Unconserved Amino Acid Sequences in V3 Domain of gp120 Show No Significant

Correlation to Altered Folding and Function

Bobak Seddighzadeh

Alex GeorgeLoyola Marymount University

BIOL398-01/S10: Bioinformatics Laboratory

March 23, 2010

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 structures4. DNA mutations can affect protein function5. Unconserved regions are predicted to serve as key sites where

functional changes occur6. 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 conserved9. Future studies involving the the conserved regions of the V3 loop

as well as other domains can provide better insight

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

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

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

Mutations in DNA Sequence Can Affect Tertiary Protein Structure

• Central Dogma:DNARNAProteinExample:• Sickle Cell Anemia

– Single point mutation changes Glutamic acid (hydrophilic) to Valine (hydrophobic)

– Results in dysfunctional folding of Hemoglobin A (Tertiary)

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

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

Multiple Sequence Alignment Reveals Key Unconserved Regions of gp120

Fully conserved Strongly conserved Weakly Conserved

More Non-conservative than Conservative Amino Acid Substitutions were Found Between Clones

6 S13 V5-3 Serine Phenylalanine Polar to Hydrophobic 10 S2 V3-4 Threonine Methionine Polar to Hydrophobic 10 S11 V4-8,6 Threonine Serine Polar to Polar10 S11 V3-5,2 Threonine Serine Polar to Polar10 S11 V2-6,1 Threonine Serine Polar to Polar15 S10 V5-10,3 V6-7,4 Isoleucine Threonine Polar to Hydrophobic 15 S13 V5-6 Isoleucine Threonine Polar to Hydrophobic 20 S2 V4-3 Leucine Proline Hydrophobic to uncharged rigid ring structure 23 S10 V5-10 Serine Alanaine Polar to Hydrophobic 23 S10 V4-6 Serine Alanaine Polar to Hydrophobic 23 S10 V6-7 Serine Alanaine Polar to Hydrophobic 23 S12 V3-3,1 Serine Threonine Polar to Polar23 S12 V4-2,1 Serine Threonine Polar to Polar23 S12 V5-6,5 Serine Threonine Polar to Polar25 S4 V3-16 Glutamic acid Glycine Negative charge to Uncharged 25 S11 V4-8,6, V3-5,2, V2-6,1Glutamic acid Valine Negative charge to Hydrophobic 39 S10 V5-10, V4-6,8, V6-4Serine Arginine Polar to Positive charge39 S10 V5-3 Serine Lysine Polar to Positive charge39 S4 V3-16, V2-13, V4-1, V3-2Serine Lysine Polar to Positive charge43 S2 V4-8 Glycine Arginine Uncharged to Positive charge 71 S12 V5-6 Aspargine Aspartate Polar to Negative charge88 S13 V2-1,2 Glutamine Histadine Polar to positive charge 88 S13 V3-3.6 Glutamine Histadine Polar to positive charge 88 S13 V5-3,6 Glutamine Histadine Polar to positive charge

Position SubjectInitial

amino acidFinal

amino acid Characteristic Change

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

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

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

Threonine to Serine is a Polar to Polar Amino Acid Substitution

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

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

-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

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

Scanprosite Analysis Reveals Possible Effects on Glycosylation and Phosphorylation

Position 10

Position 23

Position 43

Position 25

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

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

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

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

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