Transcript Structure of HIV-1 gp120 with gp41-interactive

Mutated Amino Acid Sequences in
V3 Domain of gp120 Show No Significant
Correlation to Altered Folding and Function
Bobak Seddighzadeh
Alex George
Loyola Marymount University
March 23, 2010
Outline
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Levels of protein organization
Amino acid characteristics and classifications
Determinants of Tertiary and Quaternary Structures
DNA mutations can affect protein function
What led us to our question
Multiple Sequence alignment shows key insight on unconserved regions of V3 domain
Analysis of the amino acid substitutions found in the multiple
sequence alignment
In depth structural analysis of key amino acid substitutions
Scan prosite indicates the function of amino acids at unconserved regions of V3 domain
Correlation and conclusion of mutations on structure and
function in V3 domain
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
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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
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Amino Acid Side Chains Play a Large
Role in Tertiary and Quaternary Structure
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Covalent disulfide bonds
Electrostatic Interactions
Hydrogen bonds
Van der Waals forces
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)
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Determining the Effects of Amino Acid
Alterations in Conserved Sequences
• Analysis of sequences from Markham et. al
study confirmed the presence of highly
conserved regions in the V3 loop of gp120
• Kwong et. al study hypothesize the
conserved regions were necessary for viral
cell entry
• Our question: Will amino acid mutations in
conserved regions of amino acids
alter/disrupt the function of the protein
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 for Subjects 2,4,10,11,12,13
Fully conserved
Strongly conserved
Weakly Conserved
Figure: Highlights conserved, unconserved, and similar amino acid
positions in the V3 domain of gp120 amongst these subjects
Gp120 in complex with CD4 and two antibodies
with the V3 region highlighted
gp120
Human antibody
CD4 Receptor
V3 loop is located on the periphery of the protein
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
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 doesn’t 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
• The residue substitutions on the surface don’t affect
structure significantly
At position twenty the substitution from
Leucine to Proline May Affects 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 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 protien
-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 protien
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
Post-Transcriptional modifications likely occur at
common amino acid sequences in Kwong’s sequence
• 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
– Position 25 mutation (Glutamic acid  Glycine/Valine) is at the
C-terminus of the acceptor site, thus not affecting function
Mutations in un-conserved regions of the V3 Domain
do not greatly affect function of gp120
• The structure of the V3 domain remains relatively
unaffected by un-conserved mutations
• The location of the V3 domain may serve as a
defense to mutational changes
• Analysis of the other domains of gp120 may better
suit our investigation
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
• Defined elements between
gp120 and gp41 provides
conformational diversity
necessary for viral entry
• Pancera et. al
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