2. Unconserved Amino Acid Sequences in V3 Domain of gp120 Show No Significant Correlation to Altered Folding and Function Bobak Seddighzadeh Alex George Loyola Marymount University BIOL398-01/S10: Bioinformatics Laboratory March 23, 2010

3. Outline 1.Protein organization determines folding patterns that affect function. 2.Amino acid side chains are in part responsible for protein folding 3.Molecular interactions determine tertiary and quaternary structures 4.DNA mutations can affect protein function 5.Unconserved regions are predicted to serve as key sites where functional changes occur 6.Multiple Sequence alignment reveals unconserved regions of V3 domain 7.Amino acid changes do not affect the structure due to the peripheral location of the V3 loop 8.Scan prosite indicates the function of amino acids are conserved 9.Future studies involving the the conserved regions of the V3 loop as well as other domains can provide better insight

4. Levels of Protein Organization Affect Overall Function Primary Structure –The number and sequence of amino acids Secondary Structure –Alpha Helices –Beta-pleated Sheets Tertiary Structure –3-D shape of structure Quaternary Structure –Intra-protein interactions

5. Amino Acid Characteristics Determine Molecular Interactions Classified into Four Types based on R-Group: 1.Uncharged Polar Hydrophilic 2.Nonpolar Hydrophobic 3.Acidic Positive charge 4.Basic Negative charge * Proline and Glycine are unique

6. Amino Acid Side Chains Play a Large Role in Tertiary and Quaternary Structure 1.Covalent disulfide bonds 2.Electrostatic Interactions 3.Hydrogen bonds 4.Van der Waals forces 5.Hydrophobic Side Chains

7. Mutations in DNA Sequence Can Affect Tertiary Protein Structure Central Dogma: DNA  RNA  Protein Example: Sickle Cell Anemia –Single point mutation changes Glutamic acid (hydrophilic) to Valine (hydrophobic) –Results in dysfunctional folding of Hemoglobin A (Tertiary)

8. Determining the Effects of Amino Acid Alterations in Unconserved Sequences Markham et. al study found that increased diversity and divergence in variants correlated to increased virulence Kwong et. al study solved the X-Ray structure for gp120 core complexed with CD4 and antibodies Our question: Will amino acid mutations disrupt the structure of gp120 significantly enough to alter its function? We hypothesize that specific amino acid mutations in unconserved regions of the V3 loop are responsible for structural change that alters function

9. Rapid and Non-progressors Sequences were Chosen According to High Diversity and Divergence Rapid Progressors: –Subject 4 Visit 4 - clones 4,1 Visit 3 - clones 16,2 Visit 2 - clones 13, 5 –Subject 10 Visit 6 - clones 7,4 Visit 5 - clones 10,3 Visit 4 - clones 8,5 –Subject 11 Visit 4 - clones 8,6 Visit 3 - clones 5,2 Visit 2 - clones 6,1 Non Progressors: –Subject 13 Visit 2 - clones 1,2 Visit 3 - clones 3,6 Visit 5 - clones 3,6 –Subject 12 Visit 5 - clones 6,5 Visit 4 - clones 2,1 Visit 3 - clones 3,1 –Subject 2 Visit 4 - clones 6,5 Visit 3 - clones 5,4 Visit 1 - clones 5,1

10. Multiple Sequence Alignment Reveals Key Unconserved Regions of gp120 Fully conserved Strongly conservedWeakly Conserved

11. More Non-conservative than Conservative Amino Acid Substitutions were Found Between Clones PositionSubject Initial amino acid Final amino acid Characteristic Change

12. Kwong et. al. data reveals the peripheral location of the V3 loop gp120 Human antibody CD4 Receptor Gp120 complexes with two antibodies and CD4 receptors

13. Phenylalanine Substitution at Position Six does not Interfere with CD4 Interactions Needs to be directly touching CD4 Polar to Hydrophobic change, but located on the surface which does not significantly affect structure

14. Threonine to Serine Substitution at Position Ten has minimal affect on Structure Serine and Threonine both have hydroxyl's on their side chain making them structurally similar Residue substitutions on the surface does not affect structure significantly

15. Threonine to Serine is a Polar to Polar Amino Acid Substitution

16. At Position Twenty, the Substitution from Leucine to Proline May Affect the  -sheet The amino acid sequence is buried in the peptide The direction and nature of the Beta turn can be altered

17. Amino Acid Substitution from Serine to Alanine at Position Twenty-three has Minimal Affect Serine and Alanine are very similar in size The residue is located on the surface of the protein

18.  - sheet Is Unaffected by Amino Acid Substitution to Glycine at Position Twenty-five B-pleated Sheets are very forgiving Glycine is more likely to affect Alpha helices The residue is located on the surface of the protein

19. Alpha Helices May be Affected by Substituting Glycine to Arginine at Position Forty-three Lysine to Arginine substitution is small uncharged to bulky positive charge The residue is located on the surface of the protein structure

20. Scanprosite Analysis Reveals Possible Effects on Glycosylation and Phosphorylation Position 10 Position 23 Position 43 Position 25

21. Post-Translational Modifications Are Not Affected by Amino Acid Mutations Observed N-Glycosylation –Typical Sequence = Asn-X-Ser or Asn-X-Thr –Important in folding and cell-cell interaction Phosphorylation –Increases energy so that the protein can undergo subsequent reactions spontaneously –Charged amino acids at the N-terminus affect phosphorylation rate

22. Mutations in Unconserved Regions of the V3 Domain do not Greatly Affect gp120 Function The structure of the V3 domain remains relatively unaffected by unconserved mutations The location of the V3 domain may serve as a defense to mutational changes Glycosylation and Phosphorylation are conserved functions despite amino acid mutations We reject our hypothesis that amino acid mutations in unconserved regions affect function of the V3 Loop

23. Pancera et al Study Shows Conserved Elements Between gp120 and gp41 May Play Large Role in Viral Entry Conformational changes in gp120 affect drug and antibody neutralization The association between gp120 and gp41 plays a role in determining viral cell entry Defined elements between gp120 and gp41 provides conformational diversity necessary for viral entry

24. Future Studies Looking into the significance of mutational changes and the rate at which they occur Analysis of the other domains of gp120 may better suit our investigation

25. References Kwong PD, Wyatt R, Robinson J, Sweet RW, Sodroski J, Hendrickson WAStructure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody Nature v393, p.648-659 Markham RB, Wang WC, Weisstein AE, Wang Z, Munoz A, Templeton A, Margolick J, Vlahov D, Quinn T, Farzadegan H, and Yu XF. Patterns of HIV-1 evolution in individuals with differing rates of CD4 T cell decline. Proc Natl Acad Sci U S A 1998 Oct 13; 95(21) 12568-73. pmid:9770526. Pancera M, Majeed S, Ban YE, Chen L, Huang CC, Kong L, Kwon YD, Stuckey J, Zhou T, Robinson JE, Schief WR, Sodroski J, Wyatt R, Kwong PD Structure of HIV-1 gp120 with gp41-interactive region reveals layered envelope architecture and basis of conformational mobility Proc. Natl. Acad. Sci. U. S. A. v107, p.1166-1171