of the reason it remains elusive to the human immune system. Gp120 differs because a part of it must always remain the same in order to “fit” with non-changing human immune cells. Since this site is the only unchanging, and therefore recognizable, unit on the surface of the virus, it is the location of greatest vulnerability.
Until recently, many thought that this site of attachment was hidden deep within the protein until just before binding with human immune cells. The hidden binding site created an enormous challenge for antibody mediation; namely that an antibody couldn’t bind to a site it couldn’t access, thus rendering it nearly impossible to use an antibody to block HIV infection. Researchers at the US National Institute of Allergy and Infectious Diseases, however, have now shown that the gp120 molecule doesn’t change its shape until after it has bound to human immune cells (Kwong et.al., 2007). If no shape change occurs to make the binding site available, it must be accessible all along.
Additionally, the HIV virus has been shown to bind to a human antibody called b12. The discovery of an antibody that can neutralize the HIV virus coupled with the knowledge that the antibody is able to access its site of action in the body leads to the exciting possibility of an HIV/AIDS vaccine. The structure and function of the HIV/b12 complex, both by itself and compared to the complex of HIV and human immune cells, is critical to understanding the components of infection and prevention and possibly developing a vaccine. Although more work is necessary before a vaccine becomes a reality, a more complete knowledge of the virus and its interactions with human cells is an important step toward achieving that goal.

Structure of the HIV envelope protein gp120
The surface of the HIV virus is composed of six proteins: three identical envelope glycoproteins (gp120) and three identical transmembrane proteins (gp41). Gp120 is the site of initial contact with cells that have CD4 receptors. Once contact is made, gp120 undergoes a conformational change that exposes gp41, which then aids in fusion with host’s cell. Gp120 and gp41 are non-covalently bound and thus dissociate relatively easily, resulting in a “shedding” of some gp120 molecules from the virus (Harrison 1994). Of HIV’s envelope proteins, gp120 will be the focus of the remainder of this paper.
Gp120 is comprised of five highly variable regions (V1-V5) and five relatively conserved regions (C1-C5). The V regions are heavily glycosylated and differ greatly between individual HIV molecules (Weiss, 1994). The conserved regions tend to be located more toward the core of the protein and exhibit a higher degree of sequence conservation between individual virions. Although minor differences are present among HIV molecules, a combination of residues from CD2, CD4, CD5, and V4 regions are what usually combine to form a surrounding contact site for host cells (Olshevsky et al., 1990).
Most of the binding activity of gp120 takes place on the protein’s outer domain. This domain is composed of two barrels, which are stacked end-to-end: one 7-stranded antiparallel β-barrel and one six-stranded β-barrel that surrounds an alpha-helix. At the juncture of these two barrels are two parallel loops that extend from the only two parallel beta strands in the gp120 core. These two loops comprise one of the most important structures that antibodies use for recognition (Kwong et al., 2007).
One of these two loops is the CD4 binding loop, also known as beta strand 15, which contains the CD4 binding site. The majority of CD4 and b12 binding take place at this site (as the name implies), which lies toward the carboxy-terminus end of gp120. Part of the CD4 binding loop forms a portion of a pocket that extends in toward the protein’s center; this pocket is critical in binding “aromatic fingers” on both CD4 immune cells and b12 antibodies, structures which will be discussed later. The pocket surrounds aromatic side chains of its target cells and is comprised of residues including W112, V255, T257, D368, E370, and I371.(Kwong et al., 1998). A second, minor loop is found in both b12 and CD4, and a small canyon between residues T373 and N386 is present in gp120 to accommodate this secondary loop as well. The hydrophobic pocket and canyon are separated by a ridge formed by residues S364-D368, illustrated in Figure 1 (Saphire et.al., 2001).

Figure 1. Stereo view of HIV gp120 with the CD4 binding site is oriented forward. Shown in blue are residues that are important for H3 loop/W100 binding; in maroon are residues important for H2 loop/Y53 binding. Shown in white is the ridge that separates the hydrophobic pocket from the canyon. (pdb code: 2ny7)

Structure of the human antibody IgG1 b12
Antibody b12 is in the G class of immunoglobulin antibodies, the class that is responsible for the majority of immunity against foreign invaders. Immunoglobulins are generally shaped like a ‘Y’, with the arms of the Y containing the Fab fragments (fragment, antigen binding). As the name implies, the arms of the Y are where antigens are recognized and bound. On these Fab fragments are CDRs, complementarity determining regions. These provide further specificity to the antibody and directly contact the antigen’s surface.
The H3 CDR loop of b12, a structure portrayed in Figure 2, is crucial to HIV gp120 binding. The most critical amino acid in the entire structure is W100 at the apex of this loop. The loop, with the projecting tryptophan, forms one aromatic finger that extends into the gp120 pocket. This aromatic finger is analogous to the F43 loop of CD4. The structural similarity of the aromatic side chains of phenylalanine and tryptophan, as well as the specificity of the HIV pocket for an aromatic amino acid, makes W100 absolutely essential to binding with gp120. Although one might assume that a mutation to a phenylalanine at position 100 of b12 would make the antibody more structurally similar to the immune cell and thus increase b12 binding affinity, data shows that tryptophan is actually preferable at this position on b12 for optimal binding affinity (Zwick et al., 2003).

Figure 2. Chain K of the Fab fragment of antibody b12. The critical W100 residue is shown in yellow sticks. Residues that contribute to charge repulsion and rigidity of the loop are shown in hot pink spheres. (pdb code: 1hzh)

The projection of this loop is made possible in part by the fact that it is raised 15 angstroms above the rest of the antibody. This raising is created by a charge repulsion effect of several mildly acidic residues at the base of the loop, including D100a, D100b, D100f, S99, S100c, and Y100i. These amino acids function similar to a group of magnets: when positive charges are gathered together, they push away from each other, generating charge repulsion. By pushing away from one another, these amino acids project the loop away from the rest of the protein. The charge repulsion generated among these acidic amino acids is also important due to the fact that it lends the entire loop rigidity, rigidity that is necessary to insert the “aromatic finger” loop into gp120’s pocket. Mutations in any of these amino acids resulted in a mild to moderate decrease in b12 binding (Zwick et al., 2003).
A second finger-like projection exists on the H2 CDR loop of b12, with Y53 as its insertional aromatic. Y53 inserts into a different gap than the H3 CDR; it fits into a canyon between residues 373 and 386 on gp120 (Zwick et al., 2003). W100 and Y53, along with N31, comprise approximately 40% of the b12 contact surface (Kwong et.al., 2007).
Structure of the membrane protein CD4
Although the primary focus of this paper is the binding relationship between b12 and gp120, which ideally excludes CD4 from the complex, a brief discussion of the structure of CD4 is necessary to be able to compare with that of b12. The key features of CD4 include two disulfide loops, which serve similar functions as the H2 and H3 CDR loops on b12 – insertion into pockets and canyons of gp120. The main loop is comprised of residues 31-57, which of course includes residue 43 – the phenylalanine residue that serves an analogous function to the W100 residue on b12’s H3 loop (Mikuzami et al., 1988). Both residues fill the hydrophobic pocket of the gp120 binding site. A comparison of these two loops and their critical aromatics is presented in Figure 3.
Upon binding of CD4, gp120 undergoes a conformational change that exposes gp41 and allows for fusion to the host’s cell. Gp41 also has a hydrophobic pocket that becomes exposed after the conformational change, further requiring the hydrophobic aromatic apexes of the loops on b12 and CD4 (Harrison, 1994).

Figure 3. Chain K of antibody b12 (blue) with the CD4 surface receptor protein (red). This is a comparison of the analogous loops on the b12 and CD4 molecules – the portions that insert into the gp120 binding sites. Projecting loops are shown in blue and orange, with analogous W100 and F43 residues shown in aquamarine and yellow spheres, respectively. (pdb code: 1hzh for b12 and 1cdj for CD4)

Binding of the b12-gp120 complex and vaccine potential

Recent research has shown that the site of contact for CD4 is accessible even before the conformational change in gp120. To demonstrate this, researchers at the National Institute of Allergy and Infectious Diseases stabilized gp120 in its CD4 bound state. Several alterations were made to the primary sequence (amino acid composition) of the protein, and five disulphide bonds were added to gp120, resulting in a stable mutant that maintains the CD4 bound conformation of gp120. These changes produced minimal structural differences between wild-type and mutant gp120 in its CD4 bound state. Little change in ligand binding was noted between the stabilized gp120 mutants and the wild type gp120, suggesting that the binding site is present and accessible in unliganded gp120. Therefore, it appears that the conformational change in gp120 protein serves only to lock in the ligand once initial contact has occurred (Kwong et al, 2007). Since most of the structure of gp120 is highly variable, a cohesive model was not available to serve as the basis of a figure to illustrate this process. CD4 receptors also undergo a conformational change at the same time as gp120 during the binding process; the conformational change in the CD4 receptor is displayed in Figure 4.

Figure 4. Comparison of the conformations of CD4 in its bound (blue) and unbound (ruby) states. F43 residues of each are aligned as a point of reference; other residues highlighted in green and yellow are simply colored to show the positional differences between the two conformations.

The previous belief that the gp120 binding site was hidden until just before contact with CD4 presented a huge challenge to vaccine development. Experiments showing antibody binding effectiveness in vitro had little significance if these results physically could not be reproduced in vivo. Equipped with the knowledge that access to the binding site is no longer a problem, scientists must set out to tackle other challenges in vaccine development.

Many other complex issues and challenges complicate the design of a vaccine. First and foremost, the body must begin to recognize HIV as an antigen that requires antibody response before the HIV is able to infect immune cells (currently, antibody formation may take weeks). Another challenge lies in the fact that a natural infection always elicits a greater immune cell response compared to a vaccination. The levels of b12 needed to combat a natural encounter with the HIV virus may be difficult to achieve with a vaccine.
Other major concerns that occur with any protein/ligand complex are the issues of binding affinity and preferential binding. Upon binding with CD4, gp120 undergoes a conformational change that results in a high affinity state for CD4 (Harrison, 1994). Although b12 binds at the same site as CD4, it binds slightly differently due to the difference in residues. Since b12 binds differently than CD4, it is possible that it triggers a slightly different conformational change as well, which could result in a lesser affinity for ligand. In addition, gp120’s affinity for CD4 varies drastically depending on the viral source (Daar, 1990). Free gp120 binds much more tightly than multimeric envelope gp120, regardless of the source of the virus (Ashkenazi et.al., 1991). As stated earlier, CD4 binding to gp120 causes “shedding” of other gp120 molecules from the virus envelope (Harrison, 1994). This could result in a decoy-type effect where antibodies would attack the free-floating, non-pathogenic gp120, leaving the intact gp120 free to contact and infect T cells. Lastly, it has been shown that HIV can infect the glial cells of brain tissue (as well as a few others), which do not, in fact, have a CD4 site on their surface (Christofinis et.al., 1987). Even if a vaccine could be engineered such that antibodies would preferentially and firmly bind to the correct site, a handful of specialized cells in the body could still be vulnerable to the virus.
One interesting topic for discussion is the topic of inhibitory versus regulatory vaccines. If a vaccine cannot be designed to completely prevent infection, it is still possible that one could be designed to slow disease progression – a so-called regulatory vaccine. Such a vaccine could be developed first to lessen the tragic number of lives lost while researchers search for a preventative, inhibitory version.
If a regulatory vaccine to halt the spread of the virus were developed, it would do best to mimic the response long-term non-progressors (Kalams and Walker, 2002). Long-term non-progressors’ immune systems have responded to the virus in such a way that slows the progression of disease to a near halt for years, and if an artificial version of their immune response could be copied, it would buy precious time needed for further research. A regulatory vaccine would also be a good alternative to HAART – Highly Active Antiretroviral Therapy. HAART is the main drug treatment for people living with HIV. It consists of a cocktail of drugs including reverse transcriptase inhibitors, protease inhibitors, and fusion inhibitors (fda.gov) While HAART keeps many people alive, patients undergoing this treatment pay a high price – both financially and physically. HAART can cost ten to twenty thousand dollars a year, and side effects include nausea, diarrhea, liver and kidney problems, fatty deposits, and disorders of the central nervous system (hivmedicine.com, 2006, and Garrett, 1999). Therapeutic, regulatory vaccines could lessen or eliminate the need for HAART, saving the patient the hardships of the cocktail therapy.
Disease progression is also linked to a low early viral load (Lyles et al, 2000). If a vaccine could be developed that would recognize HIV as an antigen and kill off at least some of the initial virions before they could make contact with T cells, this would also slow the progression of the disease, again providing more valuable time until a better alternative is found.
Discussion
For years, research has been accumulating about HIV and its mechanisms of disease: scientists discovered HIV’s target cell (specifically the CD4 surface protein on that cell), elucidated the binding and fusion mechanisms of gp120 and gp41, and found a way to block binding in vitro and occasionally in animal studies on a related virus. One molecule key to blocking binding of gp120 and CD4, and thus HIV infection of host cells, is IgG1 b12, a broadly-neutralizing immunoglobulin antibody. CD4 and b12 are structurally similar at their locations of attachment to gp120; most notably, they both have an extended loop capped off by a hydrophobic, aromatic amino acid. CD4 and b12 attach at similar points to gp120 and fill the same cavities on the viral protein.
While this set of knowledge certainly is a valuable tool in understanding infection by the HIV virus, these data were incredibly difficult to implement under the previous belief that the CD4 binding site was hidden until just prior to infection. According to studies done by Kwong et al., the CD4 binding site is always conformationally available, illuminating the path towards a possible vaccine. Many more challenges are yet to be overcome before the dream of a vaccine becomes a reality, such as ensuring tight, preferential binding of gp120 to antibodies and eliciting a sufficient antibody response to the presence of HIV. However, the discovery that the crucial binding site for both T cells and antibodies is always accessible is one more crucial step in the battle to beat HIV/AIDS.

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