HIV-1 Integrase Core Domain
This Jmol Exploration was created using the Jmol Exploration Webpage Creator from the MSOE Center for BioMolecular Modeling.
Beginning with a brief overview- human immunodeficiency virus, otherwise known as HIV, is a virus that attacks the body's immune system, specifically CD4 cells (also known as T-cells or T-helper cells) ('About HIV/AIDS', 2016). CD4 cells are a type of white blood cells that play a major role in protecting your body from infection. They send signals to activate your body's immune response when they detect 'intruders,' like viruses or bacteria ('CD4 Count', 2016). HIV's function of infection is characterized based on two things: it is both a retrovirus and inserts its viral DNA into the host's DNA. The following video shows an overview of how HIV enters a host cell and replicates.
Moving on, the function of viral DNA insertion into a host genome will be the focus.
If you would like to learn more about what a retrovirus is, the following website has some easy to read general information:
https://www.verywell.com/hiv-is-a-retrovirus-what-does-that-mean-3132822
Integrase:
Integrase is a 32-kDa enzyme that is transported into a targeted host cell along with other enzymes and the viral RNA. Once reverse transcriptase codes the RNA to produce viral DNA and the viral DNA is carried into the host cell's nucleus, integrase begins its vital role in the replication of the virus. The critical DNA cutting and joining events that integrate the viral DNA are carried out by the integrase protein itself(Craigie, 2001). The first step carried out by integrase is 3'-end processing. In this step, two nucleotides are removed from the 3'-end of the viral DNA, exposing the 3'-hydroxyl group that will eventually be joined to the host DNA (Craigie, 2001). The next step of integrase is called DNA strand transfer. In this step, a pair of processed viral DNA ends is inserted into the target DNA. In the case of HIV, the sites of integration on the two target DNA strands are separated by 5 base pairs. Repair of this integration intermediate results in a direct duplication of 5 base pairs flanking the integrated viral DNA. The repair step requires removal of the two unpaired nucleotides at the 5′-ends of the viral DNA, filling in the single gaps, and finally ligation. The repair steps of this DNA integration are likely to be carried out by cellular enzymes of the host cell. Due to the lack of specificity for site of integration on host DNA, insertion can occur at essentially any location. (Craigie, 2001).
HIV-1 integrase has three major domains in its structure: N-terminal domain, core domain, and C-terminal domain. All three domains of the integrase enzyme contribute to enzymatic activity, but since the core domain possess the active site for catalytic function of the enzyme it will be the focus here.
The HIV-1 Integrase Core Domain Jmol image displays both identical chains as a ribbon structure. The helices are noted in the green color, beta sheets in pink, and segments of the chain that do not have assigned secondary structure are in gray.
Primary Structure: The core domain of HIV-1 integrase has two identical molecular chains, composed of 153 residues on each chain. The sequence of amino acid residues that make the polypeptide chain is what we would consider to be the primary structure. Since the core domain is only a section of the overall enzyme, these residues begin at enzyme residue number 56 and end at residue 209. Known mutations in the primary structure have been observed, however mutations at only a few sites has shown to result in complete loss of function.
Primary StructureThe primary structure Jmol image displays the basic backbone structure and the side chains of both identical chains of HIV-1 Integrase Core Domain. The green sections represent the helices, blue sections are beta sheets, and white sections are the remainder of the protein chain without any assigned secondary structure.
Secondary Structure: Secondary structure is most readily known as the formation of alpha helices and beta sheets. The core domain of HIV-1 integrase forms a total of 8 helices, making its secondary structure 44% helical. The readily recognizable alpha helix structures account for 6 of the 8 total helices. The other two helices are known as 310 helices. A 310 helix is a type of protein secondary structure found less commonly than the well known alpha helix, differing in a single amino acid spacing (i+3 vs. i+4, where i indicates the H-bond forming residue). This slightly more condensed alignment means each amino acid corresponds to a 120° turn in the helix, [360° ÷ 3] vs. 100° for the alpha helix [360° ÷ 3.6]('310 Helix', 2013). A 310 helix is usually always following an alpha helix. Additionally, HIV-1 integrase core domain contains 5 beta sheet strands, making its secondary structure 18% beta sheet.
Secondary Structure: HelicesThe secondary structure of helices Jmol image shows both types of helices present in the HIV-1 Integrase Core Domain. The blue structures are the twelve total alpha helices (both strands). As the figure rotates, the purple structures are highlighted in green to point out the four 310 helices (both strands).
This Jmol image demonstrates the special differences in the common alpha helices and the more rare 310 helices. The initial structures are ribbon models of two alpha helices that lead into 310 helices. These ribbon structures then convert to a wireframe backbone model of the combination of helices, followed by only the backbone of the 310 helices. Here, the structures are moveable to note the spacing of the three involved amino acids. The 310 helices are then replaced with the backbone structure of the alpha helices. By interacting with the structure it is possible to observe the difference in shape and the four amino acids involved before the next turn in structure occurs.
Secondary Structure: Beta Sheet StrandsIn the beta sheet Jmol image, you can observe the two sets of five-strand beta sheet composition.
This domain also possesses a distinct super-secondary structure motif, called a Ribonuclease H-like fold. The main element of the Ribonuclease H-like structure is a beta sheet comprising five beta strands, where the beta strand is antiparallel to the other beta strands. On both sides, the central beta sheet is flanked by alpha helices (Majorek123, et al., 2014).
Although most of the structure of the core domain has been determined by x-ray crystallography of the protein, there are still various residues that do not have secondary structure assigned and one multi-residue segment (residue 140 to residue 153) where the secondary structure is still unknown (Kabsch W., Sander C., 1983). This segment of the protein displayed a significant degree of disorder, which was serious enough that it remained crystallographically invisible (Goldgur, et al., 1998).
As explained before, HIV-1 integrase is the enzyme responsible for preparing the viral DNA and then inserting it into the host cell's genome. The core domain is more specifically known as the catalytic domain of the enzyme because it possesses the active site. The previously mentioned Ribonuclease H-like fold of the core domain is the important structural feature of this enzyme that exposes the residues that are directly involved in the catalytic activity of this enzyme.
The Ribonuclease H-like fold super-secondary structure of HIV-1 integrase core domain exposes three key catalytic residues: aspartic acid residue 64, aspartic acid residue 116, and glutamic acid residue 152. In site-directed mutagenesis experiments, it was demonstrated that substitution of any one of these residues resulted in the abolition of any enzymatic activity (Goldgur, et al., 1998). The catalytic residues Asp64, Asp116, and Glu152 of HIV-1 integrase may coordinate divalent metal ion, such as Mg2+ or Mn2+, and define the active site (Craigie, 2001)(Goldgur, et al., 1998). However, the residues comprising the active site region exhibit considerable flexibility, suggesting that binding of DNA substrate is required to impose the precise configuration of residues that is required for catalysis (Craigie, 2001).
The RNase H-like motif Jmol image distinctly highlights the motif's characteristic structure of the central beta sheet strand flanked by two alpha helices. A prior image showed the other characteristic arrangement of the five beta sheet strands (see Secondary Structure: Beta Sheet Strands Jmol image). The portions of the protein chain shown here contain the important catalytic residues.
Catalytic ResiduesThe catalytic residues Jmol image first shows the entire two chain HIV-1 Integrase Core Domain in ribbon structure, alpha helices in pink and beta sheets in green, with the three catalytic residues on each chain shown in ball form. The image then shifts to showing the backbone and side chains of the three residues. First, the Asp64 residue is highlighted. Then the Asp116 residue is highlighted, followed by the Glu152 residue. In addition to pointing out the catalytic residues, while each residue flashes a different color it is possible to see if each residue is located on a helix or a beta strand.
Currently, most of the drugs used in antiretroviral therapy target processes of viral infection and replication other than the function of integrase. The first class of FDA-approved drugs used to treat HIV was reverse transcriptase inhibitors, specifically nucleoside reverse transcriptase inhibitors ('A Timeline of HIV/AIDS'). As research continued, three other drug classes that targeted different steps in HIV infection/replication were created. These drug classes included fusion inhibitors and CCR5 antagonists, which both work by preventing HIV from entering the host cell, and protease inhibitors, which prevent maturation of newly formed HIV particles, disallowing them to infect surrounding healthy CD4 cells. ('Overview of HIV Treatments', 2015)
Much like reverse transcriptase, integrase has no human counterpart, therefore it forms an attractive target for drug design. Raltegravir, the first drug in a class of integrase strand transfer inhibitors, was approved by the FDA for antiretroviral therapy in 2007 ('FDA-Approved HIV Medicines', 2016).
HIV-1 has an extremely high mutation rate per cell, the highest reported for any biological entity, which grants it the ability to escape the immune system, rapidly evolve drug resistance, and slows down development of vaccinations (Cuevas, J.M., et. al., 2015). Studies have suggested that HIV-1 integrase is comparatively sensitive to mutation (maybe even more so), as opposed to other viral components, therefore understanding the degree of tolerance of HIV-1 integrase to mutation is particularly valuable given that it is an increasingly important therapeutic target in HIV-1 infection (Cuevas, J.M., et. al., 2015).
About HIV/AIDS. (2016, November 30). Retrieved December 09, 2016, from http://www.cdc.gov/hiv/basics/whatishiv.html
CD4 Count. (2016, July 14). Retrieved December 09, 2016, from https://www.aids.gov/hiv-aids-basics/just-diagnosed-with-hiv-aids/understand-your-test-results/cd4-count/
Craigie, R. (2001, May 29). HIV Integrase, a Brief Overview from Chemistry to Therapeutics*. Retrieved December 09, 2016, from http://www.jbc.org/content/276/26/23213.full
310 Helix. (2013). Retrieved December 13, 2016, from http://biochemistri.es/the-3-10-helix
Murzin A.G., Brenner S.E., Hubbard T., Chothia C. (1995, April). SCOP: a structural classification of proteins database for the investigation of sequences and structures. Retrieved December 13, 2016, from http://www.rcsb.org/pdb/explore/remediatedSequence.do?params.showJmol=false&structureId=1BIS#DSSPRefAnchor
Majorek123, K. A., Dunin-Horkawicz1, S., Steczkiewicz4, K., Muszewska45, A., Nowotny6, M., & And, K. G. (2014, January 23). The RNase H-like superfamily: New members, comparative structural analysis and evolutionary classification. Retrieved December 13, 2016, from http://nar.oxfordjournals.org/content/early/2014/01/23/nar.gkt1414.full
Kabsch W., Sander C. (1983, December 22). Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Retrieved December 13, 2016, from http://www.rcsb.org/pdb/explore/remediatedSequence.do?structureId=1BIS
Goldgur, Y., Dyda, F., Hickman, A. B., Jenkins, T. M., Craigie, R., & Davies, D. R. (1998). Three new structures of the core domain of HIV-1 integrase: An active site that binds magnesium. Retrieved December 13, 2016, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC21307/
Overview of HIV Treatments. (2015, August 12). Retrieved December 13, 2016, from https://www.aids.gov/hiv-aids-basics/just-diagnosed-with-hiv-aids/treatment-options/overview-of-hiv-treatments/
A Timeline of HIV/AIDS. (n.d.). Retrieved December 13, 2016, from https://www.aids.gov/hiv-aids-basics/hiv-aids-101/aids-timeline/
FDA-Approved HIV Medicines | Understanding HIV/AIDS | AIDSinfo. (2016, December 13). Retrieved December 13, 2016, from https://aidsinfo.nih.gov/education-materials/fact-sheets/21/58/fda-approved-hiv-medicines
Cuevas, J.M., Geller, R., Garijo, R., López-Aldeguer, J., Sanjuán, R. (2015, September 16). Extremely High Mutation Rate of HIV-1 In Vivo. Retrieved December 13, 2016, from http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002251