Zrusso Biol 368 week 7
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Journal Club Prep
- My partners are Samantha Hurndon and Nicki Harmon and we are working on the Kwong paper.
Biological Terms
- Oligomeric - a polymer molecule consisting of a small number of monomers. Retrieved from [1] on 10/12/11
- Chemokine - any of a group of chemotactic cytokines that are produced by various cells (as at sites of inflammation), that are thought to provide directional cues for the movement of white blood cells (as T cells, monocytes, and neutrophils), and that include some playing a role in HIV infection because the cell surface receptors to which they bind are also used by specific strains of HIV for entry into cells. Retrieved from [2] on 10/12/11
- Fusogenic - Facilitating fusion, especially relating to cells. Retrieved from [3] on 10/12/11
- Prophylactic - defending or protecting from disease or infection, as a drug. Retrieved from [4] on 10/12/11
- Ternary - consisting of three different elements or groups. Retrieved from [5] on 10/12/11
- Prolate - elongated along the polar diameter, as a spheroid generated by the revolution of an ellipse about its longer axis ( opposed to oblate). Retrieved from [6] on 10/12/11
- Interfacial - included between two faces. Retrieved from [7] on 10/12/11
- Antigenic - having the properties of any substance that can stimulate the production of antibodies and combine specifically with them. Retrieved from [8] on 10/12/11
- Proteolytic - the breaking down of proteins into simpler compounds, as in digestion. Retrieved from [9] on 10/12/11
- Glycocalyx - a polysaccharide or glycoprotein covering on a cell surface. Retrieved from [10] on 10/12/11
Paper Outline
- Introduction
- HIV-1, HIV-2 and their cousin the Simian immunodeficiency viruses (SIV) destroy CD4 lymphocytes in their hosts, which results in AIDS
- Entry of HIV virus into host cells is mediated by viral envelope glycoproteins
- These glycoproteins are arranged in oligomeric, most likely trimeric spikes along the surface of the virion
- These spikes are anchored to the viral membrane by gp41 transmembrane protein
- The surface of the spike is primarily gp120
- gp120 contains five variable regions (V1-V5)
- both conserved and variable gp120 regions are heavily glycosylated
- this glycosylation probably modulates the immunogenicity and antigenicity of gp120
- gp120 is the main target for antibodies
- gp120 will bind to glycoprotein on CD4 and acts as main receptor
- gp120 binds to the most amino-terminal of the four immunoglobulin like domains of CD4
- mutagenesis has found critical regions in both gp120 and CD4 for binding
- CD4 binding induces a conformation change in gp120 which exposes/forms a chemokine receptor
- This chemokine receptor for CCR5 and CXCR4 serve as obligate secondary receptors for HIV entry into the cell
- V3 is the principle determinant of chemokine receptor specificity
- There are other more conserved regions of gp120 that seem to be involved in chemokine-receptor binding
- CD4i (CD4 induced) antibodies block the binding of the gp120-CD4 complex to the chemokine receptor
- HIV and related retroviruses belong to a class of enveloped fusogenic viruses, all which require post-translational cleavage for activation.
- Some share protein sequence similarity while others are quite distinctive.
- since gp120 is so important in receptor binding and in interactions with antibodies, info about it is important
- In this paper is reported the crystal structure at 2.5 Å detail a partially deglycosylated HIV-1 gp120 core bound to a two domain fragment of CD4 receptor and to an antigen binding fragment (Fab) 17b, which acts on a CD4i epitope.
- Structure Determination
- Due to the fact that gp120 is extensively glycosylated and shows great conformational heterogeneity, radical modification of the protein surface was devised to image it.
- truncations were made at the termini and at variable loops in various combinations from various strains. These variants were then heavily deglycosylated and produced complexes with
- This was done because a theoretical analysis showed an increase in probable crystal formation with the reduction of surface heterogeneity and trials with multiple variants.
- After many combinations, crystals were obtained of a ternary complex that contained a truncated gp120, the N-terminal of two domains of CD4, and a Fab from 17b.
- the gp120 crystallized was from HIV-1 strain HXBc2
- deletions of 52 residues from N-terminus and 19 from C-terminus.
- Gly-Ala-Gly tripeptide substitutions for 67 V1/V2 loop residues and 32 V3 loop residues
- removal of all sugar groups beyond the linkages between the two core N-acetyl-glucosamine residues.
- removal of 90% of total carbohydrate but retains 80% of non-variable loop protein
- capacity to interact with CD4 and relevant antibodies is preserved at or near wild-type levels.
- Due to the fact that gp120 is extensively glycosylated and shows great conformational heterogeneity, radical modification of the protein surface was devised to image it.
- Structure of gp120
- deglycosylated core of gp120 approximately looks like a prolate ellipsoid, though the outline is heart-shaped.
- core is made up of 25 β-sheets, 5 α-helices, and 10 defined loop segments
- the polypeptide chain is folded into two main domains along with some digressions from this body
- Inner domain (with respect to N and C termini) contains a two-helix, two-sheet bundle with a small five sheet β-sandwich at its termini-proximal end and a projection from the distal side where the V1/V2 stem originates.
- Outer domain is a stacked double barrel that lies alongside inner domain so that the both barrel axes are roughly parallel to each other.
- There is a ‘minidomain’ which is comprised of four antiparallel β-sheets that create a ‘bridging sheet’ that is in contact with both the inner and outer domains
- the structure of gp120 has no precedent
- domain-level searches revealed no similar structures, though missing terminal segments might account for this
- there is fragmentary similarity for portions of outer domain but no sequence evidence to support an evolution argument.
- This structure of core gp120 should be a prototype for the class
- structure based alignment shows conservation despite the variability in HIV strains
- HIV-2 is 35% similar
- 77% and 51% for HIV-1 clade C and O respectively
- Outer domain is more conserved with 86%, 72%, and 45% for HIV-1 C, HIV-1 O, and HIV-2 respectively
- structure based alignment shows conservation despite the variability in HIV strains
- CD4-gp120 Interaction
- CD4 is bound in a depression formed at the interface of the outer and inner domain along with the bridging sheet of gp120
- the interaction buries a total of 742 Å2 from CD4 and 802 Å2 from gp120
- the surface area actually in contact is much smaller than this due to mismatch in surface topography that creates large cavitites
- there is a general complementation of electrostatic potential at the surfaces of contact, though not perfect
- Phe43 and Arg59 of CD4 make multiple contacts with gp120
- Phe43 accounts for 23% of all interatomic contact
- several gp120 residues that are covered by CD4 are variable
- this variation is due to the fact that they are in contact with a large water filled cavity
- there are two cavities that are unusually large
- larger cavity is lined with hydrophilic residues from both gp120 and CD4
- while formally a cavity in the crystal structure, minor changes in side-chain orientation would make it accessible to solvents
- residues lining the cavity are variable, but those directly surrounding this variable patch are conserved and their substitution affect CD4 binding
- this cavity acts as a water buffer between gp120 and CD4 and is a variation island or ‘anti-hotspot’ located between conserved regions for CD4 binding, therefore might help virus escape from antibodies directed at its CD4 binding site.
- the Phe43 cavity is very different, and is roughly spherical
- located just beyond Phe43 and at the intersection of inner and outer domains and binding sheet
- deeply buried and extends into interior of gp120
- only a few water molecules are seen in this cavity and at the center of it is a large spherical density over 4 Å from any protein atom
- its density does not match any major crystallization component and is currently unidentified.
- residues that line the cavity are hydrophobic and are as highly conserved as the buried hydrophobic core of gp120
- there is no steric hindrance but no substitutions to larger residues were found
- such conservation indicates functional significance
- many of the residues that line the Phe43 cavity interact with elements of the chemokine-receptor-binding region
- larger cavity is lined with hydrophilic residues from both gp120 and CD4
- the missing structures could not conceivably have a role in filling these cavities
Journal Club Presentation
Media:Kwong_Journal_Club_Presentation_Nicki,_Samantha,_Zeb.ppt
