BIOL368/F14:Nicole Anguiano Week 9

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HIV Structure Project

Presentation Link

HIV Structure Project Presentation

Question

How does the structure of the V3 protein region affect the HIV status (diagnosed, progressing or non-trending) of the patient?

Hypothesis

We hypothesize that diagnosed groups will express greater variability in the V3 region in their protein structure, in comparison to the non-trending groups. Initial comparisons show that diagnosed groups and progressing groups expressed greater genetic variability than non-trending groups. These changes may affect the third variable region, affecting the host's ability to adapt to the changes and generate sufficient immune response.

Subject Data

  • The project will be done using the following sequences (the txt file containing all of the relevant amino acid sequences is linked to the name of the progressor group):
Group Subject Visit Sequences
AIDS Diagnosed 3


10


15		 1
6

1
6

1
4		 1, 2, 4
3, 4, 5

3, 6, 7
2, 4, 8

2, 3, 4
5, 8, 10
AIDS Progressing 7


8


14		 1
5

1
7

1
9		 2, 3, 9
2, 8, 9

1, 4, 5
1, 6, 7

2, 3, 4
9, 10, 11
No Trend 5


6


13		 1
5

1
9

1
5		 1, 3, 8
4, 5, 2

1, 2, 3
6, 7, 9

1, 3, 4
3, 5, 4

Results and Methods

Obtaining the Protein Sequences

Multiple Sequence Alignment

  • Then, I navigated to the Biology Workbench. I selected "Session Tools" and created a new session called "AIDS Diagnosed". Then I went to "Protein Tools" and hit "Add". I uploaded the file containing all of the amino acid sequences, then selected "Upload File".
  • I selected all of the visit 1 clones and selected "ClustalW" and submitted it with the default settings. Then, I ran a ClustalW on all the final visit clones, and lastly I ran a ClustalW on all the clones over all the visits.
Sequence alignment for the visit 1 clones of the AIDS diagnosed patients
Figure 1: The sequence alignment for the AIDS diagnosed patients at the first visit. There are 35 differences between the sequences. There are 17 strongly conserved regions and 4 weekly conserved regions.
Tree for the visit 1 clones of the AIDS diagnosed patients
Figure 2: Tree for the AIDS diagnosed patients at the first visit. Each clone is most closely related to clones from its own subject, as expected.
Sequence Analysis for the Final visit
Figure 3: The sequence alignment for the AIDS diagnosed patients at the final visit. There are 45 differences between the sequences. There are 24 strongly conserved regions and 4 weakly conserved regions.
Tree for the Final Visit
Figure 4: Tree for the AIDS diagnosed patients at the final visit. Each clone is most closely related to clones from its own subject, as expected.
Sequence analysis for all visits
Figure 5: The sequence alignment for the AIDS diagnosed patients over both visits. There are 49 differences between the sequences. There are 21 strongly conserved regions and 2 weakly conserved regions.
Tree for all visits
Figure 6: The tree for the AIDS diagnosed patients over both visits. Each clone is most similar to the clones of its particular subject, and typically is most similar to the clones that came from its same visit. However, one of the visit 1 subject 15 clones is most similar to the three clones from the final visit that the three clones at its same visit.
  • The number of differences between the first and final visits increased, as expected (Table 1). The clones, though they diverged, remained most similar to the other clones in their particular groups. Overall, the clones are most similar to the clones in their own group, with the exception of Subject 15, visit 1, clone 3. This clone is more similar to the clones from the final visit for subject 15 than the first visit. This li
  • Next, I switched to the "Nucleic Acid Tools". I uploaded the DNA sequences of the clones that correspond to the amino acid sequences. I then ran a clustalW on the first visit clones, the final visit clones, and then lastly all the clones to get a comparison of the number of differences between the amino acid sequences and the DNA sequences.
Sequence Analysis for the first visit DNA
Figure 7: The DNA sequence for the AIDS diagnosed patients at the first visit. Out of the 287 nucleotides, there were 69 differences.
Sequence Analysis for the final visit DNA
Figure 8: The DNA sequence for the AIDS diagnosed patients at the first visit. Out of the 287 nucleotides, there were 79 differences.
  • Also as expected, the number of differences in the DNA sequence went up between the first and final visit.
Visit Type Number of Differences Percentage Different
1 Amino Acids 37 37/95 = 38.9%
1 DNA 69 69/285 = 24.2%
Final Amino Acids 45 45/95 = 47.3%
Final DNA 79 79/285 = 27.7%
Both Amino Acids 49 49/95 = 51.6%
Both DNA 94 94/285 = 33%
  • Table 1: Table illustrating the number of differences between the amino acid sequences and the DNA sequences at both visits.
  • The increase between the visits in the amino acids was 8 (37 at the first visit and 45 at the second), and in the DNA it was 10 (69 in the first visit and 79 at the second), which is not a particularly large difference in increase. The overall percentage of Amino acids that are different is higher than the number of DNA nucleotides that are different, likely due to the fact that there are significantly more nucleotides than there are amino acids. A difference in a DNA sequence does not necessarily translate into a change into the amino acid sequence due to there being multiple codons for the same amino acid.

Secondary Structure Predictions

  • Due to the nature of the question we wanted to ask, using PsiPred would be most applicable. PsiPred returns a result of the secondary structure, which is important to what we were asking in our question, as we wanted to see if the structure influenced the progressor groups.
  • I went to the Biology Workbench, and performed a ClustalW on both visits of subject 3, then imported the alignment. I downloaded the alignment and inputted the result into PsiPred, then hit run. I repeated this with Subjects 10 and 15, then all of them at visit 1, then all of them at the final visit, then finally all together. Lastly, I ran PsiPred on the sequence in the full gp120 protein that corresponds to the region coded for by the clones of each subject.
PsiPred result for Subject 3.
Figure 10: PsiPred result for both the first and last visits of subject 3. The V3 region is located at 28-62.
PsiPred result for Subject 10.
Figure 11: PsiPred result for both the first and last visits of subject 10. The V3 region is located at 28-62.
PsiPred result for Subject 15.
Figure 12: PsiPred result for both the first and last visits of subject 15. The V3 region is located at 28-62. This is the only subject in which there is not a beta sheet at residues 47-49.
PsiPred result for the Visit 1 Aids Diagnosed Patients.
Figure 13: PsiPred result for the first visit of all the AIDS diagnosed patients. The V3 region is located at 28-62. Like subject 15, it does not have a beta sheet at residues 47-49, likely due to the influence of subject 15.
PsiPred result for the Final Visit Aids Diagnosed Patients.
Figure 14: PsiPred result for the final visit of all the AIDS diagnosed patients. The V3 region is located at 28-62. Like subject 15, it does not have a beta sheet at residues 47-49, likely due to the influence of subject 15.
PsiPred result for the Final Visit Aids Diagnosed Patients.
Figure 15: PsiPred result for both visits of all the AIDS diagnosed patients. The V3 region is located at 28-62. Like subject 15, it does not have a beta sheet at residues 47-49, likely due to the influence of subject 15.
PsiPred result for the consensus sequence.
Figure 16: PsiPred result for the sequence in the gp120 protein that corresponds to the region coded for in each of the clones. The V3 region is located at 28-62.
  • With the exception of subject 15, each of the sequences matched the consensus sequence. Perhaps due to the influence of subject 15, the first and final visits results for all the sequences also are missing the beta sheet that subject 15 is missing.

gp120 Crystal Structure

cn3d result for the protein
Figure 17: The structure of gp120, as viewed in Cn3D. Chain G is in pink, chain C is in blue, chain L is in brown, and chain H is in green.
  • To find the N and C-terminus of each polypeptide, I used the Sequence Viewer. The sequence viewer stated the beginning and end of each tertiary structure. There are 4 separate tertiary structures that make up the overall quaternary structure. They are known as the G, C, L, and H chains. I clicked the first and last amino acid of each one. The first would be the N-terminus, and the last would be the C-terminus (table 2).
Chain N-ter C-ter
G Valine Glutamic acid
C Lysine Valine
L Glutamic acid Cysteine
H Glutamine Cysteine
  • Table 2: The N and C termini of each tertiary structure in gp120.
  • Next, I selected "Style > Coloring Shortcuts > Secondary Structure". Then, in the Sequence/Alignment Viewer, I selected "EVVIRSDNFTNNAKTIIVQLKESVEINCTRPNNNTRKSIHIGPGRAFYTTGEIIGDIRQAHCNISRAKWNDTLKQIVIKLREQFENKTIVFNHSS" in the 2B4C_G row. This corresponds to the section coded for by the subject's clones (Fig. 18). After that, I selected "Select > Show Selected Residues" to show just that selection (Fig. 19).
secondary structure for the gp120 protein
Figure 18: The gp120 protein, with the secondary structure highlighted. The beta sheets are in orange, and the alpha helixes are in blue. The section coded for by the protein studied in Huang's study is highlighted in yellow. This was generated in CN3D.
section coded for by the clones in the gp120 protein
Figure 19: The section coded for by the clones being studied, viewed by CN3D. This includes the V3 region.
  • The V3 base ends and becomes the gp120 protein at the disulfide bond between the antiparallel beta sheets. Looking at the structure above, I found the region in which there was the disulfide bond. I selected the regions that extended out from the bond and came back to it, which was "CTRPNQNTRKSIHIGPGRAFYTTGEIIGDIRQAHC". This region corresponds to the V3 region (Fig. 20).
v3 loop
Figure 20: The V3 loop, as viewed by CN3D.
  • The structure of V3 found and it's location, which is stated in Figures 10-16, does not match up to the official structure. Each of the PsiPred results shows an alpha helix towards the end, but it's clear that it should be a beta sheet. The reliability and confidence in the prediction at that location is low, which would likely explain the discrepancy.

Amino Acid Analysis

  • The original sequence found by Huang, "CTRPNQNTRKSIHIGPGRAFYTTGEIIGDIRQAHC", has the following amino acid properties:
    • Nonpolar (P, I, A, F, V, L, M): 10
    • Uncharged Polar (C, T, N, S, G, Q, Y): 16
    • Basic (R, K, H): 7
    • Acidic (E, D): 2
  • The sequence found in the subject 3 clones is "CTRPGNNTRKRVTLGPGRVYYTTGQIIGDIRKAHC". It has the following amino acid properties:
    • Nonpolar (P, I, A, F, V, L, M): 9
    • Uncharged Polar (C, T, N, S, G, Q, Y): 17
    • Basic (R, K, H): 8
    • Acidic (E, D): 1
  • The sequence found in the subject 10 clones is "CTRPNNNTRRSINMGPGRAFYTTGEIIGDIRQAHC". It has the following amino acid properties:
    • Nonpolar (P, I, A, F, V, L, M): 10
    • Uncharged Polar (C, T, N, S, G, Q, Y): 17
    • Basic (R, K, H): 6
    • Acidic (E, D): 2
  • The sequence found in the subject 15 clones is "CTRPNNNTRRKIHIGPGKTFYTGDIIGNIRQAHC". It has the following amino acid properties:
    • Nonpolar (P, I, A, F, V, L, M): 9
    • Uncharged Polar (C, T, N, S, G, Q, Y): 16
    • Basic (R, K, H): 8
    • Acidic (E, D): 1
  • I think that it is possible that amino acid changes can affect the 3D structure. Especially in the case of Subject 15, who was missing an amino acid and as a result was missing the beta sheet, it is highly possible for changes in amino acid structure to cause changes to the structure. Because the V3 loop has regions that are highly conserved between regions with moderate to high amounts of diversity, I am not necessarily sure whether the 3D structure changes would be relevant in the V3 loop, with the exception of structure 15. If the changes take place in random coil, then it would not have a significant effect. There are several reasons within the protein that are highly conserved, and do not change, and I believe a change in those conserved regions would have a significant effect on 3D structure, but changes in other regions, while they may lead to changes in the 3D structure, may not lead to significant changes in the structure.

Links

Nicole Anguiano
BIOL 368, Fall 2014

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