Recurring conformation of the human immunodeficiency virus type
- Slides: 33
Recurring conformation of the human immunodeficiency virus type 1 gp 120 V 3 loop. Stanfield RL, Ghiara JB, Ollmann Saphire E, Profy AT, and Wilson IA. Virology (2003) Oct 10; 315(1) 159 -73 Bio. Informatics Lab Tuesday, March 9, 2010 Kristoffer Chin Salomon Garcia Michael Piña
Outline • Introduction – Background on HIV Structure – Background on HIV V 3 Region • Materials and Methods – Crystallography • Results and Discussion • References
HIV has an outer surface of Glycoproteins http: //www. bijvoet-center. nl/cpc/research/HIVEnv/Env. jpg • gp 120 responsible for binding to CD 4 receptors and coreceptors, CCR 5 or CXCR 4 • gp 41 anchors gp 120 to the viral membrane providing noncovalent association
Glycoproteins binding to CD 4 T-cells http: //www. dkfz. de/de/f 020/images/bosch/web_fig_1. jpg
V 3 Region as part of gp 120 http: //www. prn. org/images/uploads/86_tsibris_athe_fig 2_680. gif • V 3 important to viral infectivity • Progresses initial infection to AIDS
Introduction to the Stanfield et al. (2003) Study • V 3 regions have high propensity to elicit neutralizing antibodies • V 3 region inaccessible due to carbohydrates masking or tertiary or quaternary interactions with gp 120 complex • Is there a limited range of conformational states that gp 120 can adopt? • How is V 3 loop recognized by antibodies and how an alteration of sequence, conformation, or exposure can affect it
Finding a dominant conformation for V 3 • Fabs are antibodies that bind to V 3 conformation • Fab 83. 1, 50. 1, and 59. 1 bind to a similar conformation of V 3 region • NMR studies have shown V 3 to form similar hairpin loops • Stabilization of V 3 loops to prevent change of conformation through turns • 5 antibodies used for neutralizing and stablization
Materials and Methods • Mab 83. 1 was made by immunization of an ASW mice with cyclic peptide RP 70 • Antibody was produced in ascites fluid of a mice and purified with an immobilized protein A column • Fab was made from immunoglobin by cleavage • Fab was concentrated to 15. 0 mg/ml for crystallization studies
Fab Purification • Fab was mixed with 16 -mer peptide MP 1 in a 6: 1 mole ration • Crystals were grown using sitting-drop vapor diffusion method with a reservoir solution of 1. 6 M Na/K phosphate, 5% isopropanol, p. H 6. 0. • Crystals grow as clusters of thin plates • Crystals used for this experiments grew over a 2 -week period
Fab Crystallization procedure • Crystals were cryocooled to liquid nitrogen temperatures in order to collect the data in a rapid manner • The crystals were protected by putting them in a solution containing the following 25% glycerol, 1. 6 M Na/K phosphate, 5% isopropanol, p. H 6. 0 • HKL 2000 was used to format all of the data that was obtained
Structure determination • Matthew coefficient was obtained by two Fab molecules • Model was constructed from the constant region of Fab 58. 2 • EPMR program was used to position the model in the cell • EPMR also used to locate the first Fab molecule in the asymetric unit
Stanfield et al. 2003.
Model building and refinement • TOM/FRODO was used to rebuild the mutated hybrid model and to correct the sequence and were subsequently refined with CNS version 1. 1 • Refinement was carried with tight NCS restraints in the beginning and progressively released towards the end of refinement
Structural Analysis • Kabat convention was used to number the molecules – Light and heavy chains are labeled using “L” and “H” – Peptide labeled “P” and was numbered according to HXB 2 isolate sequence • HBPLUS was used to evaluate the Hydrogen bonds • Contacsym program was used to assign van der waals contacts
Results and Discussion • Rcryst and Rfree values were slightly higher than other structures determined at 2. 6Å resolution • Electron density maps were good quality • However, repeated refinement and manual rebuilding of the structures caused the higher Rvalues • Rcryst 28. 8%, Rfree 32. 6% • An index (0. 45*l) close to an integer value is strong, whereas close to one-half integer is weak
Stanfield et al. 2003.
Figure 1: comparison of V 3 peptide conformations determined by X-Ray crystallography and NMR Stanfield et al. 2003.
Figure 2: stereoview of electron density for the V 3 peptide bound to Fab 83. 1 Stanfield et al. 2003.
All CDR loops fall into their expected canonical classes with the exception of L 1 • The L 1 CDR loops have a 5 amino acid insertion after residue L 27 • In both Fabs, the tip of this loop bends away from the antigen binding site in an unusual manner • Comparison with other L 1 loops shows the angle is about 9Å
Figure 3: the structure of the Fab 83. 1 -V 3 peptide complex Stanfield et al. 2003.
Figure 4: Noncanonical loops from Fab 83. 1 Stanfield et al. 2003.
CDR H 3 has a “kinked” base • This was not predicted from its sequence • At least two other Fabs have kinked H 3 bases that were not predicted • Asp. H 101 normally forms a salt bridge (with Arg or Lys), but in this case it does not which is unexpected
Stanfield et al. 2003.
Figure 5: stereoview of H 3 loop conformations from Fabs 83. 1, B 02 C 11, and 26 -10 Stanfield et al. 2003.
The peptide makes contact with both the light and heavy chains from the Fab • 110 total contacts for 1 molecule • 7 are hydrogen bonds with no charge-charge interactions • 6 hydrogen bonds are to peptide main-chain atoms • 1 bond to Arg side chain • The H 3 CDR makes the most contacts
Stanfield et al. 2003.
The 83. 1 peptide structure is the 4 th crystal structure determined for a neutralizing antibody V 3 peptide complex • Analysis of the 4 peptide reveals that 3 are very similar • The 4 th differs around the V 3 region • The 4 antibody peptides were generated from related mice • The antibodies themselves do not have structural homology • The similarity among the conformation of the peptides is not due to the similarity of the Fabs
Figure 6: comparison of the V 3 peptide conformations Stanfield et al. 2003.
Figure 7: the antibody combining site of Fab 83. 1 Stanfield et al. 2003.
Figure 8: antigen binding sites of Fabs 83. 1, 59. 1, 58. 2, & 50. 1 Stanfield et al. 2003.
The peptides, although adopting the same shapes, bind in different orientations and locations in the antibody • The antibodies were chosen for ability to neutralize (bind to intact viruses) • These peptide conformations should reflect “preferred” conformations of the V 3 loop • The identified V 3 structures represent a recurring conformer on the intact virus
The X-rays of V 3 peptides in complex with antibodies help define the range of V 3 conformation • Studies suggest that V 3 interacts with coreceptors CCR 5 and CXCR 4 during cell entry • This information may be useful in the design of V 3 based inhibitors • Ultimately, a better understanding of the gp 120/gp 41 structure (and the V 3 region) is vital for understanding how HIV-1 carries out its binding and fusion activities
References • Stanfield et al. Recurring conformation of the human immunodeficiency virus type 1 gp 120 V 3 loop. Virology (2003) Oct 10; 315(1) 159 -73 • http: //www. bijvoet-center. nl/cpc/research/HIVEnv/Env. jpg • http: //www. dkfz. de/de/f 020/images/bosch/web_fig_1. jpg • http: //www. prn. org/images/uploads/86_tsibris_athe_fig 2_680. gif
- Secondary immunodeficiency diseases
- Severe combined immunodeficiency
- Bond angle in cyclohexane
- Horse conformation pictures
- E and z alkene
- Tetrahydropyran chair conformation
- Planar trans-1 2-dimethylcyclohexane
- Horse conformation judging practice
- Carbohydrates organic chemistry
- Isomerie de conformation
- Hsv encephalitis
- Roundworms are what type of biohazard virus/parasite
- Recurring decimal questions
- It looks for patterns recurring characteristics and events
- Recurring aspects of designs are called design
- Rhythm music appreciation
- Recurring decimal
- The recurring aspects of designs are called design
- The recurring aspects of designs are called design
- Recurring decimal
- Characteristically definition
- Accident chain definition
- Recurring or dominant theme
- Recount the story of pony‘s recurring dream.
- What is a steady recurring pulse called
- What is a steady recurring pulse called
- Hình ảnh bộ gõ cơ thể búng tay
- Ng-html
- Bổ thể
- Tỉ lệ cơ thể trẻ em
- Gấu đi như thế nào
- Thang điểm glasgow
- Chúa yêu trần thế alleluia
- Các môn thể thao bắt đầu bằng tiếng chạy