Janelle N. Ruiz Assignment 7

Vocabulary

 * 1) Glycoprotein: A molecule that consists of a carbohydrate plus a protein. Glycoproteins play essential roles in the body. For instance, in the immune system almost all of the key molecules involved in the immune response are glycoproteins. http://www.medterms.com/script/main/art.asp?articlekey=16842
 * 2) Oligomer: A molecule that consists of a relatively small and specifiable number of monomers (usually less than five). Unlike a polymer, if one of the monomers is removed from an oligomer, its chemical properties are altered. http://www.thefreedictionary.com/oligomeric
 * 3) Discontinuous: Meaning that more than one segment of the polypeptide is required to form the domain. This is likely to be the result of the insertion of one domain into another during the protein's evolution. It has been shown from known structures that about a quarter of structural domains are discontinuous. http://en.wikipedia.org/wiki/Protein_domain
 * 4) Ectodomain: the part of a membrane protein that extends into the extracellular space (the space outside a cell). Ectodomains are usually the part of a protein that initiate contact with surface which leads to signal transduction. en.wikipedia.org/wiki/Ectodomain
 * 5) Glycosylation: Proteins are modified by the addition of carbohydrates (glycosylation) to specific amino acids in the peptide chain. www.encyclopedia.com/doc/1G2-3400700147.html
 * 6) Immunogenicity: the ability of a particular substance, such as an antigen or epitope, to provoke an immune response. en.wikipedia.org/wiki/Immunogenicity
 * 7) Antigenicity: the ability of a chemical structure (refered to as an Antigen) to bind specifically with certain products of adaptive immunity: T cell receptors or Antibodies (a.k.a. B cell receptors). en.wikipedia.org/wiki/Antigenicity
 * 8) Chemokine: a small protein molecule that activates immune cells, stimulates their migration, and helps direct immune cell traffic throughout the body. www3.niaid.nih.gov/topics/immuneSystem/glossary.htm
 * 9) Fusogenic: Facilitating fusion of the viral envelope with the cellular plasma membrane. www.nature.com/nrg/journal/v4/n5/glossary/nrg1066_glossary.html
 * 10) Prophylactic: A medical procedure or practice that prevents or protects against a disease or condition (eg, vaccines, antibiotics, drugs). www.pandemicflu.gov/glossary/

Outline
Paper1 pmid=9641677

Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody

Introduction
CD4 structure analogous of CDR2 of IG's (2nd hypervariable region) Conserved gp120 residues Chemokine receptors: CCR5 and CXCR4 (for HIV-1) = obligate second receptors for virus entry into CD4 T cells Gp120 V3 region = principal determinant of chemokine receptor specificity, but other more conserved regions involved also How do we know this happens? Following CD4/gp120 binding, enhanced binding of gp120 Abs (called CD4i Ab) which block binding of gp120/CD4 complexes to chemokine receptor (CCR5 and CXCR4).
 * 1) HIV-1 and HIV-2 lead to destruction of CD4+ T Cells in infected hosts, resulting in the eventual development of AIDS
 * 2) Entry of HIV in host cells is mediated by viral envelope proteins, organized in oligomeric (trimeric) spikes on surface of virion.
 * These envelope complexes are anchored in the viral membrane by the gp120 transmembrane envelope glycoprotein.
 * Surface of viral envelope glycoprotein: composed of gp120 + each subunit of trimeric gp41 glycoprotein complex associated together by non-covalent interactions.
 * 1) Five variable regions exist in gp120 sequences: V1-V5
 * V1-V4: form surface-exposed loops containing disulphide bonds at bases
 * Conserved gp120 regions form discontinuous structures (important for interaction  with the gp120 ectodomain and the viral receptors on host cell)
 * Both conserved and variable regions of gp120 protein = extensively glycosylated.
 * 1) Why is this important? Probably modulates the immunogenicity and antigenicity of gp120
 * 2) Gp120 = main target for neutralizing antibodies produced during infection by host immune system.
 * 3) HIV entry into host cell:
 * Involved binding of gp120 envelope glycoprotein to CD4 glycoprotein (primary receptor to gp120: Gp120 binds to amino-terminal of four IG-like domains of CD4, Critical structures for gp120 binding to CD4:
 * CD4 binding induces conformational changes in gp120: exposure/formation of binding site for specific chemokine receptors.
 * 1) Also, CD4/gp120 binding may induce release of gp120 from complex (although importance of this process to HIV entry = uncertain)
 * 2) HIV is a retrovirus and belongs to a class of enveloped fusogenic viruses which require post-translational cleavage for activation. It is very similar to other retroviruses in terms of viral entry but also possesses differences. HIV utilizes direct modes of entry via membrane penetration, rather than endocytosis.
 * 3) Main point: Information about gp120 structure is important for understanding HIV infection and ultimately in designing therapeutics against the virus because of the important role of gp120 in CD4 receptor binding and interactions with neutralizing Abs.
 * 4) Main Result: this paper reports the crystal structure of a partially deglycosylated HIV-1 gp120bound to a two-domain fragment of the CD4 cellular receptor and to the Fab fragment of an Ab directed against the CD4i epitope at 2.5 A resolution . This structure is related to the antigenic properties of the gp120 protein.

Structure Determination
Modified gp120 maintains near wt levels of binding with N-terminal two domains (DiD2) of CD4 and Fab from neutralizing Ab Ternary structure solved by combo of molecular replacement, isomorphic replacement and density modification techniques The deglycosylated core of gp120 appears to be a prolate ellipsoid with dimensions 50x50x25 A (although seems to be more heart-shaped) Core gp120: 25 B-strands, 5 a-helices, and 10 defined loop segments Polypeptide chain of gp120 = folded into two major domains: Inner domain: has a two-helix, two-strand bundle with a small five-stranded B-sandwich at termini-proximal end and projection at distal end (from which V1/V2 stem emanates), Outer domain: stacked double barrel that lies next to inner domain so that outer barrel and inner bundle axis = parallel: Proximal barrel of outer domain: composed of six-stranded, mixed directional B-sheet twisted to embrace a helix, Proximal end of outer domain: includes variable loops V4 and V5, Distal end: includes base of V3 + excursion via additional loop which H-bonds with V1/V2 stem from inner domain, This completes an anti-parallel, four-stranded "bridging sheet" which acts of mini-domain connecting inner and outer domains as well as V1/V2 domain. Inner domain= more conserved than outer domain 7 disulphide bridges = absolutely conserved and mostly buried. Glycosylation sites all surface exposed and conserved more than avg Previously identified HIV variable segments all on loops connecting elements of secondary structure Variable segments in out domain: seems to arise from neutral mutation rather than selective pressure a: Ribbon diagram of inner and outer domain connected by bridging sheet b: Topology diagram: arranged to coincide with orientation of (a) Helices: corkscrews Strands: arrows □ Loops with high sequence variability are labeled and circled c: Stereo plot of an a-carbon trace. Every tenth Carbon is marked with a filled circle and every 20th residue is labeled. Disulphide connections = ball and stick. Ordered residues are shown. d: Structure based sequence alignment. Sequences are shown of HIV-1 B, C, HIV-2, and SIV Secondary structure assignments = arrows and cylinders
 * 1) Researchers devises a crystallization strategy to radically modify the gp120 protein surface.
 * Why? Because of the extensive glycosylation and conformational heterogeneity associated with gp120.
 * How: Made truncations at protein ends and variable loops of gp120, Extensively de-glycosylated these gp120 variants, Produced complexes with various ligands
 * 1) After screening ~20 combos of gp120 variants and ligands, obtained crystals of a ternary complex composed of truncated gp120+CD4+Fab from neutralizing Ab
 * Fig 1: Overall structure of the complex of gp120 with DiD2 (N-terminal two domains) of CD4 and Fab 17b (neutralizing Ab)
 * 1) Structure of gp120:
 * Bridging sheet also participates in interactions of gp120 with both CD4 and Fab Ab
 * Taken as whole: structure of gp120 has not been seen before: No similarity of inner domain to any other known structures of its kind; HOWEVER, do see similarity for portions of outer domain with other known structures.
 * Structure of core gp120 should a prototype for its class of envelope proteins. As seen by the structure-base alignment of other sequences, there is quite a bit of conservation b/w HIV strains.
 * Figure 2: Structure of core gp120

CD4-gp120 interaction
Variation accommodated by large interfacial cavity Gp120 residues in contact with this water-filled cavity = especially variable Half of the gp120 residues that make contacts with CD4 do so only through main-chain atoms (60% of contacts) a: Ribbon diagram of gp120 binding to CD4: Recessed nature of gp120 binding pocket is evident b: Electron density in the Phe43 cavity c: Electrostatic surfaces of CD4 and gp120 d: CD4-gp120 contact surface e: CD4-gp120 mutational hot-spots f: side-chain/main-chain contribution to the gp120 surface g: sequence variability mapped to the gp120 surface h: Phe43 cavity i: CD4-gp120 interface j: gp120 contacts around Phe43 and Arg59 of CD4
 * CD4 bound into a depression formed at the interface of the outer domain and inner domain and bridging sheet of gp120
 * Interaction buries 742 A from CD4 and 802 A from gp120
 * Surface areas in contact = smaller because of mismatches with create large, unmatched cavities
 * The binding site is devoid of carbohydrate
 * Direct inter-atomic contacts are made b/w 22 CD4 and 26 gp120 a.a. residues
 * 1) 219 van der Walls contacts
 * 2) 12 H-bonds
 * 3) Residues in contact are concentrated in the span from 25-64 of CD4, but distributed over six segments of gp120
 * 4) Residues identified as critical for binding interact with each other
 * 5) Phe 43 and Arg 59 of CD4 make multiple contacts centered on Asp 368, Glu 370, and Trp 427 of gp120 (all conserved)
 * Several gp120 residues that are covered by CD4 are variable in sequence are variable in sequence
 * Atomic details of the interaction are intricate and unusual b/w gp120 and CD4 residues Phe43 and Arg 59
 * Figure 3: CD4-gp12 interactions

Interfacial Cavities
The larger is formed at the interface b/w slightly concave middle of CD4 sheet and groove on gp120 Second is from a pocket in the gp120 surface that is plugged by Phe43 from CD4. : This pocket is at interface b/w inner and outer domains of gp120 Several other smaller cavities are also wedged at the interface b/w two gp120 domains Lined mostly by hydrophilic residues, half from gp120 and half from CD4 Not deeply buried Observed electron density and H-bonding are consistent with at least 8 water molecules in the cavity. Residues from gp120 that line cavity show sequence variability (surrounding are conserved residues and are important for effective CD4 binding) CD4 residues with line the cavity can be mutated with only moderate effect on gp120 binding This cavity serves as a water buffer b/w gp120 and CD4 Tolerance for variation in gp120 surface associated with this cavity produces a variation island or "anti-hotspot" -- centrally located b/w regions required for CD4 binding Phe43 cavity: Different from larger cavity; Roughly spherical, diameter ~8A; Positioned just beyond Phe43 of CD4 at the intersection of inner domain, outer domain, and bridging sheet; Deeply buried, extending into the hydrophobic interior of gp120; Multiple routes of solvent access possible, but only a few water molecules are seen within cavity itself; Residues that line cavity are primarily hydrophobic, highly conserved -- implies functional significance; Although residues that line this cavity provide little direct contact to CD4, they do affect gp120-CD4 interaction; This cavity may form as consequence of a CD4 induced conformations change.
 * Analysis of the solvent-accessible surface of the ternary complex reveals a number of topologically interior surfaces (or cavities)
 * 1) Two at the gp120-CD4 interface = unusually large
 * 1) Larger cavity:
 * This may help virus escape from Abs directed against the CD4 binding site.

Antibody Interface
CD4i epitopes tend to be masked from immune surveillance by V2 and V3 loops Base of V3 loop points at this gap. In gp120 -- variable loops may need to be bypassed for aces to conserved structure in bridging sheet 17b epitope may be protected from immune system by a CD4-induced conformational change
 * 17b Ab = broadly neutralizing human monclonal Ab isolated from the blood of HIV-infected individual
 * It binds to a CD4 induced (CD4i) gp120 epitope that overlaps the chemokine receptor binding site
 * Interface b/w Fab 17b and gp120 = small
 * The solvent-accessible area is largely from heavy chain of Fab 17b -- the long CDR3 region from HC dominates but light chain CDR3 also contributes
 * On gp120, the 17b epitope lies across the base of the four-stranded bridging sheet.
 * 1) All four strands of bridging sheet make substantial contact with 17b. Integrity of bridging sheet may be necessary for binding.
 * 2) Gp120 surface that contacts 17b = hydrophobic centre surrounded by basic outside
 * Interaction b/w 17b and gp120 = hydrophobic central region flanked by outside charged regions.
 * No direct CD4-17b contacts and none of gp120 residues contacts both 17b and CD4 (CD4 binds on opposite face of the bridging sheet -- specific contacts seem to stabilize conformation which may explain CD4 induction of 17b binding)
 * 1) 17b epitope= well conserved --more conserved that gp120 residues
 * In the complex structure -- a large gap exists b/w gp120 and tips of LC CHR1 and CDR2 loops
 * Why is this significant? Structure may be important for immune evasion

Chemokine-receptor site
a: Ca worm diagram of Fab 17b and gp120 b: Contact surface and V3 loop c: Contact surface and V3 loop rotated 90 degrees from (b) d: Electrostatic potential at the solvent-accessible surface e: Ca worm diagram of gp120
 * Site of interaction with the chemokine receptor CCR5 overlaps with the 17b epitope: Both are induced upon CD4 binding and involve highly conserved residues
 * Basic and polar gp120 residues that contact 17b HC also important for CCR5 interaction
 * 17b HC may mimic N-term region of CCR5 which is important for gp120 binding and HIV-1 entry
 * Site is directed at cellular membrane when gp120 engaged by CD4
 * Interactions b/w basic surface of bridging sheet and acidic chemokine receptor could drive conformational change related to virus entry
 * Figure 4: Neutralizing antibody 17b-gp120 interface

Oligomer and gp120 interactions

 * Gp120 is monomeric in isolation but probably exist as a trimeric complex with gp41 on virion surface
 * 1) Probably site of trimer binding: large electroneutral surface on inner domain. Why? Lack of glycosylation, conservation of sequence, location of CD4 and CCR5 binding sites, and immune response to this region
 * N and C term regions of full-length gp120 = most important regions for interaction with the gp41 protein
 * 1) Expts show that gp41 interacting regions extend away from core gp120 towards the viral membrane and that the conserved surface is closed off in the oligomer/gp41 interface

Conformational change in core gp120
Evolutionary algorithms of known sequence variants of gp120 gives secondary structure predictions with high reliability Compared with structure predicted by authors, theory is accurate except at three places where it is wrong: at Phe43 cavity or locations in contact with CD4 State 1: single monomer of gp120 -- V1/V2 loops = partially block the CD4 binding site Following CD4 binding -- conformational change depicted as an inner/outer domain shift and formation on Phe43 cavity Chemokine receptor binds to the bridging sheet and the V3 loop causing orientational shift of core gp120 Triggers further changes which leads to fusion of viral and target membranes
 * Although abundant evidence to suggest that CD4 binding induces a conformational change in gp120, this evidence derives from intact gp120 with V loops in place or from oligomeric gp120-gp41 complex: No evidence yet to explain the nature of the conformational change which occurs within core gp120 itself -- studying the ternary complex structure could tell us what these changes are
 * Evidence for CD4-induced conformational change:
 * 1) If the new conformation of gp120 activated by binding of CD4 were preserved in the absence of CD4 = structural dilemma with Phe43 cavity: Why? Cavity lining residues have few structural restrictions yet residues highly conserved and hydrophobic if exposed in a pocket. This pocket structure is therefore intimately connected to the bridging sheet (another structural dilemma in absence of CD4; Structures seen in presence of CD4 would be sensitive would be sensitive to orientational shifts b/w the inner and outer domains
 * 2) Characteristics of 17b binding to core gp120: Do not observe detectable binding of Fab 17b to core gp120 UNLESS CD4 is present; Because no direct CD4-17b contacts in structure, effect of CD4 must be to stabilize bridging-sheet minidoman to which 17b binds (suggesting conformational change induced by CD4 binding); Binding of CD4 to gp120 not limited to an unmasking of Ab epitope.
 * 3) Comparison with theory
 * 1) Phe43 cavity is the nexus of the CD4 interface -- lying b/w the inner and outer domain and bridging sheet = without which structure might collapse: How does CD4 binding lead to this state? Exceptional CD4 binding thermodynamics suggest answer is a large conformational change in gp120 occurs upon CD4 binding; CD4 inserts Phe43 to stabilize the cavity and the interaction b/w itself and gp120
 * Figure 5: Diagram of gp120 initiation of fusion.

Viral Evasion of Immune Response

 * How?
 * 1) Analysis of antigenic structure of gp120 shows that most of the envelope protein surface is hidden from humoral immune response by glycosylation and oligomeric blockage
 * 2) Most neutralizing antibodies access only two surfaces: one that overlaps CD4-bidnign site (shielded by V1/V2 loop) and another that overlaps the chemokine-receptor binding site (Shielded by V2/V3 loops)
 * 3) Conformational changes in core gp120
 * 4) CD4-bidngin site recognition by neutralizing Ab prevented by…: Recessed nature of the binding pocket; Topographical surface mismatch
 * 5) Chemokine receptor region recognition by neutralizing Ab prevented by…: Conformational change may hide conserved epitope; Steric blockage may take place b/w CD4 and target membrane; Surface mismatch may camouflage chemokine-receptor binding site on V3 loop

Mechanistic implications for virus entry
Gp120 functions in: POSITIONING: Locating a cell capable of productive viral infection; Anchoring virus to cell surface; Orienting viral spike next to target membrane Gp120 function in: TIMING: Holding gp41 in specific conformation and triggering release of fusion peptides of trimeric gp41; Entry process initiated by binding of HIV-1 to cellular receptor CD4; Proposed structure suggests: this binding orients viral spike: orients the N and C termini of gp120 towards viral membrane and the 17b chemokine receptor binding site on gp120 surface faces target cell. Structure described here describes state in atomic detail CD4 binding results in movement of V2 loop, which partially blocks V3 loop and CD4 interacting epitopes Also creates/stabilizes bridging sheet CD4 binding changes V3 region -- altering exposure of V3 epitopes: Uncovered V3 and CD4i epitopes create chemokine-receptor binding site; THUS: CD4 binding not only orients the gp12 surface implicated in chemokine receptor binding to face target cell but also forms and exposes site itself Gp120 contacts dominate interaction with chemokine receptor Because most of chemokine receptor encased in host membrane, binding will move gp120 closer to target membrane Chemokine receptor binding believed to trigger additional conformational changes in envelope glycoprotein trimer leading to exposure of gp120 ectodomain Perhaps chemokine receptors triggers gp41 exposure by taking gp120 protomers away from the trimer axis
 * During virus entry, HIV entry proteins fuse the viral membrane with target cell membrane. Gp120 crucial for fusion of HIV to cell surface
 * 1) Given data presented here, suggest this may be a simple process: Comprises two membrane: viral oligomer and two host receptors. Two snapshots: an intermediate stare in which gp120 bound to CD4 and a final "fusion-active" state of gp41 ectodomain.
 * CD4 binding induces conformational change in gp120
 * Next step: Interaction of gp120-CD4 complex with chemokine receptor
 * Structure of gp120/CD4/17b antibody ternary complex described here revels many ,molecular aspects of HIV-1 entry including atomic structure of gp120, explicit interaction with CD4 and conserved binding for the chemokine receptor
 * Still unknown: Details of apo state of core gp120, oligomeric structure, interactions with chemokine receptors, etc. Further understanding will require snapshots of intermediates

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