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 , 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 conformation e. Distorted type 1 turn around GPGR f. Extended KRIHI region, like a. & b. Gamma-turn around GPGR g. 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 large amount of weak data contributed to higher R values

Final statistics of the refined structure are 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 measured out of reflections present at the given 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 contactsPeptide 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 October