HJ16Complex

Neutralizing antibody HJ16 (PBD: 4YE4) is one of the many neutralizing antibodies of the human immunodeficiency virus found in Homo Sapiens. This protein is a CD4 binding site antibody that binds to the envelope glycoprotein-120 displayed on the HIV virion membrane. Neutralizing antibody HJ16 is also a CDR H3 dominated protein (1). An antibody, from the immunoglobulin family of proteins, is composed of two heavy chains and light chains of the Y shape (2). Immunoglobulins are produced by the immune system to mitigate pathogens. Neutralizing antibody HJ16 in complex with HIV-gp120 is a necessary and current avenue of therapeutic research for HIV. Of the two major subtypes of HIV, neutralizing antibody HJ16 targets the type one strain. How this antibody binds and neutralizes in complex with gp120 could provide a paradigm for vaccination thus providing a preventative measure against HIV in the future.

The isolation and crystallization of neutralizing antibody HJ16 in complex with HIV-gp120 was two-fold. First, the antibody was collected and purified and then the binding complex formed. This was done by collecting blood samples from donors already infected with HIV-1 who had not started an antiretroviral treatment. The antibody was isolated from HEHK283F cells and purified with affinity chromatography. The antigen binding fragment or Fab portion of the antibody was prepared overnight with Lys-C digestion at 37? with a IgG:Lys-C ratio of 4,000:1 and sequential purification with size exclusion columns. The antibody-gp120 complexes were formed by mixing deglycosylated HIV-1 gp120 with antibody Fab in a 1:1.2 molar ratio then purified with size exclusion chromatography. Fractions of the solution with the complex successfully formed were concentrated to ~10 mg/ml for crystallization (1).

The molecular weight of neutralizing antibody HJ16 in complex with HIV-gp120 is 310.31 Da and the isoelectric point is 4.00 (3). The general structure consists of three protein subunits. The G, H and L subunits total 802 amino acid residues. Subunit G is the glycoprotein of which HJ16 binds to in complex. Subunit H is the heavy chain, consisting of two domains, and subunit L is the light chain, also consisting of two domains (4). The generic structure of an antibody is two light chains and two heavy chains both of one unique protein structure (2). The entire antibody was crystallized but not the entire glycoprotein. Only the CD4 binding domain of gp120 was necessary to isolate. The CDR H3 dominated designation for neutralizing antibody HJ16 refers to the complementary determining region at the N-terminal consisting of the variable fragment or Fv region. This is the antigen binding site made of three complementary loops from both the heavy and light chains that connect B strands from the two Fv domains (5). For neutralizing antibody HJ16, the CDR H3 region dominates 75% of the heavy chain (1). Other important domains are the heavy and light chains. The two types of light chains are lambda or kappa, however there is no functional differentiation between the two. There are five possible heavy chains: IgM, IgD, IgG, IgA and IgE that determine functional activity, specifically at the carboxy terminal (2). Neutralizing antibody HJ16 is of the IgG subtype. Another important domain is the glycoprotein which consists of oligosaccharides N or O linked to a protein sequence. Glycoproteins serve as vital integral membrane proteins playing a role in cell to cell communication (6).

The amino acid sequence of neutralizing antibody HJ16 can be split into the three subunits. Subunit G with 356 amino acids, subunit H with 228 amino acids and subunit L with 218 amino acids (4). The primary structure of a protein consists of a peptide backbone of the repeating triple segment: N-amino group, alpha carbon and the carboxyl group with the side chains of each amino acid alternating in direction (7). Secondary structure consists of alpha helices, beta sheets, random coils and 3/10 helices. Neutralizing antibody HJ16 in complex with HIV-gp120 expresses all of the above. There are seven alpha helix regions, a majority of all antiparallel beta strands, and more random coils than 3/10 helices whose purpose is to connect beta sheets. Subunits H and L only contain beta sheets and subunit G contains all of the alpha helices (4). Alpha helices are stabilized by intrachain hydrogen bonding between the amide bond of one peptide group and the carbonyl oxygen of another causing the amino acid sequence to coil (7). Beta sheets are composed of interchain hydrogen bonds between sheets side by side (7). When forming tertiary structures alpha helices and beta sheets pack closely together. The hydrophobic amino acids face the interior and polar amino acids face the exterior toward the polar solvent, leading to hydrophobic collapse. This aids in stabilization because stabilization energies for amino acids are lower when positioned in a preferential region based on polar motif (7). All three subunits contain disulfide bonds. The heavy and light chains of the neutralizing antibody HJ16 express two disulfide bridges whereas the gp120 subunit expresses six (4). In antibodies intrachain disulfide bonds link the two heavy and light chains and interchain disulfide bonds link the heavy and light chains together (2). In terms of quaternary structure, separate subunits within a protein are bound by weak forces; for example, hydrophobic interactions, ionic interactions and van der Waals forces (7). Subunits H and L come together to make neutralizing antibody HJ16 and this protein in turn binds to subunit G, the glycoprotein, to form the antigen complex on the virion membrane of HIV-1.

Important amino acid residues can be seen where neutralizing antibody HJ16 forms the antigen binding complex with gp120. Amino acid residues Lys-123, Asn-105, Val-125, Phe-34, Ser-200 and Glu-206 from the heavy chain subunit hydrogen bond with Gln-258, Thr-283, Ala-73 Asp-98 and Gly-124 of the gp120 subunit. Amino acid residues Try-94 and Thr-81from the light chain subunit hydrogen bond with Asn-300and Gly-124 from gp120 subunit. Two important amino acids vital in forming this complex are Phe-43 and Asp-368 located in the gp120 cavity. Salt bridges form between Glu on the heavy chain and Arg on gp120 subunit (1). Neutralizing body HJ16 in complex with HIV-1 gp120 binds to multiple NAG ligands in subunit G. The IUPAC name for this molecule is 2-acetamido-2-deoxy-beta-D-glucopyranose and its common name is N-acetylglucosamine or D-GlcNAc. Individually, this monosaccharide is an enzyme cofactor essential for the urea cycle in mammals and commercially it is present in dietary supplements for the prevention of osteoarthritis (4). The function of NAG in the context of gp120 is unknown.

Multiple database searches were conducted to investigate possible comparison proteins. Psi-Blast is used to find proteins with similar primary structure to a protein query. The specific protein being examined serves as the query and the results of the Psi-Blast are referred to as subjects. An E value is assigned to each resulting subject. This value is calculated by looking at differences or gaps between query and subject. Sequence homology decreases the E value while gaps increase the E value. A value less than .05 is significant (8). After running the Psi-Blast search three proteins, also found in Homo Sapiens but of different function, were selected based on a significant E value: broadly neutralizing antibody CR8043 (PBD: 4NM4), norovirus neutralizing HBGA blocking human IgA antibody (PBD: 5KW9) and PfCSP peptide 20 with human protective antibody CIS43 (PBD: 6B5L). Their respective E values were 3*10^(-146), 3*10^(-149) and 1*10^(-152) (8). In addition to the Psi-Blast server, the Dali server was also used. The purpose of Dali is to find proteins with tertiary structure similar to a query. By using a sum-of-pairs method the intramolecular distances between tertiary components are determined, producing a Z score. Structures with significant similarities have a Z score above 2, indicating similar folds (9). Three proteins, also found in Homo Sapiens with different function, were selected: broadly neutralizing antibody CR8043 (PBD: 4NM4), norovirus neutralizing HBGA blocking human IgA antibody (PBD: 5KW9) and fab-bound human insulin degrading enzyme IDE (PBD: 4IOF). Their respective Z scores were: 28.7, 28.5 and 22.4 (9).

Of the three proteins found for each search two were the same. Out of these two, broadly neutralizing antibody CR8043 was chosen to compare to broadly neutralizing antibody HJ16 in complex with HIV-1 gp120. Neutralizing antibody CR8043 is an antibody found in the homo sapien immune system and consists of two heavy chains, subunits H and I, and two light chains, subunits L and M. The total structure weight is 96.62 kDa and the protein has a total of 880 residues (4). Although there are four subunits there are only two unique proteins of the quaternary structure. The protein structure consists of alpha helices, beta sheets, random coils and 3/10 helices. Beta sheets are the majority. There are also intrachain and interchain disulfide bonds. There is one kind of ligand present PEG, located in the I and L chains. PEG is also called di(hydroxyethyl)ether. This antibody serves to neutralize influenza strains (4).

Neutralizing antibody HJ16 in complex with HIV-1 gp120 is a CD4 super site binding immunoglobulin that forms an antigen complex with glycoprotein 120 on the virion membrane of HIV-1. Neutralizing antibody HJ16 is composed of three unique tertiary structures, two of which are the heavy and light chains and one of which is gp120. The importance of this protein lies in future vaccine development. A better understanding of how neutralizing antibody HJ16 binds and neutralizes HIV will move the medical community closer to reaching this goal. There is currently no preventative medicine or cure for HIV and over a million Americans are living with this autoimmune deficiency today. A vaccine is one step closer to eradicating this disease. A possible avenue of future investigation is to examine how neutralizing antibody HJ16 functions in other mammals. Many primates carry either HIV-1 or HIV-2. Knowledge concerning the function of a protein in an animal model can be applied to the human model.