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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
Outline
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Protein organization determines folding patterns that affect
function.
Amino acid side chains are in part responsible for protein folding
Molecular interactions determine tertiary and quaternary structures
DNA mutations can affect protein function
Unconserved regions are predicted to serve as key sites where
functional changes occur
Multiple Sequence alignment reveals unconserved regions of V3
domain
Amino acid changes do not affect the structure due to the
peripheral location of the V3 loop
Scan prosite indicates the function of amino acids are conserved
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
•
2.
Nonpolar
•
3.
Hydrophobic
Acidic
•
4.
Hydrophilic
Positive charge
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:
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)
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
Position
6
10
10
10
10
15
15
20
23
23
23
23
23
23
25
25
39
39
39
43
71
88
88
88
Initial
amino acid
Final
amino acid
Serine
S13 V5-3
Threonine
S2 V3-4
Threonine
S11 V4-8,6
Threonine
S11 V3-5,2
Threonine
S11 V2-6,1
S10 V5-10,3 V6-7,4 Isoleucine
Isoleucine
S13 V5-6
Leucine
S2 V4-3
Serine
S10 V5-10
Serine
S10 V4-6
Serine
S10 V6-7
Serine
S12 V3-3,1
Serine
S12 V4-2,1
Serine
S12 V5-6,5
Glutamic acid
S4 V3-16
Glutamic acid
S11 V4-8,6, V3-5,2, V2-6,1
Serine
S10 V5-10, V4-6,8, V6-4
Serine
S10 V5-3
V3-2
Serine
S4 V3-16, V2-13, V4-1,
Glycine
S2 V4-8
Aspargine
S12 V5-6
Glutamine
S13 V2-1,2
Glutamine
S13 V3-3.6
Glutamine
S13 V5-3,6
Phenylalanine
Methionine
Serine
Serine
Serine
Threonine
Threonine
Proline
Alanaine
Alanaine
Alanaine
Threonine
Threonine
Threonine
Glycine
Valine
Arginine
Lysine
Lysine
Arginine
Aspartate
Histadine
Histadine
Histadine
Subject
Characteristic Change
Polar to Hydrophobic
Polar to Hydrophobic
Polar to Polar
Polar to Polar
Polar to Polar
Polar to Hydrophobic
Polar to Hydrophobic
Hydrophobic to uncharged rigid ring structure
Polar to Hydrophobic
Polar to Hydrophobic
Polar to Hydrophobic
Polar to Polar
Polar to Polar
Polar to Polar
Negative charge to Uncharged
Negative charge to Hydrophobic
Polar to Positive charge
Polar to Positive charge
Polar to Positive charge
Uncharged to Positive charge
Polar to Negative charge
Polar to positive charge
Polar to positive charge
Polar to positive charge
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