Recurring conformation of the human immunodeficiency virus type 1 gp120 V3 loop

Robyn L. Stanfield, Jayant B. Ghiara, Erica O. Saphire, Albert T.

Profy, Ian A. Wilson

Biol 368

Bobby Arnold

Isaiah Castaneda

Outline

• Human immunodefiency virus type 1 make-up

• Overview of the Stanfield et. Al 2003 article

• Importance of the V3 region• Creating model structures for different

Fabs• Findings of the study

HIV-1 has an outer surface made up of glycoproteins

• These proteins are named gp120 and gp41– Result from cleavage of gp160 chain– They are noncovalently associated

• Gp120 binds to CD4– Contains 5 variable regions

• They are known as V1-V5

Outline

• Human immunodefiency virus type 1 make-up• Overview of the Stanfield et. Al 2003 article• Importance of the V3 region• Creating model structures for different Fabs• Findings of the study

The Stanfield et. al study looks closely at the V3 region

• By doing so, they hope to learn– How the V3 loop is recognized by

antibodies– How alterations affect the properties of the

virus• Sequence• Conformation• Exposure

Outline

• Human immunodefiency virus type 1 make-up• Overview of the Stanfield et. Al 2003 article• Importance of the V3 region• Creating model structures for different Fabs• Findings of the study

The V3 region is of particular interest

• Variation in this region has been linked to several factors– change in cell types that get infected– Induction of syncitia– Ability to be neutralized– Progression to AIDS

Outline

• Human immunodefiency virus type 1 make-up• Overview of the Stanfield et. Al 2003 article• Importance of the V3 region• Creating model structures for different Fabs• Findings of the study

Fabs are antibodies which bind to a V3 conformation

• 4 Fabs - 59.1, 83.1, 50.1, 58.2• Fab 83.1, 50.1, and 59.1 all bind to

similar conformations of V3• V3 loops are stabilized to prevent

conformation change• 5 antibodies chosen from over 85,000

possible for their neutralizing and stabilizing qualities

Fab models are purified, crystallized, then evaluated

• Antibodies isolated from mice

• Fab fragments collected from antibodies, then purified

• Fab mixed with 16-mer peptide MP1, and made into crystals on thin plates

• Structures determined, then models created from Fab 58.2

V3 peptides bound to Fabs

50.1 & 59.1

Conformation is least similar to others, but still binds to neutralizing antibodies

Comparing V3 Peptide Conformations

d. Type 1 turn conformatione. Distorted type 1 turn around

GPGRf. Extended KRIHI region, like

a. & b. Gamma-turn around GPGRg. Unclassified turn around

GPGR

Outline

• Human immunodefiency virus type 1 make-up• Overview of the Stanfield et. Al 2003 article• Importance of the V3 region• Creating model structures for different Fabs• Findings of the study

Analysis of structures showed higher R values

• Refinement and rebuilding of structures caused high R values

• Electron density maps were of good quality

• Rfree 32.6%, Rcryst 28.8% where average for 2.6A is typically 25-29% for Rfree

Strong data

Weak data

The relatively largeamount of weakdata contributed tohigher R values

Final statistics of the refined structureare shown in the table to the right

•Amount that atoms oscillate•By including # for different components, users can more easily detect error

•Used in structure refinement •A well refined model should not have many plots in the disallowed region

•Number of reflections measuredout of reflections present at thegiven resolution

Electron Density of V3 peptide bound to 83.1

Despite good quality maps, there was still room for refinement

Analyzing the molecular structure

• Light and heavy chains categorized into L and H groups in table 3

• CDR portion of each molecule in bold

• CDR - complementarity-determining region

Below are the sequences of the Fabs

-This table will be referred to again when the issue of kinks arises

All CDR loops normal except L1

• L1 CDR has 5 AA insertion after residue L27

• Tip of the loop bends away from antigen binding site

• L1 loops move about 9 A from corresponding spot in other Fabs

Fab 83.1-V3 Complex

•Light chains are in green•Heavy chains are in blue•V3 Peptide is in ball & stick model•Used MolScript program for the data

The L1 CDR loop of Fab 83.1bends away from the typical position

•27 other domains in grey•Fab 83.1 in red

The bending is to avoid clashing with H41 & H42, shown in blue

CDR H3 shows a kinked base

• Kinked base not predicted by sequence

• Seen in at least 2 other Fabs, also not predicted

• A salt bridge is usually formed with AspH101 and Arg/Lys, but does not for some reason

A straight torso was expected because there is no Arg or Lys at H94 for the Asp to form a salt bridge with

-This table will be referred to again when the issue of sequence homology arises

Ala Ile Asp

The sequence indicated that H3 loops would have an extended

torso

•However, they are actually kinked

Peptide complex connect with light and heavy Fab chains

• 110 total contact points

• 7 Hydrogen bonds no charge-charge interaction

• 6 Hydrogen bonds peptide-main chain

• 1 bond to Arg side chain

• H3 CDR most contacts

The table below shows all hydrogen bonds and the distance between them

Conformation of all but 58.2 similar

• 58.2 varies around the residues at the tip of V3

• Do not show structural homology

• Similarity in conformation is not due to similarity of Fabs

• All 4 antibodies generated from similar mice

Comparing the V3 peptide-Fab complexes

Overall shape is similar•Extended regions•Crowns

Peptides bind in different locations and ways on antibody• Antibodies chosen for ability to

neutralize, bind viruses

• Conformations show preferred V3 loop

• V3 structures shown show a recurring conformer on virus

•Light chains labeled in red•Heavy chains labeled in blue

Peptide makes 110 contacts Peptide makes 107 contacts

This is how the V3 peptide binds with Fab 83.1

•The antibodies are structurally and sequentially very different•Binding sites differ as well•Still, peptide conformations remain similar

The peptides adopt the same shape, but bind in different orientations and locations

Below are the sequences of the Fabs

Range of V3 conformation defined by X-ray snapshots

• V3 region contacts CCR5 and CXCR4 during infection

• To fully determine range, a more complete understanding of quaternary gp120/gp41 oligomers along with the role of V3 in protein assembly

• This will allow for understanding how HIV-1 completes receptor binding and viral fusion

References

• Stanfield et al. Recurring conformation of the human immunodeficiency virus type 1 gp120 V3 loop. Virology 315 (2003) p. 159-173. October 12 2011.